JBC Hist Persp Lipids PDF

HISTORICAL PERSPECTIVES
Lipid
Biochemistry
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The Journal of Biological Chemistry
TABLE OF CONTENTS
2010
HISTORICAL PERSPECTIVES ON LIPID BIOCHEMISTRY
PROLOGUE H20 The Prostaglandins, Sune Bergström and Bengt Samuelsson
H1 JBC Historical Perspectives: Lipid Biochemistry. Nicole Kresge, H23 Biotin-dependent Enzymes: the Work of Feodor Lynen
Robert D. Simoni, and Robert L. Hill H25 Acetyl-CoA Carboxylase and Other Biotin-dependent Enzymes:
the Work of M. Daniel Lane
CLASSICS H28 The Role of the Acyl Carrier Protein in Fatty Acid Synthesis: the
H3 The Biosynthetic Pathway for Cholesterol: Konrad Bloch Work of P. Roy Vagelos
H30 How Aspirin Interferes with Cyclooxygenase Activity: the Work
H6 The ATP Requirement for Fatty Acid Oxidation: the Early Work
of William L. Smith
of Albert L. Lehninger
H33 30 Years of Cholesterol Metabolism: the Work of Michael
H8 The Kennedy Pathway for Phospholipid Synthesis: the Work of Brown and Joseph Goldstein
Eugene Kennedy
H37 Salih Wakil’s Elucidation of the Animal Fatty Acid Synthetase
H11 Fatty Acid Synthesis and Glutamine Synthetase: the Work of Complex Architecture
Earl Stadtman
H40 N-Myristoyltransferase Substrate Selection and Catalysis: the
H14 A Role for Phosphoinositides in Signaling: the Work of Mabel R. Work of Jeffrey I. Gordon
Hokin and Lowell E. Hokin
H16 The Selective Placement of Acyl Chains: the Work of William REFLECTIONS
E. M. Lands H42 Hitler’s Gift and the Era of Biosynthesis. Eugene P. Kennedy
H18 Lipid Storage Disorders and the Biosynthesis of Inositol H55 The Biotin Connection: Severo Ochoa, Harland Wood, and
Phosphatide: the Work of Roscoe Brady Feodor Lynen. M. Daniel Lane
JOURNAL OF BIOLOGICAL CHEMISTRY i

PROLOGUE This paper is available online at www.jbc.org
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
JBC Historical Perspectives: Lipid Biochemistry*

Nicole Kresge, Robert D. Simoni, and Robert L. Hill
Lipids are often broadly, and poorly, defined as biomolecules sis. He and his graduate student Samuel Weiss eventually deter-
that are insoluble in water but soluble in organic solvents. They mined that intermediary formation of CDP-choline was occurring
are structurally quite diverse, covering pigments, vitamins, fatty in the reaction. Using 14C to label the cytidine coenzymes, the pair
acids, cholesterol, phospholipids, sphingolipids, and many oth- proved that CDP-choline and cytidine diphosphate ethanolamine
ers. The Journal of Biological (JBC) Classic articles selected for were activated forms of phosphorylcholine and phosphoryleth-
this collection fall into two general categories: lipid biosynthesis anolamine and were precursors of lecithin and phosphatidyleth-
and lipid signaling. Full accounts of the research and attribution anolamine. This scheme of phospholipid synthesis became known
can be found in each Classic. as the “Kennedy Pathway.”
In 1958, William E. M. Lands, who is best known for his
Fatty Acid Synthesis discovery of the phospholipid retailoring or “Lands” pathway,
Early work on fatty acid synthesis was done by Horace A. published results in the JBC that suggested “the diglyceride unit
Barker and Earl R. Stadtman, who, in 1949, published a JBC of the phospholipids is metabolically different in some respect
paper examining the synthesis of short chain fatty acids by a from that of the triglycerides.” This initial finding led Lands to
species of bacteria called Clostridium kluyveri. The pair took publish a series of papers in the JBC describing the selective
advantage of the newly available 14C isotope and used it to label placement of acyl chains by phospholipid acyltransferases.
acetate and demonstrate that fatty acid synthesis is accom-
plished by the multiple condensation of 2-carbon molecules. Cholesterol Synthesis and Regulation
Stadtman later developed an in vitro extract to study the enzy- The synthesis of cholesterol has a long and impressive history
mology of fatty acid synthesis. in the JBC. Nobel laureate Konrad Bloch and his colleagues
The detailed enzymology of fatty acid synthesis came, in part, made important contributions to the understanding of the syn-
from the work of P. Roy Vagelos and Salih Wakil. Using Esche- thetic pathway for cholesterol, which he published in the JBC in
richia coli extracts in which all of the enzymes of fatty acid the mid-1940s. Eventually, through the combined efforts of
synthesis were soluble, Vagelos was able to define the individual Bloch, John Cornforth, and George Popják, the origin of each of
steps in long chain fatty acid synthesis and found that acyl car- the 27 individual carbon atoms of cholesterol (from the methyl
rier protein (ACP) was the small protein to which growing acyl or carboxyl group of acetate) was established. Bloch also aided
chains were attached during the elongation cycle. He published in the identification of several important landmarks in the
his findings in 1965 as a series of JBC papers, two of which are series of more than 30 reactions in the biosynthesis of choles-
reproduced as JBC Classics. Wakil, who contributed to the terol, including the intricate and fascinating cyclization of squa-
studies using the E. coli soluble system, also went on to examine lene to lanosterol.
fatty acid synthesis in animals, where the enzyme activities The regulation of cholesterol synthesis also has a history that
are part of a multifunctional enzyme complex. This work was is reflected in the JBC. A 1974 paper by Nobel laureates and long
published as a series of JBC papers in 1983. time collaborators Michael S. Brown and Joseph L. Goldstein
Adding to the studies done by Vagelos and Wakil, M. Daniel established the role of LDL as a major regulator of cholesterol
Lane, initially working with Feodor Lynen, described the first synthesis. This pioneering work also revealed the existence of a
step in fatty acid synthesis, which involves the conversion of cell surface receptor for LDL, shed light on the process of recep-
acetyl-CoA to malonyl-CoA by the enzyme acetyl-CoA carbox- tor-mediated endocytosis, and explained the receptor defect in
ylase. He also explained the role of the vitamin biotin in carbox- the human genetic disease, familial hypercholesterolemia.
ylation reactions.
Lipid Signaling
Phospholipid Synthesis
Phosphoinostides—The role of lipid molecules in signaling
One of the pathways for phospholipid synthesis was deter-
has also enjoyed a lot of coverage in the JBC. Lowell and Mable
mined, in part, by Eugene P. Kennedy and his colleagues. However,
Hokin’s two papers, published in 1953 and 1958, demonstrated
Kennedy began his research career working not on lipid biosyn-
that the acetylcholine-stimulated secretion of amylase from
thesis but on fatty acid oxidation with Albert Lehninger. In the
pancreas slices caused the incorporation of 32P into phospho-
mid-1940s, they published results in the JBC that showed that ATP
inositides. This so-called “PI Effect” laid the groundwork for
was required to “activate” fatty acids for degradation. Later,
thousands of studies on the role of phosphoinositides in
Kennedy turned his attention to synthesis reactions and made the
signaling.
important discovery that CTP is required for phospholipid synthe-
Roscoe O. Brady studied the synthesis of inositol phospha-
tides, and in 1958, he published a JBC paper showing that an
* To cite articles in this collection, use the citation information that appears in enzyme system catalyzed the incorporation of inositol into inositol
the upper right-hand corner of the first page of the article. phosphatide in the presence of Mg2⫹ and CDP or cytidine
JOURNAL OF BIOLOGICAL CHEMISTRY

H1
PROLOGUE: Lipid Biochemistry
5⬘-phosphate. From these results, Brady proposed a mechanism Gordon, who has two Classic papers, has made significant
for the synthesis of inositol phosphatide in which CDP is contributions to our knowledge about protein myristoyla-
transphosphorylated to form CDP-D-␣,␤-diglyceride, which then tion, the post-translational process by which a myristoyl
reacts with the hydroxyl group of inositol to form inositol group is covalently attached via an amide bond to the ␣-
phosphatide. amino group of an N-terminal glycine residue of a nascent
Prostaglandins—The prostaglandins are another class of polypeptide.
important lipid signaling molecules. Nobel laureates Sune Berg-
ström and Bengt Samuelson were pioneers in establishing the bio- Reflections
synthesis and structures of several of these molecules. They pub-
This collection also contains two JBC Reflections. The
lished some of these studies in the JBC in the mid-1960s.
One of the more fascinating roles that prostaglandins play is first, titled “Hitler’s Gift and the Era of Biosynthesis,” by
in mediating inflammation. In the early 1970s, John Vane Eugene P. Kennedy, discusses how the war brought on a flow
showed that aspirin and other anti-inflammatory drugs inhibit of students to America, which caused a surge in biochemis-
prostaglandin synthesis. JBC Associate Editor William L. Smith try research and biosynthetic studies there. In the second
went on to show that aspirin blocks cyclooxygenase (COX1) by article, M. Daniel Lane talks about his research and the cir-
acylating a serine residue. cumstances that brought him into contact with three great
biochemists: Severo Ochoa, Harland Wood, and Feodor
Lipid Modification of Proteins Lynen.
Many proteins, particularly membrane proteins, are mod- We hope you enjoy this collection that we have assembled for
ified by post-translational covalent lipid attachment. Jeffrey you.
JOURNAL OF BIOLOGICAL CHEMISTRY

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 10, Issue of March 11, p. e7, 2005
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
Classics
A PAPER IN A SERIES REPRINTED TO CELEBRATE THE CENTENARY OF THE JBC IN 2005
JBC Centennial
1905–2005
100 Years of Biochemistry and Molecular Biology
The Biosynthetic Pathway for Cholesterol: Konrad Bloch

On the Utilization of Acetic Acid for Cholesterol Formation
(Bloch, K., and Rittenberg, D. (1942) J. Biol. Chem. 145, 625– 636)
The Utilization of Acetic Acid for the Synthesis of Fatty Acids
(Rittenberg, D., and Bloch, K. (1945) J. Biol. Chem. 160, 417– 424)
The Biological Conversion of Cholesterol to Pregnanediol
(Bloch, K. (1945) J. Biol. Chem. 157, 661– 666)
Konrad Emil Bloch (1912–2000) was born in Neisse, eastern Germany (now Nysa in Poland).
Growing up he was more interested in engineering and natural sciences than chemistry, but
an organic chemistry course taught by future Nobel laureate Hans Fischer at the Technische
Hochschule in Munich provided a turning point. Bloch said of Fischer’s class, “As he presented
it, the subject matter was fascinating, the organization superb, and the delivery monotonous”
(1). Despite Fischer’s monotone delivery, Bloch was influenced enough to become a chemistry
student in his laboratory.1
In early 1934, Bloch was told by Nazi authorities, in line with new racial laws, that he could
no longer study at the Technische Hochschule. Fischer managed to arrange for Bloch to work
at the Schweizerisches Höhenforschung’s Institut in Davos. There, he studied the lipids of
human tubercle bacilli and was able to show that a previous report of the presence of
cholesterol in this organism was erroneous. This was Bloch’s first encounter with cholesterol,
a subject in which he would eventually play a great role.
In 1936, with the help of R. J. Anderson at Yale University, Bloch immigrated to the United
States and started working with Hans Clarke at the College of Physicians and Surgeons (P &
S), Columbia University. He received his Ph.D. a year and one-half later, after completing a
relatively straightforward piece of research on amino acid chemistry, and was then invited by
Rudolf Schoenheimer to join his group.
Also in Schoenheimer’s laboratory at this time was a scientist named David Rittenberg who
had done his graduate work on deuterium with Harold Urey. Shortly after receiving his
degree, Rittenberg approached Schoenheimer with the prospect of using deuterium as a
biological tracer. This resulted in the publication of several seminal papers authored by
Rittenberg and Schoenheimer on the use of deuterium to study metabolism, which was the
subject of a previous Journal of Biological Chemistry (JBC) Classic (2).
Schoenheimer was eager for Bloch to apply the isotope tracer method to study the biosyn-
thesis of cholesterol and had him start by investigating whether the hydroxyl oxygen in
cholesterol came from water or oxygen. Unfortunately, Bloch was unable to solve this first
problem because no method existed at that time for the mass spectrometric analysis of stably
bound oxygen in complex organic compounds. Eventually, in 1956, Bloch’s student T. T. Tchen
would show that molecular oxygen is the source of the hydroxyl oxygen (3).
Schoenheimer died in 1941, and his laboratory’s research projects were divided up among its
members. Bloch inherited lipids, and Rittenberg acquired protein synthesis. Although he was
still working on the cholesterol oxygen problem at that time, Bloch’s attention was quickly
diverted with the publication of a paper by R. Sonderhoff and H. Thomas, which reported that
“The nonsaponifiable fraction of yeast grown in a medium supplemented with deuterated
1
All biographical information on Konrad Bloch was taken from Refs. 1, 7, 8, and 9.
This paper is available on line at http://www.jbc.org
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Classics
Konrad Bloch. Photo courtesy of the National Library of Medicine.
acetate had a deuterium content so high that a direct conversion of acetic acid to sterols has
to be postulated” (4). Schoenheimer and Rittenberg had also done experiments with D2O that
indicated that animal cholesterol is synthesized from small molecules (5). Combining their
areas of expertise, Rittenberg and Bloch did the next obvious experiment: they fed labeled
acetates to rats and mice. As reported in the first JBC Classic reprinted here, they found that
a substantial amount of deuterium was incorporated into cholesterol. However, their results
did not tell them how many of the 27 sterol carbon atoms were supplied by acetic acid. The
definitive answer came 10 years later when Bloch used an acetateless mutant of Neurospora
crassa to show that the mutant’s sterol derived all its carbon atoms from exogenous acetate (6).
Over time, Rittenberg added 15N, 13C, and 18O to the isotopes he used to study biological
processes. This led to a second labeling experiment with Bloch, this time showing that acetic
acid is used in the synthesis of fatty acids. This work is described in the second JBC Classic.
Rittenberg and Bloch fed sodium acetate labeled with 13C and deuterium to mice and rats and
found that the animals’ lipids and cholesterol contained both labeled carbon and hydrogen.
From this they concluded that both carbon atoms in acetic acid were used for the synthesis of
fatty acids and cholesterol.
Eventually Rittenberg became director of the isotope laboratory at P & S in 1941 and
remained there until he retired. His research with isotope tracers encompassed a wide variety
of subjects, including the study of hippuric acid metabolism, the dynamics of red blood cell
survival in patients with blood abnormalities, the development of a method to assay amino
acids in protein hydrolysates, and investigations into the synthesis of porphyrin, which will be
the subject of a future JBC Classic. Rittenberg’s many contributions to the isotope tracer
technique were recognized when he was awarded the Eli Lilly Award in Biological Chemistry
from the American Chemical Society and also with his election to the National Academy of
Sciences in 1953.2
In addition to using isotopes to study the biosynthesis of cholesterol, Bloch also used the
tracers to examine the precursor role of cholesterol in bile acids and steroid hormones. In the
final JBC Classic reprinted here, Bloch demonstrates that cholesterol is converted into pro-
gesterone. However, Bloch encountered several logistical problems when starting this exper-
iment. First, labeled cholesterol was unavailable commercially so he had to spend much of his
time introducing deuterium into cholesterol by platinum-catalyzed exchange in heavy water-
acetic acid mixtures. Second, the only practical source for isolating the progesterone metabo-
lite pregnanediol in sufficient quantity was from human pregnancy urine, and his request to
the P & S department of obstetrics and gynecology for permission to administer labeled
cholesterol to one of its patients was denied. Bloch eventually managed to obtain his preg-
nanediol due to a “willingness to cooperate at home” (1) and proved that progesterone was
indeed synthesized from cholesterol.
In 1946 Bloch moved to the Department of Biochemistry at the University of Chicago and
then to Harvard University in 1954. He continued to study fatty acids and cholesterol as well
as the enzymatic synthesis of the tripeptide glutathione. Eventually, through the combined
2
All biographical information on David Rittenberg was taken from Ref. 10.
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Classics
efforts of Bloch, John Cornforth, and George Popják, the origin of each of the 27 individual
carbon atoms of cholesterol (from the methyl or carboxyl group of acetate) was established.
Bloch also aided in the identification of several important landmarks in the series of more than
30 reactions in the biosynthesis of cholesterol, including the cyclization of squalene to lanos-
terol. His work on fatty acids and cholesterol was eventually rewarded when he shared the
1964 Nobel Prize in Physiology or Medicine with Feodor Lynen “for their discoveries concern-
ing the mechanism and regulation of the cholesterol and fatty acid metabolism.”
The elucidation of the pathway from acetic acid to cholesterol was not only a tremendous
achievement for biochemistry but also of great importance to medicine. Knowledge of the
biosynthetic pathway for cholesterol eventually aided in the discovery of statins, drugs that
interfere with cholesterol synthesis, which are now widely used to treat high cholesterol.
REFERENCES
1. Bloch, K. (1987) Summing up. Annu. Rev. Biochem. 56, 1–19
2. JBC Classics: Schoenheimer, R., and Rittenberg, D. (1935) J. Biol. Chem. 111, 163–168; Rittenberg, D., and
Schoenheimer, R. (1937) J. Biol. Chem. 121, 235–253 (http://www.jbc.org/cgi/content/full/277/43/e31)
3. Tchen, T. T., and Bloch, K. (1956) On the mechanism of cyclization of squalene. J. Am. Chem. Soc. 78, 1516 –1517
4. Sonderhoff, R., and Thomas, H. (1937) Ann. Chem. 530, 195–213
5. Rittenberg, D., and Schoenheimer, R. (1937) Deuterium as an indicator in the study of intermediary metabolism.
XI. Further studies on the biological uptake of deuterium into organic substances, with special reference to fat
and cholesterol formation. J. Biol. Chem. 121, 235–253
6. Ottke, R. C., Tatum, E. L., Zabin, I., and Bloch, K. (1951) Isotopic acetate and isovalerate in the synthesis of
ergosterol by Neurospora. J. Biol. Chem. 189 429 – 433
7. Kennedy, E. P. (2001) Hitler’s gift and the era of biosynthesis. J. Biol. Chem. 276, 42619 – 42631
8. Goldfine, H., and Vance, D. E. (2001) Obituary: Konrad E. Bloch (1912–2000) Nature 409, 779
9. Kennedy, E. P. (2003) Biographical memoirs: Konrad Bloch. Proc. Am. Philos. Soc. 147, 65–72
10. Shemin, D., and Bentley, R. (2001) Biographical Memoir of David Rittenberg, Vol. 80, pp. 256 –275, National
Academy of Sciences, Washington, D. C.
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 14, Issue of April 8, p. e11, 2005
Classics
JBC Centennial
1905–2005
The ATP Requirement for Fatty Acid Oxidation: the Early

Work of Albert L. Lehninger
The Relationship of the Adenosine Polyphosphates to Fatty Acid Oxidation in
Homogenized Liver Preparations
(Lehninger, A. L. (1945) J. Biol. Chem. 157, 363–382)
Albert Lester Lehninger (1917–1986) was born in Bridgeport, Connecticut. In 1935 he
enrolled at Wesleyan University as an English major. Although his interests soon changed
to chemistry, Lehninger would later make use of his writing talents to author three classic
textbooks: Biochemistry, The Mitochondrion, and Bioenergetics. Inspired by the work of
Otto Warburg and Hans Krebs, Lehninger went on to graduate school at the University of
Wisconsin and received his Ph.D. in 1942. His graduate research with Edgar J. Witzemann
was on the metabolism of acetoacetate and the oxidation of fatty acids by disrupted liver
preparations.
The Journal of Biological Chemistry (JBC) Classic reprinted here concerns Lehninger’s
work on fatty acid oxidation. At the time, much of what was known about glycolysis and the
citric acid cycle had been elucidated from minced tissue and tissue extracts. However, similar
studies on fatty acid oxidation had been hampered by the fact that ruptured liver cells lost
their ability to oxidize fatty acids. Luis F. Leloir and Juan M. Muñoz had some success with
liver homogenates at low temperatures in the presence of oxygen, inorganic phosphate,
fumarate, cytochrome c, adenylic acid, and magnesium ions (1), but these experiments were
not always reproducible. When the reaction was carried out successfully, Leloir and Muñoz
noted that there was a decrease in ATP phosphorus and phosphopyruvic acid phosphate and
an increase in inorganic phosphate, indicating the reaction was somehow coupled with
phosphorylation.
In examining these early experiments, Lehninger realized that high concentrations of ATP
or ADP might be required to activate or facilitate oxidation by the liver extracts. He came to
this conclusion for a number of reasons including the fact that ATP is rapidly dephosphoryl-
ated when cells are disrupted and that the fumarate needed for the reaction might provide a
substrate for oxidations capable of phosphorylating adenylic acid to ATP. In the Classic,
Lehninger proves his hypothesis by adding ATP to a homogenized liver preparation and
demonstrating that it consistently and reproducibly carried out fatty acid oxidation. Subse-
quent work would show that fatty acids are activated by the formation of a thioester linkage
between the carboxyl group of the fatty acid and the sulfhydryl group of coenzyme A. This
reaction is driven by ATP.
With the start of the war, Lehninger abandoned his fatty acid studies and joined the
wartime research effort of the Plasma Protein Fractionation Program led by Edwin Joseph
Cohn, who was the author of a previous JBC Classic (2). During this time, Lehninger
discovered several papers on oxidative phosphorylation, and from then on the mechanisms of
energy capture and transduction in cells became the central focus of his research.
In 1945 Lehninger accepted a faculty position at the University of Chicago. During his 6
years in Chicago, Lehninger and two of his students would make two significant discoveries
that would contribute greatly to the study of metabolism. First, Lehninger and Eugene P.
Kennedy would discover that virtually all of the cell’s oxidative activity occurred in the
mitochondria. Second, Lehninger and Morris E. Friedkin would show that electron transport
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Classics
Albert L. Lehninger. Photo courtesy of the National Library of Medicine.
from NADH to oxygen is an immediate and direct energy source for oxidative phosphorylation.
Lehninger’s work with Kennedy and the latter part of his research career will be the subject
of a future JBC Classic.1
REFERENCES
1. Muñoz, J. M., and Leloir, L. F. (1943) Fatty acid oxidation by liver enzymes. J. Biol. Chem. 147, 355–362
2. JBC Classics: Cohn, E. J., Hendry, J. L., and Prentiss, A. M. (1925) J. Biol. Chem. 63, 721–766 (http://www.jbc.org/
cgi/content/full/277/30/e19)
3. Lane, M. D., and Talalay, P. (1986) Albert Lester Lehninger 1917–1986. J. Membr. Biol. 91, 194 –197
1
All biographical information on Albert Lester Lehninger was taken from Ref. 3.
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 25, Issue of June 24, p. e22, 2005
Classics
JBC Centennial
1905–2005
The Kennedy Pathway for Phospholipid Synthesis: the

Work of Eugene Kennedy
Oxidation of Fatty Acids and Tricarboxylic Acid Cycle Intermediates by Isolated Rat
Liver Mitochondria
(Kennedy, E. P., and Lehninger, A. L. (1949) J. Biol. Chem. 179, 957–972)
The Function of Cytidine Coenzymes in the Biosynthesis of Phospholipides
(Kennedy, E. P., and Weiss, S. B. (1956) J. Biol. Chem. 222, 193–214)
Eugene Patrick Kennedy was born in Chicago in 1919. He enrolled at De Paul University in
1937 as a chemistry major and then went to the University of Chicago in 1941 for graduate
training in organic chemistry. To pay his tuition, Kennedy also got a job in the chemical
research department of Armour and Company, one of the large meat packers in Chicago. As
part of the war effort, his job at Armour was to assist in the large scale fractionation of bovine
blood to obtain pure bovine serum albumin. It was believed that the bovine serum albumin
might be useful for treating shock in soldiers on the battlefield. However, by the end of 1942,
hope had faded that bovine serum albumin would be an effective treatment, and the Red Cross
started to collect blood from volunteers instead. Armour opened a new facility in Fort Worth,
Texas for the fractionation of human blood from donors, and Kennedy was sent to Fort Worth
to assist in this effort. He remained in Texas until 1945, when the war was clearly nearing its
end and large amounts of human plasma proteins had been stockpiled.
Returning to the University of Chicago, Kennedy immediately transferred from the Depart-
ment of Chemistry to the Department of Biochemistry. His experience on the plasma project
had led to a new appreciation of biochemistry. When he was ready to begin research for his
dissertation, Kennedy approached Albert Lehninger, a young faculty member whose earlier
research was the subject of a previous Journal of Biological Chemistry (JBC) Classic (1). At
that time, Lehninger was studying oxidative phosphorylation and fatty acid oxidation.
Kennedy writes, “With staggering naiveté, I suggested to him that the proper approach would
be to purify the various enzymes undoubtedly involved in fatty acid oxidation and crystallize
them. He agreed that this would be desirable, but went on to point out rather gently that fatty
acid oxidation had not yet been demonstrated in a soluble extract from which individual
enzymes might be isolated. To reach that stage, it would first be necessary to discover the
nature of the energy-requiring activation or ”sparking“ of fatty acid oxidation and the special
dependence of the process on particulate structures” (2).
Despite this initial incident, Lehninger agreed to take Kennedy on as a graduate student,
and he began to work on the problem of fatty acid oxidation in 1947. Lehninger had observed
that both fatty acid oxidation and oxidative phosphorylation were inhibited in a strikingly
parallel fashion when particulate enzyme preparations of homogenized rat livers were exposed
to hypotonic buffers. The activity could be preserved by adding either salts or iso-osmotic
amounts of sucrose to the buffers.
Kennedy’s first project in the laboratory was a detailed study of these effects (3). These
studies led Lehninger and Kennedy to surmise that fatty acid oxidation, oxidative phospho-
rylation, and the Krebs cycle must all be taking place in one organelle, bounded by a
membrane impermeable to certain solutes. Although their enzyme preparations were quite
crude, they were convinced that the organelle was the mitochondrion, even though function-
ally and morphologically intact mitochondria had not yet been isolated.
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Classics
Around this time George Palade and his collaborators were developing methods for the
separation and identification of organelles. As reported in a previous JBC Classic (4), Palade
worked out a method for the isolation of purified mitochondria by differential centrifugation in
0.88 M sucrose. Kennedy immediately tested mitochondria isolated by this method and ob-
tained convincing evidence that oxidative phosphorylation, fatty acid oxidation, and the
reactions of the Krebs cycle did indeed occur in the mitochondria. This is the subject of the first
JBC Classic reprinted here.
After finishing graduate school, Kennedy went to the University of California, Berkeley, to
work with Horace A. Barker. Barker and his graduate student Earl Stadtman, both of whom
will be featured in future JBC Classics, had just discovered that soluble extracts of Clostrid-
ium kluyveri cells could produce short-chain fatty acids from ethyl alcohol. Although the initial
discovery had already been made, there was much to be learned about these extracts and
Kennedy aided in this effort.
In 1950, Kennedy joined Fritz Lipmann, author of a previous JBC Classic (5), at Harvard
Medical School.1 He then returned to the University of Chicago in 1951, after being given a
joint appointment in the Department of Biochemistry and the newly organized Ben May
Laboratory for Cancer Research.
In Chicago, Kennedy started to study the origins of the phosphodiester bond of phos-
phatidylcholine using labeled choline. He found that free choline, but not phosphocholine,
was converted to lipid in a reaction dependent on ATP generated by oxidative phosphoryl-
ation (6). At the same time, Kornberg and Pricer (7) reported experiments in which
phosphocholine was converted to a lipid (later identified as lecithin) in a reaction that
required ATP. Determined to understand why he and Kornberg had obtained contradicting
results, Kennedy, along with his graduate student Samuel Weiss, undertook a detailed
examination of the differences between the two studies. They discovered that they could
reproduce Kornberg’s results using commercially available ATP. However, large amounts
of ATP were needed, suggesting that an impurity, rather than ATP, might be involved in
the reaction.
Kennedy and Weiss’ discovery of the cofactor involved in the conversion of phosphocholine
to lecithin is the subject of the second JBC Classic reprinted here. After testing several
nucleoside triphosphates, they realized that cytidine triphosphate (CTP) was the active
cofactor in the phosphocholine reaction. They formulated a number of schemes to account
for the involvement of CTP in phospholipid synthesis and eventually decided that inter-
mediary formation of cytidine diphosphate choline (CDP-choline) was occurring in the
reaction. Although they had no evidence for its involvement, they synthesized CDP-choline
and cytidine diphosphate ethanolamine and tested their abilities to act as cofactors in lipid
biosynthesis. Using 14C to label the cytidine coenzymes, Kennedy and Weiss proved that
CDP-choline and cytidine diphosphate ethanolamine were activated forms of phosphoryl-
choline and phosphorylethanolamine and were precursors of lecithin and phosphatidyleth-
anolamine. They also showed that the two cytidine coenzymes were present in high
quantities in liver and yeast.
In 1959, Kennedy was invited to become a Hamilton Kuhn Professor and head of the
Department of Biological Chemistry at the Harvard Medical School. He continued his research
on phospholipid biosynthesis and was able to formulate a detailed picture of the pathways of
biosynthesis of the principal glycerophosphatides and of triacylglycerol by 1961. Kennedy’s
interests also led him to investigate membrane biogenesis and function in bacteria, the
translocation of membrane phospholipids, and periplasmic glucans and cell signaling in
bacteria. Kennedy is currently at Harvard as the Hamilton Kuhn Professor of Biological
Chemistry and Molecular Pharmacology, Emeritus.2
REFERENCES
1. JBC Classic: Lehninger, A. L. (1945) J. Biol. Chem. 157, 363–382 (http://www.jbc.org/cgi/content/full/280/14/e11)
2. Kennedy, E. (1992) Sailing to Byzantium. Annu. Rev. Biochem. 61, 1–28
3. Lehninger, A. L, and Kennedy, E. P. (1948) The requirements of the fatty acid oxidase complex of rat liver. J. Biol.
Chem. 173, 753–771
1
Please see Ref. 8 for Kennedy’s JBC Reflection on Fritz Lipmann, Rudolf Schoenheimer, and Konrad Bloch.
2
All biographical information on Eugene P. Kennedy was taken from Ref. 2.
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4. JBC Classic: Hogeboom, G. H., Schneider, W. C., and Palade, G. E. (1948) J. Biol. Chem. 172, 619 – 635
(http://www.jbc.org/cgi/content/full/280/22/e19)
5. JBC Classic: Lipmann, F. (1945) J. Biol. Chem. 160, 173–190 (http://www.jbc.org/cgi/content/full/280/21/e18)
6. Kennedy, E. P. (1953) The synthesis of lecithin in isolated mitochondria. J. Am. Chem. Soc. 75, 249 –250
7. Kornberg, A., and Pricer, W. E. (1952) Fed. Proc. 11, 242
8. Kennedy, E. P. (2001) Hitler’s gift and the era of biosynthesis. J. Biol. Chem. 276, 42619 – 42631
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 26, Issue of July 1, p. e23, 2005
Classics
JBC Centennial
1905–2005
Fatty Acid Synthesis and Glutamine Synthetase: the Work

of Earl Stadtman
Fatty Acid Synthesis by Enzyme Preparations of Clostridium kluyveri. I. Prepara-
tion of Cell-free Extracts That Catalyze the Conversion of Ethanol and Acetate to
Butyrate and Caproate
(Stadtman, E. R., and Barker, H. A. (1949) J. Biol. Chem. 180, 1085–1093)
Allosteric Regulation of the State of Adenylylation of Glutamine Synthetase in
Permeabilized Cell Preparations of Escherichia coli. Studies of Monocyclic and
Bicyclic Interconvertible Enzyme Cascades, in Situ
(Mura, U., Chock, P. B., and Stadtman, E. R. (1981) J. Biol. Chem. 256, 13022–13029)
Earl Reece Stadtman was born in 1919 in Carrizozo, a small town in New Mexico. When he
was 10, his family moved to San Bernardino, California, where he attended high school. After
graduating from high school in 1937, Stadtman enrolled in several science courses at San
Bernardino Valley College, hoping to eventually set up a soil-testing laboratory. However, he
soon realized that he needed a more rigorous education and enrolled at the University of
California, Berkeley. He earned a B.S. in soil science in 1942.
After spending a year in Alaska, involved in a wartime project of mapping the Alaskan-
Canadian (Al-Can) Highway, Stadtman returned to Berkeley looking for work. He paid a visit
to Horace A. Barker, a Berkeley biochemist for whom he had worked as a laboratory technician
(and author of a future Journal of Biological Chemistry (JBC) Classic). At that time, Barker
was directing various war efforts in the Department of Food Technology and offered Stadtman
a job as principal investigator on a project studying the “Browning of Dried Apricots,” the goal
of which was to find a way to slow the deterioration of dried fruits during storage. Around this
time, Stadtman also met his future wife, Thressa Campbell, who was working as a laboratory
assistant in the food technology department.
After the war, Stadtman started graduate studies in the Department of Biochemistry
working in Barker’s laboratory. Barker had spent a year as a postdoc in Albert J. Kluyver’s
laboratory at the Technical School in Delft in the Netherlands before coming to Berkeley and
had isolated a species of bacteria called Clostridium kluyveri (named after Kluyver) from the
Delft canal mud. Since then, Barker had been searching for an explanation for the observation
that C. kluyveri could produce short-chain fatty acids from ethyl alcohol. He made a break-
through when he obtained some 14C and used the isotope to label acetate and demonstrate that
fatty acid synthesis is accomplished by the multiple condensation of 2-carbon molecules (1). He
deduced that ethanol is first oxidized to “active” acetate (a 2-carbon compound), which is
condensed with acetate to form a 4-carbon compound that is reduced to form butyrate. Active
acetate can then be condensed with butyrate to form caproate. Barker surmised that acetyl
phosphate might be the active acetate formed in the above reaction.
It was at this point that Stadtman joined Barker’s laboratory and started working on fatty
acid synthesis. Initially, like Barker, Stadtman used 14C to trace the metabolic pathways in
whole cell preparations of C. kluyveri. However, he abandoned this approach after a visit to
Irwin C. Gunsalus’s laboratory at Cornell University. Gunsalus showed Stadtman how to dry
bacterial cells and grind the dried preparations to break open cell walls, producing a cell-free
extract. Applying this method to C. kluyveri, Stadtman was able to produce extracts that could
catalyze all of the reactions involved in the conversion of ethanol and acetate to fatty acids of
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Classics
Photo courtesy of the Office of NIH History, National Institutes of Health.
4- and 6-carbon atoms. His preparations also catalyzed the aerobic oxidation of ethanol and
butyrate. These experiments are reported in the first JBC Classic reprinted here. This
discovery was especially significant because up until that time most biochemists believed that
the capacity to make fatty acids was a unique property of specialized cellular systems or
particulate organelles.
In a series of additional papers (2– 6), all published in the JBC, Stadtman and Barker used
the enzyme extracts to study the individual reactions involved in fatty acid synthesis and
confirmed that ethanol is oxidized to acetyl phosphate, which condenses with acetate and
forms butyric acid. They also discovered that C. kluyveri contained an acetyl-transferring
enzyme (phosphotransacetylase) and an enzymatic system for using acetyl phosphate to
activate other fatty acids. Stadtman later showed that acetyl-CoA was the source of active
acetate in the synthesis of butyric acid from acetyl phosphate (7) while working as postdoctoral
fellow with Fritz Lipmann (author of a previous JBC Classic (8)).
In 1950, Stadtman began to look for an academic position. However, because his wife
Thressa also had a Ph.D., they were looking for an institution at which they could both work
at the same professional level. Unfortunately, at that time, most universities had anti-
nepotism rules that did not allow more than one family member to work in the same
department. Intended to protect universities from charges of favoritism, the rules often had
the effect of discriminating against married women. No one seriously challenged the rules
until the 1960s, when the American Association of University Women began to protest their
unfairness. Fortunately, these polices were not in effect at the National Institutes of Health
(NIH), and in September 1950, the Stadtmans moved to Bethesda, Maryland. Both continue to
do research at the NIH today.
At the NIH, Stadtman continued his research on fatty acid metabolism. In 1952, he
successfully carried out the first in vitro net synthesis of acetyl-CoA using only basic materials
(acetyl phosphate, CoA, and phosphotransacetylase). Stadtman and his postdoc P. Roy Vagelos
also demonstrated that long-chain fatty acid synthesis is catalyzed by an enzyme complex in
which malonyl-CoA is the source of active acetate.
Another topic of long term research in Stadtman’s laboratory was glutamine synthetase, the
enzyme that catalyzes the conversion of glutamate to glutamine. The activity of glutamine
synthetase is subject to feedback inhibition by 7 different end products of glutamine metab-
olism. Stadtman discovered that this end product inhibition was cumulative (the presence of
more end products resulted in more inhibition) and that susceptibility to feedback inhibition
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Classics
only occurred when glutamine synthetase was adenylated by adenylyltransferase (ATase). He
later found that adenylation was regulated by uridylyltransferase (UTase), which, depending
on the cellular concentration of various metabolites, catalyzed the covalent attachment of a
uridylyl group to the regulatory protein, PII. The uridylated form of PII stimulates ATase to
catalyze glutamine synthetase deadenylation, whereas the unmodified form of PII stimulates
ATase catalysis of the adenylation reaction.
In view of these results, Stadtman surmised that glutamine synthetase activity was con-
trolled by a cascade system in which two systems of reversible covalent modification were
tightly linked. Each system was composed of two reversible reactions, or two interconvertible
enzyme cycles, the linkage of which resulted in the formation of a bicyclic cascade system. This
cascade system allowed enzyme activity to be shifted gradually in response to metabolite
availability.
In the late 1970s and early 1980s, Stadtman and P. Boon Chock carried out a theoretical
analysis of this bicyclic cascade system to understand its implications in enzyme regulation.
However, it was not until 1981 that they were able to study the cascade in vivo. These
experiments are discussed in the second JBC Classic reprinted here. Stadtman had discovered
that after a freeze-thaw cycle, treatment of Escherichia coli cells with a nonionic detergent
rendered them permeable to small metabolites but allowed the cells to retain the protein
components of the cascade system. Furthermore, permeabilized cells from cultures containing
10 mM glutamine retained all their cascade enzymes whereas 5 mM glutamine-grown cells had
inactivated UTase. Using these cells, they were able to study the effects of different substrates
and allosteric effects on the cascade system and to confirm the previous theoretical and in vitro
studies. A more complete description of Stadtman’s work on glutamine synthetase can be
found in his JBC Reflections (9).
Stadtman has received many awards and honors for his numerous research discoveries
including the 1979 National Medal of Science, the 1983 ASBC-Merck Award, and the 1991
Robert A. Welch Award in Chemistry. Stadtman was also President of the American Society
for Biological Chemists (now American Society for Biochemistry and Molecular Biology) from
1982 to 1983 and has been a member of the National Academy of Sciences since 1969.1
REFERENCES
1. Barker, H. A., Kamen, M. D., and Bornstein, B. T. (1945) The synthesis of butyric and caproic acids from ethanol
and acetic acid by Clostridium kluyveri. Proc. Natl. Acad. Sci. U. S. A. 31, 373–381
2. Stadtman, E. R., and Barker, H. A. (1949) Fatty acid synthesis by enzyme preparations of Clostridium kluyveri.
II. The aerobic oxidation of ethanol and butyrate with the formation of acetyl phosphate. J. Biol. Chem. 180,
1095–1115
III. The activation of molecular hydrogen and the conversion of acetyl phosphate and acetate to butyrate. J. Biol.
Chem. 180, 1117–1124
IV. The phosphoroclastic decomposition of acetoacetate to acetyl phosphate and acetate. J. Biol. Chem. 180,
1169 –1186
V. A consideration of postulated 4-carbon intermediates in butyrate synthesis. J. Biol. Chem. 181, 221–235
VI. Reactions of acyl phosphates. J. Biol. Chem. 184, 769 –794
7. Stadtman, E. R., Novelli, G. D., and Lipmann, F. (1951) Coenzyme A function in and acetyl transfer by the
phosphotransacetylase system. J. Biol. Chem. 191, 365–376
8. JBC Classic: Lipmann, F. (1945) J. Biol. Chem. 160, 173–190 (http://www.jbc.org/cgi/content/full/280/21/e18)
9. Stadtman, E. R. (2001) The story of glutamine synthetase regulation. J. Biol. Chem. 276, 44357– 44364
10. Park, B. S. The Stadtman way: A tale of two biochemists at NIH. http://history.nih.gov/exhibits/stadtman/
index.htm (An online exhibit produced by the Office of NIH History in collaboration with the National Heart,
Lung, and Blood Institute)
1
All biographical information on Earl R. Stadtman was taken from Ref. 10.
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 30, Issue of July 29, p. e27, 2005
Classics
JBC Centennial
1905–2005
A Role for Phosphoinositides in Signaling: the Work of

Mabel R. Hokin and Lowell E. Hokin
Enzyme Secretion and the Incorporation of P32 into Phospholipides of Pancreas Slices
(Hokin, M. R., and Hokin, L. E. (1953) J. Biol. Chem. 203, 967–977)
Phosphoinositides and Protein Secretion in Pancreas Slices
(Hokin, L. E., and Hokin, M. R. (1958) J. Biol. Chem. 233, 805– 810)
Mabel R. Hokin (born Mabel Neaverson) and Lowell E. Hokin met in Hans Kreb’s depart-
ment at the University of Sheffield and married soon after. During their time in Sheffield, the
Hokins started investigating what they thought was an increase in the incorporation of 32P
into RNA caused by the acetylcholine-induced stimulation of pancreatic slices. However,
before they could purify the RNA, they moved to McGill University in Montréal, Québec, and
brought their radiolabeled samples with them. Once established in Montréal, they continued
with their experiments but noticed that as they purified the RNA the radioactivity was lost.
Investigating this phenomenon further, they found that most of the radioactivity was incor-
porated into the phospholipid fraction. This was a surprising discovery as up until then
phospholipids were regarded as inert structural components of membranes.
The Hokins’ studies on 32P uptake into phospholipids during enzyme secretion in pancreas
slices are published in the first Journal of Biological Chemistry (JBC) Classic reprinted here. The
Hokins incubated pigeon pancreas slices with various compounds along with 32P to see the effects
on phosphate incorporation into phospholipids. They found that when enzyme secretion was
stimulated by acetylcholine or carbamylcholine, both of which induce amylase secretion, the
incorporation of 32P into phospholipids was on average 7.0 times greater than in control tissue.
Separating individual phospholipids for analysis was difficult at that time, but fortunately Rex
Dawson devised a method that permitted the analysis of diacylglycerophospholipids by deacyla-
tion and two-dimensional separation of the water-soluble backbone (1). The Hokins used this
method to show that hormone stimulation of pancreatic slices mainly increased the rate of 32P
incorporation into phosphoinositide but that phosphatidylcholine, phosphatidylserine, and phos-
phatidic acid also contained radiolabeled phosphate (2). This was the first demonstration of
receptor-stimulated lipid turnover, and it later became known as the “PI effect.”
The second JBC Classic reprinted here presents the details of the Hokins’ study of phos-
phoinositide metabolism in relation to protein secretion in the pancreas. They incubated
pigeon pancreas slices with either NaH2P32O4, [2-3H]inositol, or [1-14C]glycerol and extracted
the lipids from the tissue and separated them by paper chromatography. They were able to
identify seven phospholipids containing 32P as well as two radioactive monophosphoinositides.
From these data they concluded, “the present work indicates that phosphoinositides are
involved in the secretion of protein from the inside of the pancreatic acinar cell into the
lumen . . . It is tempting to think that the active transport out of the cell of many other types
of molecules may involve phosphoinositides.”
In 1957 the Hokins moved to Madison, Wisconsin, where they both joined the faculty of the
University of Wisconsin-Madison Medical School. There they showed that other tissues exhibit
similar responses when provoked to secrete. In 1964 the Hokins suggested that phospholipase
C-catalyzed phosphatidylinositol hydrolysis might initiate the PI effect. Later it was confirmed
that the initiating event was the phospholipase C-catalyzed hydrolysis of phosphatidylinositol
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4,5-bisphosphate and that 3-kinase-catalyzed formation of phosphatidylinositol 3,4,5-triphos-
phate was a second widespread signaling reaction.
The Hokins’ initial work on stimulated phosphoinositide turnover in secretory tissues
motivated a large number of other investigators to focus their research on the PI effect and
second messengers. Eventually they would discover that the Hokins’ inositol phospholipids
play important roles in transmembrane signaling and many other cell regulatory processes.1
REFERENCES
1. Dawson, R. M. C. (1954) The measurement of 32P labeling of individual kephalins and lecithin in a small sample
of tissue. Biochim. Biophys. Acta 14, 374 –375
2. Hokin, L. E., and Hokin, M. R. (1955) Effects of acetylcholine on the turnover of phosphoryl units in individual
phospholipids of pancreas slices and brain cortex slices. Biochim. Biophys. Acta 18, 102–110
3. Michell, B. (2003) Obituary: Mabel R. Hokin (1924 –2003). The Biochemist. December
4. Irvine, R. F. (2003) 20 years of Ins(1,4,5)P3, and 40 years before. Nat. Rev. Mol. Cell. Biol. 4, 586 –590
1
All biographical information on Mabel R. Hokin and Lowell E. Hokin was taken from Refs. 3 and 4.
H15
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 284, No. 20, Issue of May 15, p. e3, 2009
Classics
JBC Centennial
1905–2005
The Selective Placement of Acyl Chains: the Work of

William E. M. Lands
Metabolism of Glycerolipids: A Comparison of Lecithin and Triglyceride Synthesis
(Lands, W. E. M. (1958) J. Biol. Chem. 231, 883– 888)
William E. M. Lands
William E. M. Lands was born in Chillicothe, Missouri in 1930. He earned his B.S. in
chemistry from the University of Michigan in 1951 and his Ph.D. in biological chemistry from
the University of Illinois in 1954. After graduating, he spent a year as a postdoctoral fellow at
the California Institute of Technology and then joined the faculty of the University of Michigan
as an instructor in biological chemistry. Lands spent the next 25 years at Michigan, eventually
becoming professor in 1967.
In 1980, Lands left Michigan to head the Department of Biological Chemistry at the
University of Illinois. He spent 10 years there and then moved to Bethesda, Maryland to
become Senior Scientific Advisor to the Director of the National Institute on Alcohol Abuse and
Alcoholism (NIH). In 2002, Lands retired from his position at the NIH.
Lands spent the majority of his scientific career studying fatty acids and has made many
significant contributions to this field. One such contribution is his discovery of the phospho-
lipid retailoring or “Lands” pathway. His initial paper showing the likelihood of acyl chain
turnover is reprinted here as a Journal of Biological Chemistry (JBC) Classic.
In the paper, Lands incubates various tissues with [14C]acetate and [14C]glycerol and
measures the value R, which is the ratio of [14C]acetate to [14C]glycerol in the diglyceride unit
of the triglycerides and phospholipids produced by the tissues. Lands reasoned that if diglyc-
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Classics
eride is the sole precursor of phos-
pholipids, these two compounds
should have the same R value. Also,
because a third fatty acid molecule is
added to the diglyceride unit to form
triglycerides, they should have a 3/2
R value (see Fig. 1).
However, Lands’ results showed
that R is 2– 4 times higher in phos-
pholipids than in triglycerides, sug-
FIGURE 1 gesting to him that “the diglyceride
unit of the phospholipids is metabol-
ically different in some respect from that of the triglycerides.” This initial finding led to a series
of papers published in the JBC describing the selective placement of acyl chains by phospho-
lipid acyltransferases (1– 4).
In addition to the above research which helped to explain the metabolic process that
regulates the mixture of acyl chains found in lipids, Lands is also credited with discovering the
beneficial effects of balancing excess ␻-6 fatty acids with dietary ␻-3 fatty acids.
In recognition of his contributions to science, Lands received the Glycerine Research Award
(1969), the Canadian Society of Nutritional Science Lectureship (1991), and the American Oil
Chemists’ Society Supelco Lipid Research Award (1997). The University of Michigan’s Depart-
ment of Biological Chemistry has also endowed a lectureship in honor of Lands. He has also
served on the editorial boards of several journals, including those of the Journal of Lipid
Research, Biochimica et Biophysica Acta, Lipids, and Prostaglandins.
REFERENCES
1. Lands, W. E. M. (1960) Metabolism of glycerolipids. II. The enzymatic acylation of lysolecithin. J. Biol. Chem. 235,
2233–2237
2. Lands, W. E. M., and Merkl, I. (1963) Metabolism of glycerolipids. III. Reactivity of various acyl esters of coenzyme
A with ␣⬘-acylglycerol phosphorylcholine and positional specificities in lecithin synthesis. J. Biol. Chem. 238,
898 –904
3. Merkl, I., and Lands, W. E. M. (1963) Metabolism of glycerolipids. IV. Synthesis of phosphatidylethanolamine.
J. Biol. Chem. 238, 905–906
4. Lands, W. E. M., and Hart, P. (1965) Metabolism of glycerolipids. VI. Specificities of acyl-CoA:phospholipid
acyltransferases. J. Biol. Chem. 240, 1905–1911
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 48, Issue of December 2, p. e45, 2005
Classics
JBC Centennial
1905–2005
Lipid Storage Disorders and the Biosynthesis of Inositol

Phosphatide: the Work of Roscoe Brady
The Enzymatic Synthesis of Inositol Phosphatide
(Agranoff, B. W., Bradley, R. M., and Brady, R. O. (1958) J. Biol. Chem. 233, 1077–1083)
Roscoe Owen Brady was born in 1923 in Philadelphia. He attended Pennsylvania State
University from 1941 to 1943 and then received his medical degree from Harvard Medical
School in 1947. After interning at the Hospital of the University of Pennsylvania for 1 year,
Brady did a postdoctoral fellowship in the Department of Physiological Chemistry at the
University of Pennsylvania School of Medicine (1948 to 1950) and then was a fellow in clinical
medicine in the Department of Medicine (1950 to 1952). In 1954, following 21⁄2 years on active
duty in the U.S. Naval Medical Corps, he joined the National Institutes of Health (NIH) to
become section chief of the National Institute of Neurological Diseases and Blindness. He
remained in this position until 1967 when he became assistant laboratory chief of neurochem-
istry at the National Institute of Neurological Diseases and Blindness. Currently, Brady is
Chief of the Developmental and Metabolic Neurology Branch of the National Institute of
Neurological Disorders and Stroke, a position he has held since 1972.
Early in his career at the NIH, Brady started studying lipids, specifically inositol and the
synthesis of inositol phosphatide. This is the subject of the Journal of Biological Chemistry
(JBC) Classic reprinted here. Although inositol had been isolated more than 100 years before
the Classic was published, little was known about its metabolism. In his Classic, Brady uses
tritium-labeled inositol and a preparation from guinea pig kidney mitochondria to study
inositol metabolism. He found that the enzyme system catalyzed the incorporation of inositol
into inositol phosphatide in the presence of Mg2⫹ and cytidine diphosphate-choline (CDP) or
cytidine 5⬘-phosphate. From these results, Brady proposed a mechanism for the synthesis of
inositol phosphatide in which CDP is transphosphorylated to form CDP-D-␣,␤-diglyceride,
which then reacts with the hydroxyl group of inositol to form inositol phosphatide.
These early studies stimulated Brady’s interest in lipid storage disorders, in particular,
Gaucher disease. In 1967, he showed that people with Gaucher disease had low levels of
glucocerebrosidase and thus were unable to break down the lipid glucocerebroside and clear it
out of their bodies. He also developed a diagnostic test for Gaucher disease, which worked by
measuring glucocerebrosidase activity in white blood cells. A year later, Brady suggested a
therapy for Gaucher disease based on replacing the enzyme. Using human placentas, his team
isolated a tiny sample of purified glucocerebrosidase and gave it to two patients. The patients’
health improved, and Brady soon developed large scale purification and targeting methods for
glucocerebrosidase to use in further clinical trials. Eventually, in 1991, Brady’s macrophage-
targeted glucocerebrosidase enzyme replacement therapy was approved as a specific treat-
ment for Gaucher disease by the Food and Drug Administration.
In addition to studying Gaucher disease, Brady has discovered the metabolic basis of
Niemann-Pick disease, Fabry disease, and the specific biochemical defect in Tay-Sachs dis-
ease. He has applied this knowledge to developing diagnostic tests, carrier identification
procedures, and the prenatal detection of such conditions. Currently his research is focused on
examining enzyme replacement therapy and gene therapy for patients with these other
hereditary metabolic disorders.
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Classics
In recognition of his scientific achievements, Brady received the Gairdner International

Award (1973), the Cotzias Award from the American Academy of Neurology (1980), the
Passano Foundation Award (1982), the Lasker Foundation Clinical Medical Research Award
(1982), and the Kovalenko Medal from the National Academy of Sciences (1991). He is a
member of the National Academy of Sciences and a member of the Institute of Medicine of the
National Academy of Sciences. He has served on the editorial boards and advisory boards of
many journals and organizations.
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 281, No. 9, Issue of March 3, p. e9, 2006
Classics
JBC Centennial
1905–2005
The Prostaglandins, Sune Bergström

and Bengt Samuelsson
Prostaglandins and Related Factors. 15. The Structures of Prostaglandin E1,
F1␣, and F1␤
(Bergström, S., Ryhage, R., Samuelsson, B., and Sjövall, J. (1963) J. Biol. Chem. 238,
3555–3564
On the Mechanism of the Biosynthesis of Prostaglandins E1 and F1␣
(Hamberg, M., and Samuelsson, B. (1967) J. Biol. Chem. 242, 5336 –5343)
Sune Karl Bergström (1916 –2004) was born in Stockholm, Sweden. Upon completing high
school he went to work at the Karolinska Institute as an assistant to Erik Jorpes where he did
research on the biochemistry of fats and steroids. Jorpes was sufficiently impressed with
Bergström that in 1938 he sponsored a year-long research fellowship for him at the University
of London. Then, in 1940, Bergström received a Swedish-American Fellowship, which allowed
him to study for 2 years at Columbia University and to conduct research at the Squibb
Institute for Medical Research in New Jersey. He returned to Sweden in 1942 and received
doctorates in medicine and biochemistry from the Karolinska Institute 2 years later. He was
then appointed assistant in the biochemistry department of Karolinska’s Medical Nobel
Institute.
Bergström’s involvement with prostaglandins started in 1945 at a meeting of the Physio-
logical Society of the Karolinska Institute. There he met Ulf von Euler who had been doing
research on prostaglandins. Von Euler asked Bergström if he might be interested in studying
some of his lipid extracts of sheep vesicular glands. Using Lyman Craig’s countercurrent
extraction device, which was the subject of a previous Journal of Biological Chemistry (JBC)
Classic (1), Bergström was able to purify the crude extract about 500 times. However, his work
was interrupted for a few years when he was appointed chair of physiological chemistry at the
University of Lund in 1948.
When Bergström resumed his research on prostaglandins, he was aided by his graduate
student Bengt Ingemar Samuelsson. Samuelsson, who was born in Halmstad, Sweden, in
1934, had enrolled at the University of Lund to study medicine when he came under the
mentorship of Bergström. Using countercurrent fractionations and partition chromatography,
Bergström was able to isolate small amounts of prostaglandin E1 and F1␣ by 1957 (2). A year
later, Bergström was appointed professor of chemistry at Karolinska, and he moved his
research group with him to Stockholm. Samuelsson received his doctorate in medical science
from the Karolinska Institute in 1960 and his medical degree in 1961.
At Karolinska, Bergström started to collaborate with Ragnar Ryhage who had built a
combination gas chromatograph and mass spectrometer. Using this instrument, Bergström,
Samuelsson, and Ryhage were able to deduce the structures of prostaglandins E1, F1␣, and F1␤
from mass spectrometric identification of the products formed when the prostaglandins were
treated with a weak acid or base. These structure determinations are discussed in the first
Classic reprinted here. By 1962, Bergström and his colleagues had isolated and determined
the structures of six different prostaglandins.
After completing the structural work on the prostaglandins, Samuelsson spent a year as a
postdoctoral fellow with E. J. Corey in the Department of Chemistry at Harvard University,
where he was able to study theoretical and synthetic organic chemistry. He returned to the
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Classics
Sune Karl Bergström. Photo courtesy of the Bengt Ingemar Samuelsson. Photo courtesy of
National Library of Medicine. the National Library of Medicine.
Karolinska Institute as assistant professor of medical chemistry and resumed work on the
prostaglandins. The second JBC Classic deals with some of Samuelsson’s research on the
biosynthesis of prostaglandins, an area in which he contributed considerable knowledge. In
the paper, Samuelsson follows the conversion of 8,11,14-eicosatrienoic acid to prostaglandin E1
and prostaglandin F1␣, using 3H and 14C labeling, focusing especially on the initial step of the
process.
In 1967, Samuelsson joined the faculty of the Royal Veterinary College in Stockholm as
Professor of Medical Chemistry to explore the veterinary and livestock breeding applications
of prostaglandins. However, he returned to the Karolinska Institute in 1972 to become
Professor and Chairman of the Department of Physiological Chemistry. He was Dean of the
Medical Faculty from 1978 to 1983 after which he was appointed Rector of the Karolinska
Institute.
Bergström remained at Karolinska, serving as dean of its medical school from 1963 to 1966
and as Rector of the Institute from 1969 to 1977. He was chairman of the Nobel Foundation’s
Board of Directors from 1975 to 1987, and from 1977 to 1982 he served as chairman of the
World Health Organization’s Advisory Committee on Medical Research. He retired from
teaching in 1981, choosing to devote his full time to research at Karolinska.
Independently, both Bergström and Samuelsson continued to investigate prostaglandins
and related compounds throughout their scientific careers. In honor of their contributions to
this field, they were awarded the 1982 Nobel Prize in Physiology or Medicine with John R.
Vane “for their discoveries concerning prostaglandins and related biologically active
substances.”
Samuelsson’s research has been recognized by numerous awards and honors in addition to
the Nobel Prize. These include the A. Jahres Award in Medicine from Oslo University (1970),
the Louisa Gross Horwitz Prize from Columbia University (1975), the Albert Lasker Medical
Research Award (1977), the Ciba-Geigy Drew Award for biomedical research (1980), the
Gairdner Foundation Award (1981), the Bror Holberg Medal of the Swedish Chemical Society
(1982), and the Abraham White Distinguished Scientist Award (1991). Samuelsson was
elected to the National Academy of Sciences in 1984.1
Bergström also received many awards, including the Albert Lasker Award in 1977, Oslo
University’s Anders Jahre Prize in Medicine in 1970, and Columbia University’s Louisa Gross
Horwitz Prize in 1975. He was a member of the Royal Swedish Academy of Science (and served
1
Biographical information on Bengt Samuelsson was taken from Ref. 3.
H21
Classics
as its president from 1983 to 1985), the American Philosophical Society, the National Academy
of Sciences (1973), and the American Academy of Arts and Sciences.2
REFERENCES
1. JBC Classics: Craig, L. C. (1943) J. Biol. Chem. 150, 33– 45; Craig, L. C. (1944) J. Biol. Chem. 155, 519 –534
2. Bergström, S., and Sjövall, J. (1957) The isolation of prostaglandin. Acta Chem. Scand. 11, 1086
3. Samuelsson, B. I. (1993) Studies of biochemical mechanisms to novel biological mediators: prostaglandin endoper-
oxides, thromboxanes and leukotrienes. In Nobel Lectures, Physiology or Medicine 1981–1990 (Frängsmyr, T.,
ed) World Scientific Publishing Co., Singapore
4. Bergström, S. K. (1993) The prostaglandins: from the laboratory to the clinic. In Nobel Lectures, Physiology or
Medicine 1981–1990 (Frängsmyr, T., ed) World Scientific Publishing Co., Singapore
2
Biographical information on Sune Bergström was taken from Ref. 4.
H22
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 284, No. 23, Issue of June 5, p. e6, 2009
Classics
JBC Centennial
1905–2005
Biotin-dependent Enzymes: the Work of Feodor Lynen

The Enzymatic Synthesis of Holotranscarboxylase from Apotranscarboxylase and
(ⴙ)-Biotin. I. Purification of the Apoenzyme and Synthetase; Characteristics of
the Reaction
(Lane, M. D., Young, D. L., and Lynen, F. (1964) J. Biol. Chem. 239, 2858 –2864)
Feodor Felix Konrad Lynen (1911–1979) was born
in Munich, Germany. He was undecided about his
career during his early education and even consid-
ered becoming a ski instructor. Ultimately, he en-
rolled in the Department of Chemistry at the Uni-
versity of Munich where he studied with Nobel
laureate Heinrich Wieland and received his doctor-
ate degree in 1937. Three months later, he married
Wieland’s daughter, Eva.
After graduating, Lynen remained at Munich
University as a postdoctoral fellow. He was ap-
pointed lecturer in 1942 and assistant professor in
1947. When World War II broke out, Lynen was
exempt from military service because of a knee
injury resulting from a ski accident in 1932. How-
ever, the war made it difficult to continue to do
research in Munich, and Lynen moved his labora-
tory to the small village of Schondorf on the Am-
mersee. This was lucky because in 1945 Munich
Feodor Lynen
University’s Department of Chemistry was de-
stroyed. Lynen continued his work at various lab-
oratory facilities and eventually returned to the rebuilt Department of Chemistry in 1949.
During the 1940s, Lynen began studying the biosynthesis of sterols and lipids. He eventu-
ally initiated a collaboration with Konrad Bloch, whose cholesterol research was featured in a
previous Journal of Biological Chemistry (JBC) Classic (1). Working together, Bloch and
Lynen were able to elucidate the steps in the biosynthesis of cholesterol. An especially
significant finding made by Lynen was that acetyl coenzyme A (previously discovered by JBC
Classic author Fritz Lipmann (2)) was essential for the first step of cholesterol biosynthesis.
Lynen later determined the structure of acetyl-CoA. This work on cholesterol resulted in Bloch
and Lynen being awarded the 1964 Nobel Prize in Physiology or Medicine.
In 1953, Lynen was made full professor at the University of Munich. A year later, he was
named director of the newly established Max Planck Institute for Cell Chemistry. He contin-
ued to work on fats but also turned his focus to biotin-dependent enzymes. In 1962, he was
joined by JBC Classic author M. Daniel Lane (3), who had come to Munich to work with Lynen
on a sabbatical leave. Lane was studying the biotin-dependent propionyl-CoA carboxylase and
had previously determined that its biotin prosthetic group was linked to the enzyme through
an amide linkage to a lysyl ⑀-amino group.
Before leaving for Munich, Lane developed an apoenzyme system with which to investigate
the mechanism by which biotin became attached to propionyl-CoA carboxylase. This system
made use of Propionibacterium shermanii, which expressed huge amounts of methylmalonyl-
H23
Classics
CoA:pyruvate transcarboxylase, another biotin-dependent enzyme. The organism also had an
absolute requirement for biotin in its growth medium and produced large amounts of the
apotranscarboxylase when grown at very low levels of biotin.
As reported in the JBC Classic reprinted here, Lane and Lynen were able to resolve and
purify both the apotranscarboxylase and the synthetase that catalyzed biotin loading onto the
apoenzyme. Dave Young, a postdoctoral fellow who had recently completed his medical
training at Duke University, collaborated with them on these studies. In a second paper
reprinted in the Lane Classic (4), Lane and Lynen showed that the synthetase catalyzed a
two-step reaction. The first step involved the ATP-dependent formation of biotinyl-5⬘-AMP and
pyrophosphate after which the biotinyl group was transferred from the AMP derivative to the
appropriate lysyl ⑀-amino group of the apotranscarboxylase. Lane and Lynen also showed that
the covalently bound biotinyl prosthetic group, like free biotin, was carboxylated on the 1⬘-N
position (5).
In 1972, Lynen moved to the recently founded Max Planck Institute for Biochemistry.
Between 1974 and 1976, he was acting director of the Institute while continuing to oversee a
lab at the University of Munich. He remained at the Institute until his death in 1979.
In addition to the Nobel Prize, Lynen received many honors and awards. These include the
Neuberg Medal of the American Society of European Chemists and Pharmacists (1954), the
Liebig Commemorative Medal of the Gesellschaft Deutscher Chemiker (1955), the Carus
Medal of the Deutsche Akademie der Naturforscher Leopoldina (1961), and the Otto Warburg
Medal of the Gesellschaft für Physiologische Chemie (1963).
REFERENCES
1. JBC Classics: Bloch, K., and Rittenberg, D. (1942) J. Biol. Chem. 145, 625– 636; Rittenberg, D., and Bloch, K. (1945)
J. Biol. Chem. 160, 417– 424; Bloch, K. (1945) J. Biol. Chem. 157, 661– 666
2. JBC Classics: Lipmann, F. (1945) J. Biol. Chem. 160, 173–190 (http://www.jbc.org/cgi/content/full/280/21/e18)
3. JBC Classics: Lane, M. D., Rominger, K. L., Young, D. L., and Lynen, F. (1964) J. Biol. Chem. 239, 2865–2871;
Gregolin, C., Ryder, E., Warner, R. C., Kleinschimdt, A. K., Chang, H.-C., and Lane, M. D. (1968) J. Biol. Chem.
243, 4236 – 4245; Guchhait, R. B., Polakis, S. E., Hollis, D., Fenselau, C., and Lane, M. D. (1974) J. Biol. Chem. 249,
6646 – 6656; Polakis, S. E., Guchhait, R. B., Zwergel, E. E., Lane, M. D., and Cooper, T. G. (1974) J. Biol. Chem. 249,
6657– 6667 (http://www.jbc.org/cgi/content/full/281/49/e40)
4. Lane, M. D., Rominger, K. L., Young, D. L., and Lynen, F. (1964) The enzymatic synthesis of holotranscarboxylase
from apotranscarboxylase and (⫹)-biotin. II. Investigation of the reaction mechanism. J. Biol. Chem. 239,
2865–2871
5. Lane, M. D., and Lynen, F. (1963) The biochemical function of biotin. VI. Chemical structure of the carboxylated
active site of propionyl carboxylase. Proc. Natl. Acad. Sci. U. S. A. 49, 379 –385
H24
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 281, No. 49, Issue of December 8, p. e40, 2006
Classics
JBC Centennial
1905–2005
Acetyl-CoA Carboxylase and Other Biotin-dependent

Enzymes: the Work of M. Daniel Lane
The Enzymatic Synthesis of Holotranscarboxylase from Apotranscarboxylase and
(ⴙ)-Biotin. II. Investigation of the Reaction Mechanism
(Lane, M. D., Rominger, K. L., Young, D. L., and Lynen, F. (1964) J. Biol. Chem. 239,
2865–2871)
Liver Acetyl-CoA Carboxylase. II. Further Molecular Characterization
(Gregolin, C., Ryder, E., Warner, R. C., Kleinschimdt, A. K., Chang, H.-C., and Lane,
M. D. (1968) J. Biol. Chem. 243, 4236 – 4245)
Acetyl Coenzyme A Carboxylase System of Escherichia coli. Site of Carboxylation of
Biotin and Enzymatic Reactivity of 1ⴕ-N-(Ureido)-Carboxybiotin Derivatives
(Guchhait, R. B., Polakis, S. E., Hollis, D., Fenselau, C., and Lane, M. D. (1974) J. Biol.
Chem. 249, 6646 – 6656)
Acetyl Coenzyme A Carboxylase System of Escherichia coli. Studies on the Mecha-
nisms of the Biotin Carboxylase- and Carboxyltransferase-catalyzed Reactions
(Polakis, S. E., Guchhait, R. B., Zwergel, E. E., Lane, M. D., and Cooper, T. G. (1974)
J. Biol. Chem. 249, 6657– 6667)
Malcolm Daniel Lane was born in Chicago in 1930. He received both his B.S. and M.S. from
Iowa State University in 1951 and 1953, respectively. Lane then went to the University of
Illinois for graduate school and was awarded his Ph.D. in 1956. He joined the faculty of the
Virginia Polytechnic Institute and State University in Blacksburg, Virginia in 1956 as Asso-
ciate Professor and was promoted to Professor of Biochemistry in 1963.
Upon joining the faculty at Virginia Polytechnic Institute, Lane decided to try to determine
how propionate was metabolized in the bovine liver. About this time, Journal of Biological
Chemistry (JBC) Classics author Severo Ochoa (1, 2) reported that propionyl-CoA was carbox-
ylated to form methylmalonyl-CoA, which was then was converted to succinyl-CoA. Lane was
able to purify propionyl-CoA carboxylase from bovine liver mitochondria.
Then in 1959 a paper by Lynen and Knappe appeared in Angewandte Chemie (3) indicating
that ␤-methylcrotonyl-CoA carboxylase, a biotin-dependent carboxylase, catalyzed the ATP-
dependent carboxylation of “free” biotin in the absence of its acyl-CoA substrate. Lynen
proposed that the free biotin had accessed the active site of the carboxylase and by mimicking
the biotinyl prosthetic group it had been carboxylated. Lane determined that propionyl-CoA
carboxylase was also a biotin-dependent enzyme and determined that the biotin prosthetic
group was linked to propionyl-CoA carboxylase through an amide linkage to a lysyl ⑀-amino
group.
In 1962 Lane decided to take a sabbatical leave in Munich with Feodor Lynen at the
Max-Planck Institüt Für Zellchemie where he continued to work on the enzymatic mechanism
by which biotin became attached to propionyl-CoA carboxylase. Before leaving for Munich,
Lane developed an apoenzyme system with which to investigate the “biotin loading” reaction.
This system made use of Propionibacterium shermanii, which expressed huge amounts of
methylmalonyl-CoA:pyruvate transcarboxylase, another biotin-dependent enzyme. The orga-
nism also had an absolute requirement for biotin in its growth medium and produced large
amounts of the apotranscarboxylase when grown at very low levels of biotin.
H25
Classics
M. Daniel Lane
In Munich, Lane was able to resolve and purify both the apotranscarboxylase and the
synthetase that catalyzed biotin loading onto the apoenzyme (4). Dave Young, a postdoctoral
fellow who had recently completed his medical training at Duke University, and Karl
Rominger, a Ph.D. candidate under Lynen’s direction, collaborated with him on these studies.
In a second paper, which is the first JBC Classic reprinted here, Lane and his colleagues
showed that the synthetase catalyzed a two-step reaction. The first step involved the ATP-
dependent formation of biotinyl-5⬘-AMP and pyrophosphate after which the biotinyl group was
transferred from the AMP derivative to the appropriate lysyl ⑀-amino group of the apotrans-
carboxylase. Lane and Lynen also showed that the covalently bound biotinyl prosthetic group,
like free biotin, was carboxylated on the 1⬘-N position (5).
Shortly after he returned from Munich, Lane left Virginia Polytechnic Institute to become
Associate Professor of Biochemistry at the New York University School of Medicine. He was
later promoted to Professor of Biochemistry in 1969. In New York, Lane and his colleagues
isolated acetyl coenzyme A carboxylase from chicken liver (6). The biotin-containing enzyme
catalyzes the carboxylation of acetyl-CoA to malonyl-CoA in a 2-step process involving a
carboxybiotin intermediate. In an accompanying paper, Lane described the molecular char-
acteristics of the enzyme, including its reversible inter-conversion between protomeric and
polymeric forms. The paper is reprinted here as the second JBC Classic by Lane. He deter-
mined that the carboxylase has a binding site for citrate and another for acetyl-CoA and that
citrate binding might be involved in regulating the enzyme.
Lane left New York in 1970 to become Professor of Biological Chemistry at the Johns
Hopkins University School of Medicine. Right around the time Lane took up his new post at
Johns Hopkins, Thomas C. Bruice and A. F. Hegarty published a paper (7) that called into
question Lane’s conclusion that biotin was carboxylated on the 1⬘-N position. They pointed out
that carboxylation could occur at the ureido-O and result in the same derivative. In the third
JBC Classic, Lane uses the acetyl coenzyme A carboxylase system from Escherichia coli to
provide definitive evidence that the ureido-N of biotin is the site of carboxylation.
In the final JBC Classic reprinted here, Lane presents a thorough analysis of the acetyl
coenzyme A carboxylase system from Escherichia coli. He defines the requirements and
properties of isotopic exchange and stoichiometric reactions representative of the two half-
reactions in acetyl-CoA carboxylation and also describes studies using prosthetic group and
intermediate model derivatives as substrates to elucidate the mechanisms of the partial
reactions.
Lane was eventually promoted to Director and DeLamar Professor in 1978. He is currently
Distinguished Service Professor in the Department of Biological Chemistry at Johns Hopkins.
More information about Lane’s early work on biotin can be found in his JBC Reflections (8).
Lane’s honors and awards include the American Institute of Nutrition’s Mead-Johnson
award in 1966, the American Society of Biological Chemists’ William C. Rose award in 1981,
and the Johns Hopkins University School of Medicine Professor’s Award for Distinction in
H26
Classics
Teaching in 1986. He was elected to the American Academy of Arts and Sciences in 1982, the
American Society for Nutritional Sciences in 1996, and the National Academy of Sciences in
1987. In addition to serving as president of the American Society for Biochemistry and
Molecular Biology in 1990, Lane served on the Society’s Program Committee, Membership
Committee, and Public Affairs Committee. He has served on the Editorial Boards of several
journals, including those of the Journal of Biological Chemistry, Biochemistry et Biophysica
Acta, the Archives Biochemistry and Biophysics, and Annual Reviews of Biochemistry. He also
served on the editorial board and was Executive Editor of Biochemical and Biophysical
Research Communications in 1986.
REFERENCES
1. JBC Classics: Stern, J. R., and Ochoa, S. (1951) J. Biol. Chem. 191, 161–172; Korkes, S., del Campillo, A.,
Gunsalus, I. C., and Ochoa, S. (1951) J. Biol. Chem. 193, 721–735 (http://www.jbc.org/cgi/content/full/280/11/e8)
2. JBC Classics: Salas, M., Smith, M. A., Stanley, W. M., Jr., Wahba, A. J., and Ochoa, S. (1965) J. Biol. Chem. 240,
3988 –3995 (http://www.jbc.org/cgi/content/full/281/21/e16)
3. Lynen, F., Knappe, J., Lorch, E., Jutting, G., and Ringelmann, E. (1959) Die biochemische Funktion des Biotins.
Angew. Chem. 71, 481– 486
4. Lane, M. D., Young, D. L., and Lynen, F. (1964) The enzymatic synthesis of holotranscarboxylase from apotrans-
carboxylase and (⫹)-biotin. I. Purification of the apoenzyme and synthetase; characteristics of the reaction.
J. Biol. Chem. 239, 2858 –2864
5. Lane, M. D., and Lynen, F. (1963) The biochemical function of biotin. VI. Chemical structure of the carboxylated
active site of propionyl carboxylase. Proc. Natl. Acad. Sci. U. S. A. 49, 379 –385
6. Gregolin, C., Ryder, E., and Lane, M. D. (1968) Liver acetyl coenzyme A carboxylase. I. Isolation and catalytic
properties. J. Biol. Chem. 243, 4227– 4235
7. Bruice, T. C., and Hegarty, A. F. (1970) Biotin-bound CO2 and the mechanism of enzymatic carboxylation
reactions. Proc. Natl. Acad. Sci. U. S. A. 65, 805– 809
8. Lane, M. D. (2004) The biotin connection: Severo Ochoa, Harland Wood, and Feodor Lynen. J. Biol. Chem. 279,
39187–39194
H27
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 35, Issue of September 2, p. e32, 2005
Classics
JBC Centennial
1905–2005
The Role of the Acyl Carrier Protein in Fatty Acid

Synthesis: the Work of P. Roy Vagelos
Acyl Carrier Protein. III. An Enoyl Hydrase Specific for Acyl Carrier Protein
Thioesters
(Majerus, P. W., Alberts, A. W., and Vagelos, P. R. (1965) J. Biol. Chem. 240, 618 – 621)
Acyl Carrier Protein. VII. The Primary Structure of the Substrate-binding Site
(Majerus, P. W., Alberts, A. W., and Vagelos, P. R. (1965) J. Biol. Chem. 240, 4723– 4726)
P. Roy Vagelos was born in Westfield, NJ in 1929. He received an A.B. degree from the
University of Pennsylvania in 1950 and an M.D. from Columbia University’s College of
Physicians and Surgeons in 1954. Following an internship and residency at the Massachusetts
General Hospital in Boston, he joined the National Institutes of Health. There he launched a
career as a research scientist under the guidance of Earl Stadtman, who authored a previous
Journal of Biological Chemistry (JBC) Classic (1). With Stadtman, Vagelos demonstrated that
long-chain fatty acid synthesis is catalyzed by an enzyme complex in which malonyl-CoA is the
source of active acetate.
From 1956 to 1966, Vagelos served as Senior Surgeon and then Section Head of Comparative
Biochemistry in the National Heart Institute’s Laboratory of Biochemistry. During this time,
he continued to study fatty acid synthesis, focusing on the role of acyl carrier protein (ACP).
He discovered that the intermediates in fatty acid synthesis in Escherichia coli are linked to
an acyl carrier protein via a thioester linkage. Vagelos published a series of papers on acyl
carrier protein in the JBC, two of which are reprinted here as Classics.
During fatty acid synthesis, the acyl groups of acetyl-CoA and malonyl-CoA are initially
transferred by acetyl and malonyl transacylases to the sulfhydryl group of ACP. Acetyl-ACP
and malonyl-ACP are then condensed to form acetoacetyl-ACP, which is reduced to D(⫺)-␤-
hydroxybutyryl-ACP. The transacylases, condensing enzyme (acyl-malonyl-ACP condensing
enzyme), and reductase (␤-ketoacyl-ACP reductase) were characterized by Vagelos. This first
Classic focuses on the purification and properties of the enol hydrase (3-hydroxyacyl-ACP
dehydratase) that catalyzes the dehydration of D(⫺)-␤-hydroxybutyryl-ACP to crotonyl-S-ACP.
The second Classic deals with how substrates are linked to ACP. Vagelos had previously
reported that, similar to CoA, substrates are bound to ACP via the sulfhydryl group of
4⬘-phosphopantetheine. However, he noticed that despite this similarity between the two
carriers, thioesters of CoA could not substitute effectively for ACP in fatty acid synthesis. Upon
further study of the structure of ACP, as reported in the second Classic, Vagelos discovered
that 4⬘-phosphopantetheine is bound to ACP through a phosphodiester linkage to the hydroxyl
group of a serine residue.
In 1966, Vagelos assumed the chairmanship of the Department of Biological Chemistry at
Washington University’s School of Medicine in St Louis, MO. He continued to work on fatty
acid biosynthesis and metabolism and expanded his research to the synthesis of complex lipids
and the role of cholesterol in the biochemistry of the cell. In 1973 he became Director of the
University’s Division of Biology and Biomedical Sciences, which he founded. This Division
eventually became a model for other universities. It included both the undergraduate Depart-
ment of Biology and the Medical School in one umbrella unit, which was unheard of at the
time.
H28
Classics
Vagelos left academia in 1975 to join Merck Sharp & Dohme Research Laboratories as Senior
Vice President for Research. In 1984 he was named an Executive Vice President of Merck and was
elected to its Board of Directors, and in 1984 he became Merck’s Chief Executive Officer. He
served as CEO and Chairman of the Board until 1994. Under his direction, the company
expanded its philanthropic efforts as well as its pharmaceutical research. He is perhaps best
known for his decision to make Merck’s Invermectin (Mectizan) available free to millions of people
in Africa and Central America for the treatment of river blindness, a disease spread by black flies
that causes chronic rashes, itching, weight loss, and blindness.
In recognition of his contributions to science, Vagelos received the American Chemical
Society’s Enzyme Chemistry Award in 1967. He was elected to both the National Academy of
Sciences and the American Academy of Arts and Sciences in 1972 and to the American
Philosophical Society in 1993. In 1989 he received the Thomas Alva Edison Award from then
New Jersey Governor Thomas Kean. He is currently Chairman of the Board of Regeneron
Pharmaceuticals, Inc. as well as a member of the Board of Directors of the Prudential
Insurance Company.1
Vagelos’ coauthors on several of the JBC acyl carrier protein papers, including the two
reprinted here, are Philip W. Majerus and Alfred W. Alberts. Majerus went on to become a
Professor at Washington University School of Medicine and has been a leader in phosphoi-
nositide metabolism and signaling, platelet physiology, and blood coagulation. He is a member
of the National Academy of Sciences and has won numerous awards for his research, including
the 1998 Bristol-Myers Squibb Award for Distinguished Achievement in Cardiovascular/
Metabolic Research. Alberts moved from Washington University to Merck with Vagelos and
was the lead scientist in Merck’s development of the statin drugs Lovastatin and Zocor.
REFERENCES
1. JBC Classics: Stadtman, E. R., and Barker, H. A. (1949) J. Biol. Chem. 180, 1085–1093; Mura, U., Chock, P. B.,
and Stadtman, E. R. (1981) J. Biol. Chem. 256, 13022–13029 (http://www.jbc.org/cgi/content/full/280/26/e23)
2. Hawthorne, F. (2003) The Merck Druggernaut, John Wiley & Sons, Inc., Hoboken, NJ
3. Park, B. S. The Stadtman Way: a Tale of Two Biochemists at NIH. http://history.nih.gov/exhibits/stadtman/
index.htm (An online exhibit produced by the Office of NIH History in collaboration with the National Heart,
Lung, and Blood Institute)
1
All biographical information on P. Roy Vagelos was taken from Refs. 2 and 3.
H29
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 283, No. 20, Issue of May 16, p. e12, 2008
Classics
JBC Centennial
1905–2005
How Aspirin Interferes with Cyclooxygenase Activity:

the Work of William L. Smith
Stimulation and Blockade of Prostaglandin Biosynthesis
(Smith, W. L., and Lands, W. E. M. (1971) J. Biol. Chem. 246, 6700 – 6702)
The Aspirin and Heme-binding Sites of Ovine and Murine Prostaglandin Endoper-
oxide Synthases
(DeWitt, D. L., el-Harith, E. A., Kraemer, S. A., Andrews, M. J., Yao, E. F., Armstrong,
R. L., and Smith, W. L. (1990) J. Biol. Chem. 265, 5192–5198)
William L. Smith, Jr. was born in
Tulsa, Oklahoma in 1945. He and his
family moved to the Chicago area
when he was an infant and then to
Fort Collins, Colorado when he was in
high school. After graduating from
high school, Smith enrolled in a pre-
med program at the University of Col-
orado in the fall of 1963, even though
he had no idea what sort of career he
wanted to pursue. However, this
changed during the first couple years
of college when he took several chem-
istry courses that were taught by ex-
cellent teachers. His admiration for
these professors led to his decision to
attend graduate school. Originally, he
planned to go into physical organic
chemistry but chose biological chem-
istry when he was told that physical
organic chemistry was already a
highly populated field. “Little did I
know that the same thing would
happen in biochemistry by the time I
was searching for a position,” recalls
Smith.
After graduating with a B.A. in
1967, Smith decided to attend the
University of Michigan and work with
William L. Smith William E. M. Lands. This decision
was influenced by three papers by
Mats Hamberg and Bengt Samuelsson (1–3) published in the Journal of Biological Chemistry
(JBC) that Lands gave him to read.“These papers dealt with the mechanism of oxygenation of
dihomo-␥-linolenic acid and other fatty acids by soybean lipoxygenase and sheep seminal
vesicle cyclooxygenase (i.e. prostaglandin H2 synthase 1 or PGHS-1),” recalls Smith. “In the
course of these studies they labeled the ␻8 carbon (C-13) of the substrate with tritium in the
H30
Classics
proS and proR orientations and found a distinct kinetic isotope effect in the removal of the
proS hydrogen. This indicated that the rate determining step was abstraction of this hydrogen
from the fatty acid. To this day, I find this to be a brilliant experiment and an exciting outcome.
Almost 25 years later we provided evidence that Tyr-385 of PGHSs abstracted this hydrogen
atom from the fatty acid (4).” More information on Samuelsson’s work on the prostaglandins
can be found in his JBC Classic (5).
Just as Smith was finishing up his thesis work with Lands, John Vane reported that
acetylsalicylic acid (aspirin) and another well known nonsteroidal anti-inflammatory drug
called indomethacin blocked the biosynthesis of prostaglandins from arachidonic acid (6, 7).
Smith had just set up an O2 electrode assay to measure prostaglandin production in acetone
powder preparations of sheep seminal vesicle microsomes, and he and Lands immediately
tested aspirin and indomethacin in this assay. As reported in the first JBC Classic reprinted
here, they found that aspirin and indomethacin blocked arachidonic acid-induced O2 uptake
and concluded that these drugs were blocking oxygenase activity. They also observed that the
two drugs acted in a time-dependent manner, suggesting that they were causing a chemical
modification of their target. Subsequently, it was shown that the acetyl group of aspirin was
incorporated into a protein (8) that Smith later purified (9) in his first JBC paper as an
independent scientist. The protein is now known as prostaglandin endoperoxide H synthase-1
(PGHS-1) or cyclooxygenase-1 (COX-1).
Smith completed his Ph.D. work in under 4 years, and in 1971 went to the University of
California at Berkeley to work with Clinton E. Ballou. There he changed the focus of his
research and worked on polysaccharide structures. Although he enjoyed this work, he decided
he was more interested in solving problems that he felt were more biomedically relevant. He
also realized that he still had an intense interest in prostaglandins. In 1974, Smith took a job
as a senior scientist at Mead Johnson in Evansville, Indiana where he spent his time studying
platelet aggregation, prostaglandin formation, and arachidonic acid mobilization.
A year later, he accepted a position in the Department of Biochemistry at Michigan State
University where he remained for 28 years and served as chair for the last 8 years of his time
there. At Michigan State, Smith continued to work on prostaglandins, focusing, among other
things, on the acetylation of PGHS-1. Work in other laboratories suggested that aspirin was
acetylating a serine residue on the enzyme (10, 11). In 1988 Smith and his long time colleague
David DeWitt were able to clone and sequence PGHS-1 cDNA derived from seminal vesicles
(12). They observed that the acetylated serine corresponded to Ser-530. Two years later, as
reported in the second JBC Classic reprinted here, Smith and his colleagues showed that
substitution of Ser-530 with alanine rendered the protein refractory toward aspirin but had
relatively little effect on the kinetic properties of the cyclooxygenase. They concluded that the
Ser-O-acetyl protrudes into the cyclooxygenase active site thereby interfering with arachidonic
acid binding. This is the most definitive biochemical work on how aspirin works at the
molecular level to interfere with cyclooxygenase activity. Smith later showed that substitu-
tions of Ser-530 with bulkier residues such as threonine and asparagine gradually reduced
cyclooxygenase activity (13).
In 2002 Smith decided to step down as Chair of Biochemistry at Michigan State, saying “My
opinion is that administrators should serve no longer than American presidents.” Shortly
thereafter he was given the opportunity to become Chair of the Biological Chemistry Depart-
ment at the University of Michigan. Still at the University of Michigan today, he continues to
work on prostaglandins.
In recognition of his many contributions to science, Smith has received several awards and
honors. These include the 1991 Treadwell Award from George Washington University, the
1992 Distinguished Faculty Award from Michigan State University, the 1996 Abraham White
Distinguished Scientific Achievement Award from George Washington University, the 1997
Senior Aspirin Award from Bayer Corporation, the 1999 Michigan Universities Association of
Governing Boards Award, the 2004 State of Michigan Scientist of the Year Award, the 2004
Berzelius Lectureship from the Karolinska Institute, the 2004 Avanti Award from the Amer-
ican Society for Biochemistry and Molecular Biology, the 2006 William C. Rose Award from the
American Society for Biochemistry and Molecular Biology, and the 2006 Hayaishi Lectureship
from Hamamatsu University.1
1
We thank William L. Smith for providing background information for this introduction.
H31
Classics
Smith’s co-author on the first Classic, William E. M. Lands, was a young faculty member at
the University of Michigan when Smith joined his laboratory. Lands subsequently left Mich-
igan in 1980 to become Chair of Biochemistry at the University of Illinois, Chicago, and later
moved to the National Institutes of Health in 1990, where he served as the Senior Scientific
Advisor to the Director of the National Institute on Alcohol Abuse and Alcoholism. Lands is the
discoverer of the “retailoring” pathway for membrane phospholipid synthesis. He is an au-
thority on essential fatty acids and is credited with recognizing the beneficial effects of
balancing excess ␻-6 fatty acids with dietary ␻-3 fatty acids. In recognition of his work, the
University of Michigan’s Department of Biological Chemistry endowed a lectureship in nutri-
tional biochemistry in honor of Lands.
REFERENCES
1. Hamberg, M., and Samuelsson, B. (1967) On the specificity of the oxygenation of unsaturated fatty acids catalyzed
by soybean lipoxidase. J. Biol. Chem. 242, 5329 –5335
2. Hamberg, M., and Samuelsson, B. (1967) On the mechanism of the biosynthesis of prostaglandins E1 and F1␣.
J. Biol. Chem. 242, 5336 –5343
3. Hamberg, M., and Samuelsson, B. (1967) Oxygenation of unsaturated fatty acids by the vesicular gland of sheep.
J. Biol. Chem. 242, 5344 –5354
4. Shimokawa, T., Kulmacz, R. J., DeWitt, D. L., and Smith, W. L. (1990) Tyrosine 385 of prostaglandin endoperoxide
synthase is required for cyclooxygenase catalysis. J. Biol. Chem. 265, 20073–20076
5. JBC Classics: Bergström, S., Ryhage, R., Samuelsson, B., and Sjövall, J. (1963) J. Biol. Chem. 238, 3555–3564;
Hamberg, M., and Samuelsson, B. (1967) J. Biol. Chem. 242, 5336 –5343 (http://www.jbc.org/cgi/content/full/
281/9/e9)
6. Vane, J. R. (1971) Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nat. New
Biol. 231, 232–235
7. Ferreira, S. H., Moncada, S., and Vane, J. R. (1971) Indomethacin and aspirin abolish prostaglandin release from
the spleen. Nat. New Biol. 231, 237–239
8. Roth, G. J., Stanford, N., and Majerus, P. W. (1975) Acetylation of prostaglandin synthase by aspirin. Proc. Natl.
Acad. Sci. U. S. A. 72, 3073–3076
9. Hemler, M., Lands, W. E., and Smith, W. L. (1976) Purification of the cyclooxygenase that forms prostaglandins.
Demonstration of two forms of iron in the holoenzyme. J. Biol. Chem. 251, 5575–5579
10. Van Der Ouderaa, F. J., Buytenhek, M., Nugteren, D. H., and Van Dorp, D. A. (1980) Acetylation of prostaglandin
endoperoxide synthetase with acetylsalicylic acid. Eur. J. Biochem. 109, 1– 8
11. Roth, G. J., Machuga, E. T., and Ozols, J. (1983) Isolation and covalent structure of the aspirin-modified,
active-site region of prostaglandin synthetase. Biochemistry 22, 4672– 4675
12. DeWitt, D. L., and Smith, W. L. (1988) Primary structure of prostaglandin G/H synthase from sheep vesicular
gland determined from the complementary DNA sequence. Proc. Natl. Acad. Sci. U. S. A. 85, 1412–1416
13. Shimokawa, T., and Smith, W. L. (1992) Prostaglandin endoperoxide synthase. The aspirin acetylation region.
J. Biol. Chem. 267, 12387–12392
H32
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 281, No. 31, Issue of August 4, p. e25, 2006
Classics
JBC Centennial
1905–2005
30 Years of Cholesterol Metabolism: the Work of

Michael Brown and Joseph Goldstein
Binding and Degradation of Low Density Lipoproteins by Cultured Human
Fibroblasts. Comparison of Cells from a Normal Subject and from a Patient
with Homozygous Familial Hypercholesterolemia
(Goldstein, J. L., and Brown, M. S. (1974) J. Biol. Chem. 249, 5153–5162)
Joseph Leonard Goldstein was born in 1940 in Sumter, South Carolina. He attended
Washington and Lee University in Lexington, Virginia and received a B.S. degree in chemistry
in 1962. Goldstein then went to the Southwestern Medical School at the University of Texas
Health Science Center in Dallas where he was inspired to pursue a career in academic
medicine by the Chairman of the Department of Internal Medicine, Donald W. Seldin.
During Goldstein’s last year in medical school, Seldin offered him a future faculty appoint-
ment if he agreed to specialize in genetics and return to Dallas to establish a division of
medical genetics in the Department of Internal Medicine. Goldstein initially declined, and
after receiving his M.D. in 1966, he moved to Boston where he was an intern and resident at
the Massachusetts General Hospital. It was there that he first met and developed a friendship
with Michael Stuart Brown, his long term scientific collaborator.
Brown, who was born in 1941 in Brooklyn, New York, graduated in 1962 from the University
of Pennsylvania, with a major in chemistry. He received his M.D. degree from the University
of Pennsylvania School of Medicine in 1966, after which he became an intern and resident at
the Massachusetts General Hospital.
After they completed their training in 1968, both Brown and Goldstein obtained positions at the
National Institutes of Health in Bethesda, Maryland. Brown joined the Laboratory of Biochem-
istry at the National Heart, Lung, and Blood Institute (NHLBI). Goldstein worked with Nobel
laureate Marshall W. Nirenberg and also worked as a clinical associate at NHLBI, serving as
physician to the patients of Donald S. Fredrickson, who was an expert on disorders of lipid
metabolism. Several of these patients had homozygous familial hypercholesterolemia, a condition
that causes severe elevations in cholesterol levels. Goldstein discussed these patients extensively
with Brown and, in view of their common interest in metabolic disease, convinced Brown to join
him as a faculty member at the University of Texas Health Science Center at Dallas, where they
would work collaboratively on the genetic regulation of cholesterol metabolism.
In 1971 Brown joined the division of Gastroenterology in the Department of Internal
Medicine at the University of Texas Southwestern Medical School. Before going back to Dallas,
Goldstein spent 2 years with Arno G. Motulsky at the University of Washington in Seattle,
studying human genetics. He returned to the University of Texas Health Science Center in
1972 and was appointed Assistant Professor in the Department of Internal Medicine and head
of the medical school’s first Division of Medical Genetics.
Together, Brown and Goldstein began to address the task of identifying the genetic defect in
familial hypercholesterolemia. They started by observing tissue cultures of fibroblasts har-
vested from healthy individuals and individuals with familial hypercholesterolemia. They set
up a microassay to measure the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase,
the rate-determining enzyme of cholesterol biosynthesis, in the fibroblasts. Soon it became
clear that the cholesterol transport protein, low density lipoprotein (LDL), suppressed the
activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Because high density lipopro-
H33
Classics
Fig. 1
tein (HDL) was unable to do this, Brown and Goldstein suspected that a receptor might be
involved in the control of 3-hydroxy-3-methylglutaryl coenzyme A reductase.
The existence of an LDL receptor was confirmed when Brown and Goldstein radiolabeled LDL
with 125I and incubated it with normal and familial hypercholesterolemial fibroblasts. As re-
ported in the JBC Classic reprinted here, their studies showed that normal cells had high affinity
binding sites for 125I-LDL whereas familial hypercholesterolemial cells did not. Binding of LDL
to the high affinity membrane receptor sites suppressed the synthesis of 3-hydroxy-3-methylglu-
taryl coenzyme A reductase and also facilitated the degradation of LDL when it was present at
low concentrations. Cells from subjects with familial hypercholesterolemia not only lacked the
binding sites but were also resistant to suppression of 3-hydroxy-3-methylglutaryl coenzyme A
reductase activity by LDL and were deficient in high affinity degradation of LDL.
However, the question of how LDL generated the signal that suppressed 3-hydroxy-3-methyl-
glutaryl coenzyme A reductase still remained. The answer to this question came from studies of
surface-bound 125I-LDL. Brown and Goldstein found that the receptor-bound LDL remained on
the cell’s surface for less than 10 min. Within this time most of the surface-bound LDL particles
entered the cell. Within another 60 min the protein component of 125I-LDL was digested com-
pletely. The only cellular organelle that could have degraded LDL so completely and rapidly was
the lysosome. This was eventually confirmed, and Brown and Goldstein also showed that the
cholesterol that was generated from lysosomal degradation of LDL acted as the second messenger
responsible for suppressing 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.
As is often the case with truly novel, groundbreaking discoveries, the work presented in this
JBC Classic was not met with great enthusiasm by journal reviewers. The initial review of this
paper by the JBC is presented in Fig. 1, and the decision letter from Associate Editor Eugene
Kennedy is shown in Fig. 2. This paper, the basis of the Nobel Prizes awarded to Brown and
Goldstein, was eventually accepted.
By 1974 Brown and Goldstein had merged their laboratories. They continued their work on
the LDL receptor and eventually purified and sequenced it. In recognition of their work, they
were awarded the 1985 Nobel Prize in Physiology or Medicine “for their discoveries concerning
the regulation of cholesterol metabolism.”
Goldstein eventually became Associate Professor of Internal Medicine at the University of
Texas Southwestern Medical School (1974) and then Professor (1976). In 1977, he was made
Chairman of the Department of Molecular Genetics at the University of Texas Health Science
Center and Paul J. Thomas Professor of Medicine and Genetics, a position that he still holds
today. In 1985, he was named Regental Professor of the University of Texas, and in 1983 he
became a Non-resident Fellow of The Salk Institute for Biological Sciences.
H34
Classics
Fig. 2
In 1974, Brown was promoted to the rank of Associate Professor of Internal Medicine at the
University of Texas Southwestern Medical School. He became a Professor in 1976. In 1977 he
was appointed Paul J. Thomas Professor of Medicine and Genetics and Director of the Center
for Genetic Disease at the same institution. In 1985, Brown was named Regental Professor of
the University of Texas.
In addition to the Nobel Prize, Brown and Goldstein’s work has been recognized by their receipt
of numerous awards, including the Heinrich Wieland Prize for Research in Lipid Metabolism
(1974), the American Chemical Society’s Pfizer Award for Enzyme Chemistry (1976), the Passano
Award (1978), the National Academy of Sciences’ Lounsbery Award (1979), the Gairdner Foun-
dation International Award (1981), the Lita Annenberg Hazen Award (1982), the Association of
American Medical Colleges’ Distinguished Research Award (1984), the American Heart Associ-
ation’s Research Achievement Award (1984), the FASEB 3M Life Sciences Award (1985), the
Albert D. Lasker Award in Basic Medical Research (1985), the U. S. National Medal of Science
(1988), the Albany Medical Prize in Biomedical Sciences (2003), and the Herbert Tabor/Journal
of Biological Chemistry Lectureship (2005).
Both Brown and Goldstein were elected to the National Academy of Sciences and the
American Academy of Arts and Sciences. Goldstein is, or has been, a member of the editorial
board of several journals including the Annual Review of Genetics, Arteriosclerosis, Cell, the
Journal of Biological Chemistry, the Journal of Clinical Investigation, and Science. Brown has
served on the editorial boards of the Journal of Lipid Research, the Journal of Cell Biology,
and Arteriosclerosis and Science.1,2
1
All biographical information on Joseph L. Goldstein was taken from Refs. 1 and 2.
2
All biographical information on Michael S. Brown was taken from Refs. 2 and 3.
H35
Classics
Joseph L. Goldstein Michael S. Brown
REFERENCES
1. Goldstein, J. L., and Brown, M. S. (1993) A receptor-mediated pathway for cholesterol homeostasis. From Nobel
Lectures, Physiology or Medicine 1981–1990 (Frängsmyr, T., ed) World Scientific Publishing Co., Singapore
2. Goldstein, J. L. (1986) Joseph L. Goldstein—Biography. In The Nobel Prizes 1985 (Odelberg, W., ed) Nobel
Foundation, Stockholm
3. Brown, M. S. (1986) Michael S. Brown—Biography. In The Nobel Prizes 1985 (Odelberg, W., ed) Nobel Foundation,
Stockholm
H36
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 281, No. 5, Issue of February 3, p. e5, 2006
Classics
JBC Centennial
1905–2005
Salih Wakil’s Elucidation of the Animal Fatty Acid

Synthetase Complex Architecture
The Architecture of the Animal Fatty Acid Synthetase. I. Proteolytic Dissection and
Peptide Mapping
(Mattick, J. S., Tsukamoto, Y., Nickless, J., and Wakil, S. J. (1983) J. Biol. Chem. 258,
15291–15299)
The Architecture of the Animal Fatty Acid Synthetase. II. Separation of the Core and
Thioesterase Functions and Determination of the N-C Orientation of the Subunit
(Mattick, J. S., Nickless, J., Mizugaki, M., Yang, C. Y., Uchiyama, S., and Wakil, S. J.
(1983) J. Biol. Chem. 258, 15300 –15304)
The Architecture of the Animal Fatty Acid Synthetase. III. Isolation and Character-
ization of Beta-Ketoacyl Reductase
(Wong, H., Mattick, J. S., and Wakil, S. J. (1983) J. Biol. Chem. 258, 15305–15311)
The Architecture of the Animal Fatty Acid Synthetase Complex. IV. Mapping of
Active Centers and Model for the Mechanism of Action
(Tsukamoto, Y., Wong, H., Mattick, J. S., and Wakil, S. J. (1983) J. Biol. Chem. 258,
15312–15322)
Salih Jawad Wakil was born in 1927 in Kerballa, Iraq. Because he placed third in the nation
on the baccalaureate examination out of high school, he received a scholarship to the American
University in Beirut. While at the American University he met Stanley Kerr, who introduced
him to biochemistry and gave him the opportunity to work in his laboratory. After graduating
in 1948, Wakil was accepted at the University of Washington, which he assumed was located
in the U. S. capital. However, he arrived in the United States only to learn that he would have
to take a 3-day train journey from New York to his university in Washington State. In Seattle,
Wakil worked with Donald Hanahan and finished his graduate studies in biochemistry in 31⁄2
years. Next, he decided to do postdoctoral training at the Enzyme Institute of the University
of Wisconsin, where he began to work on fatty acid oxidation. It was there that he helped to
elucidate the steps by which fatty acids are oxidized and showed that fatty acids are synthe-
sized and oxidized by different pathways.
Wakil was named assistant professor in 1956, but joined the Department of Biochemistry at
the Duke University School of Medicine in 1959 and rose to the rank of professor there (1965).
At Duke, Wakil investigated fatty acid synthesis in Escherichia coli. He and Roy Vagelos, who
was featured in a previous Journal of Biological Chemistry (JBC) Classic (1), independently
studied the role of acyl carrier protein as well as several of the individual reactions of fatty acid
elongation. Wakil left Duke in 1971 to become professor and chairman of the Verna and Marrs
McLean Department of Biochemistry and Molecular Biology at Baylor College of Medicine in
Houston,Texas. At Baylor, Wakil studied the multifunctional enzyme, fatty acid synthetase.
The characterization of this enzyme complex is the subject of the four JBC Classics reprinted
here.
In vertebrates, the fatty acid synthetase complex exists as a dimer of what Wakil believed
were identical subunits derived from a single large mRNA. The complex contains the seven
enzymatic activities needed for the assembly of fatty acids: (i) acetyl transacylase, (ii) malonyl
transacylase, (iii) ␤-ketoacyl synthetase, (iv) ␤-ketoacyl reductase, (v) ␤-hydroxyacyl dehy-
H37
Classics
Salih J. Wakil
dratase, (vi) enoyl reductase, and (vii) palmitoyl thioesterase, as well as an acyl carrier peptide
to which the nascent chain is attached. These Classics, which were printed as a back-to-back
series in one issue of the JBC, present Wakil’s comprehensive proteolytic analysis of chicken
fatty acid synthetase in which he assigned relative locations for the enzymatic activities in the
complex.
In the first Classic, Wakil and his colleagues used seven different proteases to digest the
synthetase. They found that the sum of the molecular weights of each set of fragments
generated by the proteases corresponded to the size of the synthetase subunit rather than the
native dimer, indicating that the synthetase was indeed a homodimer. The researchers also
reported that the subunit is arranged into three major domains of Mr ⫽ 127,000, 107,000, and
33,000.
Wakil describes the cleavage of chicken fatty acid synthetase by ␣-chymotrypsin in the
second Classic. The complex was cleaved into two fragments. The larger 230-kDa fragment
contained all the core activities involved in the assembly of the fatty acyl chain whereas the
smaller 33-kDa fragment retained the thioesterase activity which releases the complete
product. Using amino acid sequence analysis, Wakil showed that the thioesterase domain is
located at the carboxyl terminus of the synthetase monomer.
In the third Classic, Wakil used trypsin and subtilisin to cleave fatty acid synthetase and
isolated a polypeptide containing only ␤-ketoacyl reductase activity. Using a kallikrein/sub-
tilisin double digestion, Wakil and his colleagues also isolated another fragment containing
␤-ketoacyl reductase activity as well as the phosphopantetheine prosthetic group. From this,
Wakil concluded that the acyl carrier protein moiety is located in the 15-kDa segment that
separates the ␤-ketoacyl reductase from the thioesterase domain.
In the fourth and final Classic, Wakil presents an architectural model for the synthetase
based on his results from the previous three papers. In Wakil’s model, domain I functions as
a site for acetyl and malonyl substrate entry and acts as the site of carbon-carbon condensa-
tion. Thus, this domain contains the amino terminus of the polypeptide and the ␤-ketoacyl
synthetase and acetyl and malonyl transacylases. Domain II, the reductive domain, contains
the ␤-ketoacyl and enoyl reductases, probably the dehydratase, and the 4⬘-phosphopanteth-
eine prosthetic group of the acyl carrier protein. Finally, domain III contains the thioesterase
activity. Based on his observations, Wakil concluded that even though each subunit contains
all the activities needed for fatty acyl synthesis, the actual synthesizing unit consists of
one-half of one subunit interacting with the complementary half of the other subunit. This is
shown in the model in Fig. 2. Wakil, along with Bornali Chakravarty, Ziwei Gu, Subrah-
manyam S. Chirala, and Florante A. Quiocho, subsequently solved the crystal structure of the
H38
Classics
Fig. 2
thioesterase domain of human fatty acid synthetase (2). Recent crystallographic analysis of
both the animal and fungal fatty acid synthases demonstrates that the structure is of a
head-to-head coiled dimer (3, 4).
Today, Wakil remains at Baylor where he is Distinguished Service Professor and Bolin
Professor in the Department of Biochemistry and Molecular Biology. Most recently, his focus
has been on acetyl-CoA carboxylase (ACC), which exists in two forms, ACC1 and ACC2. He has
developed a transgenic mouse, which does not produce ACC2, and as a result can eat 20 –30%
more food and weighs 10% less than mice that produce the enzyme.
In honor of Wakil’s contributions to the field of fatty acid metabolism, he has received many
awards and honors. These include the Paul Lewis Award from the American Chemical Society
(1967), the Chilton Award of the University of Texas Southwestern Medical Center (1985), the
Kuwait Prize of the Kuwait Foundation for the Advancement of Sciences (1988), the Yaman-
ouchi USA Foundation Award (2001), and the Bristol-Myers Squibb Freedom to Discover
Award (2005). In 1990, Wakil was the first Baylor College of Medicine faculty member to be
elected to the National Academy of Sciences.
REFERENCES
1. JBC Classics: Majerus, P. W., Alberts, A. W., and Vagelos, P. R. (1965) J. Biol. Chem. 240, 618 – 621; Majerus,
P. W., Alberts, A. W., and Vagelos, P. R. (1965) J. Biol. Chem. 240, 4723– 4726 (http://www.jbc.org/cgi/
content/full/280/35/e32)
2. Chakravarty, B., Gu, Z., Chirala, S. S., Wakil, S. J., and Quiocho, F. A. (2004) Human fatty acid synthase:
structure and substrate selectivity of the thioesterase domain. Proc. Natl. Acad. Sci. U. S. A. 101, 15567–15572
3. Smith, S. (2006) Architectural options for a fatty acid synthase. Science 311, 1251–1252
4. Maier, T., Jenni, S., and Ban, N. (2006) Architecture of mammalian fatty acid synthase at 4.5 Å resolution. Science
311, 1258 –1262
H39
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 283, No. 2, Issue of January 11, p. e2, 2008
Classics
JBC Centennial
1905–2005
N-Myristoyltransferase Substrate Selection and Catalysis:

the Work of Jeffrey I. Gordon
Myristoyl CoA:Protein N-Myristoyltransferase Activities from Rat Liver and Yeast
Possess Overlapping Yet Distinct Peptide Substrate Specificities
(Towler, D. A., Adams, S. P., Eubanks, S. R., Towery, D. S., Jackson-Machelski, E.,
Glaser, L., and Gordon, J. I. (1988) J. Biol. Chem. 263, 1784 –1790)
Isothermal Titration Calorimetric Studies of Saccharomyces cerevisiae Myristoyl-
CoA:Protein N-Myristoyltransferase. Determinants of Binding Energy and Catalytic
Discrimination among Acyl-CoA and Peptide Ligands
(Bhatnagar, R. S., Jackson-Machelski, E., McWherter, C. A., and Gordon, J. I. (1994)
J. Biol. Chem. 269, 11045–11053)
Jeffrey I. Gordon obtained his A.B. from
Oberlin College in 1969 and his M.D. from
the University of Chicago in 1973. He then
became an intern and junior assistant resi-
dent at Barnes Hospital in St. Louis before
spending 3 years as a research associate in
the Laboratory of Biochemistry at the Na-
tional Cancer Institute (National Institutes
of Health). Gordon returned to Barnes Hos-
pital as a Senior Assistant Resident in 1978
and was concurrently a Chief Medical Res-
ident at John Cochran VA Hospital in St.
Louis. He became an assistant professor at
Washington University in St. Louis in 1981
and has remained there ever since. He is
currently Director of the Center for Genome
Sciences as well as the Dr. Robert J. Glaser
Distinguished University Professor.
Gordon is probably best known for his
research on gastrointestinal development
and how gut bacteria affect normal intesti-
nal function and predisposition to health
and to certain diseases. However, he has
also made significant contributions to our
knowledge about myristoylation, the post-
translational process by which a myristoyl
group is covalently attached via an amide
Jeffrey I. Gordon bond to the ␣-amino group of an N-terminal
glycine residue of a nascent polypeptide.
Some of Gordon’s work on N-myristoyltransferase, the enzyme responsible for myristoylation,
is the subject of the two Journal of Biological Chemistry (JBC) Classics reprinted here.
Gordon started doing research on N-myristoyltransferase when Luis Glaser, a fellow scien-
tist at Washington University, accepted a position at the University of Miami and transferred
H40
Classics
the project, along with his student Dwight Towler, to Gordon’s laboratory. Glaser and Towler
had already partially purified N-myristoyltransferase and were using peptides with altered
sequences to test as enzyme substrates in an in vitro assay system (1, 2). From their studies
they had determined that residues occupying positions 1, 2, and 5 in peptide substrates (where
the myristoylated glycine is residue 1) have important effects on protein-ligand interactions.
In Gordon’s laboratory, Towler worked out a purification scheme for N-myristoyltransferase
from yeast (3) and continued to investigate the primary structural characteristics of the
enzyme’s peptide substrates. As reported in the first JBC Classic, Towler and Gordon screened
over 80 synthetic peptides and discovered that a substrate hexapeptide contains much of the
information necessary for recognition by N-myristoyltransferase. They also identified a num-
ber of potential N-myristoyl proteins from searches of available protein data bases.
In the second JBC Classic, Gordon and his colleagues report on the kinetics of the reaction
in which N-myristoyltransferase transfers myristate from myristoyl-CoA to the amino-termi-
nal glycine nitrogen of its substrate. Using isothermal titration calorimetry they quantified the
effects of varying acyl chain length and removing the 3⬘-phosphate group of CoA on the
energetics of interaction between N-myristoyltransferase and acyl-CoA ligands. From these
studies, they were able to gain insights into how the enzyme selects its substrates and about
its catalytic mechanism.
In recognition of his contributions to science, Gordon has received many awards and honors.
These include the 1990 American Federation for Clinical Research Young Investigator Award,
the 1990 National Institute of Diabetes and Digestive and Kidney Diseases Young Scientist
Award, the 1992 American Gastroenterological Association (AGA) Distinguished Achievement
Award, the 1994 Marion Merrell Dow Distinguished Prize in Gastrointestinal Physiology, the
2003 Janssen/AGA Award for Sustained Achievement in Digestive Sciences, and the 2003
Horace W. Davenport Distinguished Lectureship from the American Physiological Association.
Gordon is also a member of the National Academy of Sciences and the American Academy of
Arts and Sciences.
Luis Glaser, who was responsible for initiating Gordon’s investigations of N-myristoyltrans-
ferase, has also led a fruitful career in science. Glaser, who grew up in Mexico, attended the
University of Toronto for his undergraduate education and earned his Ph.D. from Washington
University. After graduating, he joined the faculty of Washington University where he re-
mained for the next 30 years, spending the last 10 years as Chairman of the Department of
Biology and Chemistry and Director of the Division of Biology and Biomedical Science. In 1986,
Glaser accepted an offer to become Executive Vice President and Provost at the University of
Miami. He retired from that position in July of 2005 and is currently a Professor of Biology and
Special Assistant to the President.
REFERENCES
1. Towler, D., and Glaser, L. (1986) Protein fatty acid acylation: enzymatic synthesis of an N-myristoylglycyl peptide.
Proc. Natl. Acad. Sci. U. S. A. 83, 2812–2816
2. Towler, D. A., Eubanks, S. R., Towery, D. S., Adams, S. P., and Glaser, L. (1987) Amino-terminal processing of
proteins by N-myristoylation. Substrate specificity of N-myristoyltransferase. J. Biol. Chem. 262, 1030 –1036
3. Towler, D. A., Adams, S. P., Eubanks, S. R., Towery, D. S., Jackson-Machelski, E., Glaser, L., and Gordon, J. I.
(1987) Purification and characterization of yeast myristoyl CoA:protein N-myristoyltransferase. Proc. Natl.
Acad. Sci. U. S. A. 84, 2708 –2712
H41
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 46, Issue of November 16, pp. 42619 –42631, 2001
Reflections
A PAPER IN A SERIES COMMISSIONED TO CELEBRATE THE CENTENARY OF THE JBC IN 2005
JBC Centennial
1905–2005
Hitler’s Gift and the Era of Biosynthesis

Published, JBC Papers in Press, September 14, 2001, DOI 10.1074/jbc.R100051200
Eugene P. Kennedy‡
From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School,
Boston, Massachusetts 02115
Before the Second World War biochemistry in the United States had a strong flavor of
clinical chemistry. It was much occupied with problems of analysis of blood and tissues and the
determination of the structures of body constituents. This was important and indeed essential
work, but American students had to go abroad to Germany or to England for training in what
came to be called dynamic aspects of biochemistry. After the war, the flow of students was
largely reversed. This transformation was in considerable part the result of new insights and
new approaches brought to America by immigrant scientists.
It is a remarkable fact that as late as 1945 when I began graduate studies in biochemistry
at the University of Chicago almost nothing was known about the linked reactions leading to
the biosynthesis of any of the major types of cell constituents, carbohydrates, lipids, proteins,
or nucleic acids. However, this picture was about to change with dramatic rapidity. The latter
half of the 20th century became the era of biosynthesis. Now, in 2001, we know in great detail
the patterns of reactions leading to the formation of each of these classes of cellular materials,
although to be sure much remains to be learned about the regulation and integration of
biosynthetic processes in living organisms.
The achievements of three biochemists, Fritz Lipmann, Rudolf Schoenheimer, and Konrad
Bloch, greatly stimulated this flowering of biosynthetic studies in the United States at the
mid-20th century. Each had been driven out of Germany by the brutal anti-Semitism of the
Nazi regime. Each was an important part of what has been called Hitler’s gift (1) to American
and British science.
In helping to bring about the transition to the era of biosynthesis, Fritz Lipmann made clear
the crucial role of “energy-rich” phosphates in driving biosynthetic reactions and showed how
this principle operated in the formation of the much sought and highly elusive “active acetate”
involved in so many pathways. Rudolf Schoenheimer helped put into the hands of biochemists
their most subtle and versatile approach, that of the isotope tracer technique, and with its aid
revealed the dynamic state of body constituents. Konrad Bloch’s work on the formation of
cholesterol illustrated how the insights of Lipmann and Schoenheimer could be combined in a
masterpiece of biochemistry to solve a problem of great medical as well as biological significance.
Fritz Lipmann: The Energetics of Biosynthesis
Fritz Lipmann (Fig. 1), who helped to shape the development of modern biochemistry, was
born in Koenigsberg, East Prussia in 1899 into a Jewish family of the professional class (2). In
1917, he began the study of medicine. In 1918, while still a medical student, he was drafted
into the German army and spent the rest of the war in the medical corps in France. Released
from the army, Lipmann resumed his medical studies and received the M.D. degree in 1921.
He soon abandoned plans for the practice of medicine in favor of biochemical research, but he
always valued the broad view of biology his medical education had given him, concluding: “The
‡ To whom correspondence may be addressed. E-mail: eugene_kennedy@hms.harvard.edu.
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Reflections: Hitler’s Gift and the Era of Biosynthesis
FIG. 1. Fritz Lipmann. Photo by John Brook, made available through the courtesy of Ms. Freda Hall Lipmann.
biological education to which the observant student is exposed in medicine is a superior

preparation for any career.” Indeed, the study of medicine offered the most comprehensive
view of biology then available. Many of the greatest figures in biochemistry early in the 20th
century, including Warburg, Meyerhof, and Krebs, were trained as physicians. The breadth of
his background helped give Lipmann the confidence that nothing in biology was beyond his
range. Again and again, he proved ready to tackle new problems, no matter how far removed
from previous work in his laboratory.
Turning to a career in research rather than the practice of medicine, Lipmann realized that
the most fruitful approach to biological problems was through chemistry. He began a program
leading to a Ph.D. in chemistry. His work for the dissertation, begun in 1927, was carried out
in the laboratory of Otto Meyerhof. Meyerhof, whose work on glycolysis in muscle earned him
a Nobel Prize, had a laboratory on the first floor of the Kaiser-Wilhelm Institute for Biology in
Berlin, a city that was then the leading center of science in the world. Lipmann felt that his
experience in Meyerhof’s laboratory was in many ways the origin of all his later work. His most
intense admiration, however, was reserved for Warburg. As Lipmann later recalled (3): “At the
top of everything, on the uppermost floor, was Otto Warburg. Warburg already had a mystery
about him. We admired him boundlessly but saw little of him . . . ”
In Meyerhof’s laboratory, Lipmann worked on the role of creatine phosphate in muscle
contraction. It was of course known that muscle contraction, with its attendant production of
lactic acid, is intimately linked to glycolysis. The energetics of this linkage, however, remained
obscure. Lipmann (3) commented on “ . . . the vagueness of the understanding, then prevalent,
of both the intermediary path of glycolysis and the mechanism of action of energy-rich
phosphate.” This work did much to turn Lipmann’s thinking to the role of phosphorylated
intermediates in energy transduction.
In 1930, Lipmann was already aware that a career for a Jewish scientist in Germany was
fraught with difficulty and peril. There began a period of Wanderjahre before he finally found
a position that offered both independence and scope. However, he wished to remain in Berlin
at least for a time to be near his fiancee Freda Hall (3). He became an assistant to Albert
Fischer, working on problems of tissue culture. In 1931 during a hiatus in the work of Fischer’s
laboratory caused by its move from Berlin to Copenhagen, Lipmann, newly married to Freda
Hall, traveled to the United States to work at the Rockefeller Institute in New York in the
laboratory of Phoebus A. Levene on the biochemistry of phosphoproteins. Here he succeeded in
isolating phosphoserine from partial acid hydrolysates of egg phosphoprotein.
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In 1932, Lipmann rejoined Fischer’s group in its new quarters in the Carlsberg Laboratories
in Copenhagen, where he was to remain until 1939. He was free to work independently in
pursuit of his own ideas. At about this time, Otto Warburg was making his great discoveries
elucidating the central mechanisms of glycolysis. Why the splitting of glucose involved phos-
phorylated intermediates had long been a great puzzle, which Warburg now solved.
In 1905 Arthur Harden, working in London, had discovered that glycolysis requires a
heat-stable, organic cofactor, which he termed “cozymase.” This cofactor proved to be remark-
ably elusive. In 1929, more than two decades later, Hans von Euler received a Nobel Prize for
his work on its isolation and characterization, but it is clear from his Nobel lecture that he had
at that time no real idea of its true structure and function. It was Warburg and his collabo-
rators (4) who isolated “cozymase” and showed that it contains a pyridine ring that undergoes
alternate reduction and re-oxidation. It is of course the famous coenzyme NAD now known to
function in many hundreds of enzyme-catalyzed redox reactions. Warburg also discovered that
the oxidation of 3-phosphoglyceraldehyde by NAD is linked to the uptake of orthophosphate
and the formation of 1,3-diphosphoglyceric acid. This acyl phosphate may then react with ADP
to form ATP. For the first time, the bioenergetic function of glycolysis became clear. A portion
of the free energy released during the breakdown of glucose is made available to the cell as
ATP.
Lipmann followed these developments closely and they deeply influenced his thinking. In
1939, he turned to an investigation of the role of phosphate in the oxidation of pyruvate in
extracts of the organism then called Bacterium acidificans longissimum (Delbrueckii). He
discovered (5) that the oxidation of pyruvate was coupled to the uptake of orthophosphate and
the phosphorylation of AMP (presumably with the formation of ATP).
By analogy with the role of 1,3-diphosphoglyceric acid in glycolysis, he formulated the
following reactions.
Pyruvate ⫹ phosphate ¡ acetyl phosphate ⫹ 2关H]
Acetyl phosphate ⫹ AMP ¡ “adenosine polyphosphate”
REACTIONS 1 AND 2
The isolation from these enzyme preparations of highly labile acetyl phosphate present only in
very small amounts was really not feasible with the methods then available. Lipmann neatly
got around this difficulty by synthesizing acetyl phosphate from acetyl chloride and trisilver
phosphate. He then showed that this synthetic compound, like the presumed intermediate,
was effectively utilized for the formation of “adenosine polyphosphate” in these extracts.
(Much later, when I worked in Lipmann’s laboratory and read his early papers, I was greatly
taken by this strategy. I learned from it that it is sometimes easier to synthesize a suspected
intermediate in an enzyme system than to isolate it, a lesson that led me to synthesize
CDP-choline first and then demonstrate its role as coenzyme.)
The work on acetyl phosphate marked the beginning of Lipmann’s long and productive
engagement with both the role of phosphate esters in energy transduction and the problem of
“active acetate.” In July of 1939, with the Nazi menace ever more threatening, Fritz and Freda
Lipmann left Copenhagen for the United States. There followed a difficult period in which he
sought without success a position that would offer him security and scope commensurate with
his talents. In 1940, he was invited to present a talk in a symposium at the University of
Wisconsin in Madison attended by many of the leading figures in American biochemistry.
Lipmann, never a facile or polished speaker, vastly underestimated the time needed for the
material he wished to present and finally, midway through his discourse, had to be interrupted
by the chairman of the session (3). Later he felt that this painful episode was one of the factors
that made it difficult for him to secure a suitable position.
In 1940, Lipmann was invited by F. F. Nord to contribute a chapter to the first volume of the
series, Advances in Enzymology. As Lipmann later (3) wrote: “ . . . I was happy when he
accepted my suggestion that I write about the role of phosphate bonds as carriers in energy
transformations and in biosynthesis. This had begun to impress me as an extension of my
experience with acetyl phosphate. Some of the propositions made in that article must have
been more novel than I realized.”
Now, in 2001, it is very difficult to realize the impact of this article (6), particularly on
American biochemists who had not closely followed the work in European laboratories.
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Lipmann clearly distinguished between two classes of phosphate compounds in living cells.
The first class, phosphate esters of alcohols such as glycerophosphate with a free energy of
hydrolysis of 2– 4 kcal, was termed by Lipmann as “energy-poor” phosphates, designated in the
shorthand which he introduced as (⫺ph). These were to be sharply distinguished from another
class comprising pyrophosphates, acyl phosphates, enol phosphates, and nitrogen-linked phos-
phates such as phosphocreatine. The free energy of hydrolysis of phosphates of this class is of
the order of 8 –12 kcal. In Lipmann’s terminology these are energy-rich phosphate bonds,
designated with a symbol that was to become famous as the “wiggle bond” (⬃ph).
A great generalization was stressed in his essay. Photosynthesis and the breakdown of
organic foodstuffs provide energy to living cells, some part of which is captured in useful form
as “energy-rich” phosphates, leading to the formation of ATP. Lipmann pointed out: “Indica-
tions are found that the phosphate current can be utilized to carry out mechanical work . . .
(and) to synthesize protoplasmic material as lecithin, nucleic acid, and so forth.” Lipmann
made it clear that the energy needed to drive biosynthetic processes must come from ATP
either directly or, as was soon to be found, indirectly. Before this time biosynthetic processes
could be studied only in intact animals or in preparations such as tissue slices (developed by
Warburg) in which cellular structure remained intact. The principal conceptual barrier to the
study of cell-free systems was now removed. Biochemists began to add ATP (of varying degrees
of purity!) to their enzyme systems when searching for biosynthetic reactions.
The 1941 essay is revealing in many ways of Lipmann’s style, which had a personal flavor
even when dealing with chemical thermodynamics. These were problems about which he had
thought deeply, and he conveyed his ideas in striking and forceful metaphors. Thus he spoke
of a “phosphate potential” in analogy to an electrical potential and a “phosphate current” that
conveys energy as “energy-rich” phosphates are hydrolyzed. Critics pointed out that his use of
the term “bond energy” to denote the energy released in breaking a bond was the opposite of
the conventional use to denote the energy of bond formation, but Lipmann (3) tended to wave
aside such criticism. “The physical chemist remains aloof. He may be forced to accept the
usage, but he usually refrains from referring to the dilettante who originated it.”
Lipmann’s search for a suitable position now found a happy outcome in a rather unusual
way. In 1941 Dr. Oliver Cope offered him an appointment in the Department of Surgery at the
Massachusetts General Hospital. Although the space made available was at first quite limited,
he was given complete freedom to follow his own ideas. Lipmann’s years at the Massachusetts
General Hospital were highly productive and led him to a Nobel Prize in 1953.
In 1941 the identity of “active acetate,” also described as the “two-carbon unit,” was one of
the most pressing problems in intermediary metabolism. A growing body of evidence suggested
that “active acetate” was the fundamental building block for the synthesis of sterols and fatty
acids. Derived from the oxidation of pyruvate or of fatty acids, it could also react with
oxalacetate to form citrate and thus enter the Krebs cycle for the final common pathway of
oxidative metabolism. Strongly encouraged by his success in identifying acetyl phosphate as
an intermediate in the bacterial oxidation of pyruvate, Lipmann set out to examine its possible
role as the elusive “active acetate” in animal tissues.
He chose to study the acetylation of sulfanilamide, known to occur in liver, because of the
ease with which this aromatic amine could be diazotized and coupled with a chromogen to form
an intensely colored dye. The conversion of sulfanilamide to the unreactive N-acetyl derivative
could thus be easily measured. He succeeded in obtaining preparations from pigeon liver that
actively acetylated sulfanilamide but to his considerable disappointment found that acetyl
phosphate did not stimulate acetylation but instead was rapidly hydrolyzed (7). Significantly,
however, he found that ATP as well as acetate was required for acetylation and further
reported that enzyme preparations, inactivated by storage overnight at 7 °C, could be restored
to activity by the addition of boiled liver extract.
Nachmansohn and Machado had previously described a cofactor needed for the acetylation
of choline. With the arrival of Kaplan in his laboratory, Lipmann’s cofactor was purified about
100-fold and shown to be active also in the acetylation of choline (8). It appeared to be a general
coenzyme for acetylation and hence the designation coenzyme A or CoA. The next step was the
discovery (9) in 1947 that CoA, by then purified about 700-fold, contains the vitamin panto-
thenic acid. This was a very great advance.
A little later, in 1950, recommended by H. A. Barker, I entered Lipmann’s laboratory as a
postdoctoral fellow following the footsteps of Earl Stadtman, who had just departed to take up
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a position at the National Institutes of Health. Stadtman had also come to Lipmann from
Barker’s laboratory. Lipmann’s group at this time included David Novelli, John Gregory,
Morris Soodak, Harold Klein, Charles Du Toit, and Lipmann’s research assistant, Ruth Flynn.
We were crammed into a single, tiny laboratory in the Massachusetts General Hospital next
to the famous Ether Dome, scene of the first (or so it was claimed) use of diethyl ether as an
anesthetic. In the course of the year we were to move into spacious, even rather elegant,
quarters in a newly constructed research building.
With abundant hair just turning gray and usually wearing a soft bow tie and a dark blue
shirt, Lipmann presented a figure closer to that expected of an artist rather than a scientist.
He spoke softly, and his sentences often trailed off into the distance. Lipmann’s manner
toward those who worked in his laboratory was rather formal. He was friendly but a little
aloof. He inspired nevertheless not only loyalty and admiration but also lasting affection in
those who worked under his direction.
At this time, Lipmann’s chief goal was the final purification of coenzyme A, which was
proving very difficult, and the determination of the structure of “active acetate,” the interme-
diate with so many crucial roles in metabolism. Because acetyl phosphate, shown to be an
activated form of acetate in bacteria, was so labile, we surmised that acetyl-CoA, whatever its
structure might be, would be even more labile, and this supposed lability was assumed to
explain the failure of our efforts to isolate it.
One day in 1951, I came upon an article in Angewandte Chemie (10) from the laboratory of
Feodor Lynen. He and his student Ernestine Reichert reported evidence for an essential
sulfhydryl residue in CoA. They had isolated acetyl-CoA and proved it to be a thioester! I
brought the article at once to Lipmann who had not learned previously of this development. He
was generous in praise of the work although Lynen had stolen some of his thunder. He was
particularly impressed by the fact that in isolating acetyl-CoA from yeast, they had begun by
boiling the yeast. We should have realized, Lipmann pointed out, that an intermediate that
plays such varied roles is unlikely to be so extremely labile as we had feared. Lipmann also
noted that thioesters must be added to the list of biologically active “energy-rich” compounds.
“Yes,” he mused in a discussion at this time, “there is a world of sulfur, like the world of
phosphorus, only smaller!”
In 1953 Lipmann shared the Nobel Prize with H. A. Krebs. Although the citation for the
prize emphasized his work on CoA, Lipmann placed greater stress on his contributions to
bioenergetics. “In my own judgment,” he wrote (3), “there was greater scope in the recognition
that ⬃P, as I had dubbed it, was acting as a biological energy quantum, carrying energy
packages to metabolic function and biosynthesis.”
In 1957, he moved to the Rockefeller Institute. He continued to be remarkably productive in
a wide variety of biosynthetic problems, further developing his grand themes of group activa-
tion and the energetics of biosynthesis until his death in 1986 at the age of eighty-seven.
Rudolf Schoenheimer and the Dynamic State of Body Constituents

The single most important technical advance that transformed biochemistry in the 20th
century was the isotope tracer technique. Without it, the rapid growth of our knowledge of
biosynthesis would be simply inconceivable. Georg Hevesy was the first to explore the biolog-
ical usefulness of radioactive tracers in studies of the uptake of radiolead and its movement
into tissues of plants (11). It is to Rudolf Schoenheimer (Fig. 2), however, that we owe the
brilliant exploitation of the concept of isotopic tagging, that is the introduction of isotopes into
specific positions of organic molecules, whose metabolic transformations could then be traced.
Valuable accounts of Schoenheimer’s career have been published by Kohler (12) and by
Young and Ajami (13). He was born in Berlin in 1898 (12). Like Lipmann, he studied medicine
and received the M.D. degree from the University of Berlin in 1922. Again like Lipmann, he
recognized the need for deeper knowledge of chemistry and spent 3 years in the laboratory of
Karl Thomas in Leipzig, working largely on problems such as the chemical synthesis of
peptides.
In 1926, Schoenheimer went to the Institute of Pathological Anatomy in Freiburg as
assistant to Ludwig Aschoff, a leading expert on atherosclerosis (12). Schoenheimer began an
investigation on the deposition of cholesterol into the arteries of rabbits fed a high level of
cholesterol in the diet. He was to pursue his interests in cholesterol metabolism for the rest of
his life.
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FIG. 2. Rudolf Schoenheimer. From Ref. 13 with permission. Photo made available through the courtesy of Mrs.
Peter Klein.
It was here in Freiburg in 1930 that Schoenheimer encountered Hevesy, who wished to
study the partition of labeled lead between normal and tumor tissue (12). Realizing his
inadequate background in biology, Hevesy asked Aschoff to suggest a collaborator for this
work. Aschoff suggested Schoenheimer. Later, Hevesy (14) wrote: “It was in the course of these
investigations that Schoenheimer became familiar with the method of isotopic indicators,
which he applied several years later with such great success . . . Never were more beautiful
investigations carried out with isotopic indicators than those of the late Professor Schoenhei-
mer . . . ”
Although the collaboration with Hevesy was undoubtedly significant for Schoenheimer’s
thinking, his development of the use of isotopes was to go far beyond the scope of Hevesy’s
approach. In 1933 Schoenheimer, like so many others, was forced to leave Germany. The
Josiah Macy Foundation in the United States had begun in 1931 to support Schoenheimer’s
research, and the director of the foundation, Ludwig Kast, now arranged an appointment for
Schoenheimer in the Department of Biological Chemistry at Columbia, with salary and
research funds supplied by the Foundation (12).
Hans T. Clarke, an organic chemist by training, had assumed the direction of the Depart-
ment of Biological Chemistry in 1928, and he proceeded to make it the finest department in the
United States. In an account of his career (15), Clarke stated: “Among the many benefits which
accrued to Columbia University from the racial policy adopted by the Germans under the
Third Reich was the arrival in our laboratory of various European-trained biochemists,
notably Erwin Chargaff, Zacharias Dische, Karl Meyer, Rudolf Schoenheimer and Heinrich
Waelsch. Erwin Brand, who joined our group during the same period, reached this country
somewhat earlier. The scientific achievements subsequently made by these men are so well
known that their enumeration is unnecessary.” Clarke modestly omitted to mention that his
own vision and humane instincts in welcoming these gifted refugees were by no means to be
found in every American academic institution.
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In 1932, also at Columbia University in the Department of Chemistry, Harold Urey discov-
ered deuterium, the heavy isotope of hydrogen, by demonstrating the presence of new bands
in the positions predicted for a form of hydrogen of mass 2, in the spectrum of a sample of
hydrogen enriched in the heavier isotope by fractional distillation of liquid hydrogen. In 1934,
Urey received a Nobel Prize for this work. Because separation of the isotopes of an element is
a function of the ratio of their masses, isotopes of the heavier elements are very difficult to
separate. Deuterium, however, has twice the mass of ordinary hydrogen, and its preparation
in pure form or as D2O (immediately dubbed “heavy water”) is comparatively straightforward
and was very soon undertaken in the laboratories of Urey and G. N. Lewis among many others.
The discovery of a completely new form of a substance of such universal importance as water
immediately attracted great public interest all over the world. When Urey received his Nobel
Prize in 1934, Palmer, in his laudatory introduction of Urey, mentioned that large amounts of
heavy water were already being produced by an electrolytic process at the Norsk Hydro
Concern in Norway at the rate of about a half-liter per day (16). In 1940 after a more sinister
use of heavy water as the moderator for atomic piles had emerged, this Norwegian heavy water
production facility was taken over by the German army of occupation. It then became the
target for heroic and tragic efforts of Norwegian patriot saboteurs and the allied air forces to
destroy it. The Germans finally dismantled it in 1945. The first biological experiments with
D2O were relatively crude. For example, Lewis (17) reported that tobacco seeds suspended in
pure D2O failed to germinate, and flatworms died when placed in water containing more than
90% D2O. In these and other early experiments, the emphasis was on replacement of H2O as
a medium for growth by D2O and not on the specific replacement of hydrogen by deuterium in
molecules of biological importance.
Urey, a physical chemist, stated that he was a biologist at heart. Indeed, at a later stage of
his career at the University of Chicago he turned to fundamental biological research. With his
gifted collaborator Stanley Miller, he designed experiments that demonstrated the ready
synthesis (under conditions that simulated the atmosphere of the early earth) of molecules
that might plausibly be considered to be building blocks for the formation of cell substances.
These studies greatly influenced many later investigations of the origin of life.
To promote the applications of the deuterium isotope to biological research, Urey persuaded
Warren Weaver, head of the Rockefeller Foundation, to provide funds to permit David Rit-
tenberg, a recent Ph.D. in physical chemistry in Urey’s department, to come to the Department
of Biological Chemistry (12). As Hans Clarke commented (15): “In 1934, Schoenheimer made
a new contact which proved to exert a fundamental influence on the nature of his work . . .
David Rittenberg came from Urey’s group to the laboratory in which Schoenheimer had been
working for a year. From their association there developed the idea of employing a stable
isotope as a label in organic compounds, destined for experiments in intermediary metabolism,
which should be biochemically indistinguishable from their natural analogs . . . ”
This new conception of Schoenheimer and his collaborators was a far cry from the simple
measurement of the movement of a radioactive ion from one part of a plant or animal to
another, as had been done by Hevesy. In the new approach, the fate of the molecule into which
the isotope had been incorporated was studied, not simply the isotope itself. Perhaps the
nearest intellectual predecessor of this idea was the approach of Knoop, who in 1904 “labeled”
fatty acids by the attachment of a phenyl residue to the ␻-carbon atom. Knoop found that if the
fatty acid had an even number of carbon atoms, phenylacetic acid (linked to glycine in a
so-called detoxification reaction) was recovered from the urine of dogs to which it had been fed.
If on the other hand, the fatty acid had an odd number of carbon atoms, benzoic acid was
similarly recovered. Knoop concluded that the phenyl residue could not be cleaved from the ␻
carbon to which it was linked and more significantly correctly concluded that fatty acid
oxidation in animal tissues must involve oxidation at the ␤-position. This result strongly
influenced later studies of fatty acid oxidation, but the work was subject to the objections that
phenyl-substituted fatty acids are very different from natural fatty acids, and a more serious
limitation was that this type of labeling was not generally suitable for substances other than
fatty acids.
Schoenheimer was well aware of Knoop’s work. In a brief review in 1935 (18), Schoenheimer
and Rittenberg pointed out: “Many attempts have been made to label physiological substances
by the introduction of easily detectable groups such as halogens and benzene nuclei. However,
the physical and chemical properties of the resulting compounds differ so markedly from those
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of their natural analogs that they are treated differently by the organism. The interpretation
of metabolic experiments involving such substances is therefore strictly limited. We have
found the hydrogen isotope deuterium to be a valuable indicator for this purpose . . . We have
prepared several physiological compounds (fatty acids and sterol derivatives) containing one or
more deuterium atoms linked to carbon, as in methyl or methylene groups . . . The number of
possible applications of this method appear to be almost unlimited.”
At this period, mass spectrometers were still rare and finicky instruments. It was an
advantage of these early experiments that the content of deuterium in organic compounds
could be determined comparatively simply by combustion of the compound and very precise
measurement of the density of the water so produced.
In 1935, it was a widely held doctrine that the bodily constituents of an adult animal were
quite stable, while foodstuffs in the diet were immediately metabolized to provide energy and
the end products excreted. In their earliest experiments, Schoenheimer and Rittenberg found
evidence to overturn this doctrine. When fatty acids labeled with deuterium were fed to mice,
most of the deuterated fat was first deposited in the fat depots. The fat burned in the body was
not taken directly from the diet but from adipose tissue. Schoenheimer (19) concluded: “These
first experiments with isotopes showed that the fats of the depots are not inert storage
materials but are constantly involved in metabolic reactions.”
To study the synthesis of fatty acids, Bernhard and Schoenheimer (20) administered D2O to
mice and later measured the isotope content of their fatty acids. The saturated fatty acids were
found to contain relatively high levels of deuterium, but the polyunsaturated linoleic and
linolenic acids, known to be essential components of the diet, contained only traces. They
concluded that the mice carried out a very active de novo synthesis of saturated but not of
essential fatty acids. Because the total fat content of the mice did not change, the results
indicated a rapid breakdown of body fats, equal to the rate of synthesis.
As might be expected, an important objective of Schoenheimer’s new program was an
investigation of the metabolism of cholesterol. When cholesterol was isolated from mice given
D2O, Rittenberg and Schoenheimer (21) found from the rate of incorporation of deuterium into
it that cholesterol must be continually renewed with a half-time of the order of 3 weeks. To
account for the extensive incorporation of stably bound deuterium into the cholesterol mole-
cule, it was concluded that its synthesis, like that of fatty acids, must involve the condensation
of many small molecules.
A major extension of the range Schoenheimer’s investigations came with the concentration
of the isotope 15N by Urey and his collaborators in 1937. It was immediately applied to studies
of the metabolism of amino acids and proteins. In 1938, Schoenheimer et al. (22) reported the
first experiments in which an amino acid in the diet, tyrosine, was labeled with 15N. “The
original aim of this exploratory experiment was merely to find out whether in nitrogen
equilibrium, the nitrogen in the urine is derived from the food proteins directly, or whether
dietary nitrogen is deposited, with liberation of an equivalent amount of tissue nitrogen for
excretion . . . The results indicate that in our rat the nitrogen of at least one amino acid,
tyrosine, was only partly excreted in the urine, while almost half of it was retained in the body
proteins.”
Here was another blow at the doctrine that ingested foods were immediately metabolized
and the products promptly excreted. Schoenheimer now found this view very naı̈ve. If one puts
a penny into a gumball machine, he asked, and a gumball comes out, does the machine turn
copper into gum?
Schoenheimer had now become the central figure in Clarke’s Department of Biological
Chemistry. New and larger laboratory facilities were made available for him. His enthusiasm
and vision attracted collaborators and students. As Kohler (12) has pointed out, he had become
the leader of perhaps the first multidisciplinary biochemical laboratory. A physicist was
needed for the preparation and measurement of isotopes. An organic chemist was employed for
the synthesis of isotopically labeled compounds, because of course none were available com-
mercially. Biochemists were required for the separation and analysis of cell constituents.
Technicians for animal care were also needed. Schoenheimer’s background in chemistry as
well as in biology and medicine made him especially effective in the leadership of this
disparate group.
Schoenheimer’s investigations of protein metabolism, carried out with amino acids contain-
ing 15N in the amino group and deuterium on the carbon chains provided results that had the
H49
greatest impact on biochemical thought. Briefly summarized (19), body proteins were found to
be in a state of continuous turnover. “The peptide bonds have to be considered as essential
parts of the proteins and one may conclude that they are rapidly and continually opened and
closed in the proteins of normal animals. The experiments give no direct indication as to
whether the rupture is complete or partial.” The work thus raised questions that were to
challenge the next generation of biochemists.
Together with the earlier work on fat metabolism, a new and remarkable picture of the
overall metabolism of animals emerged. Schoenheimer summarized his conclusions (19): “The
large and complex molecules and their component units, fatty acids, amino acids, and nucleic
acids, are constantly involved in rapid chemical reactions. Ester, peptide, and other linkages
open; the fragments thereby liberated merge with those derived from other large molecules
and with those absorbed from the intestinal tract to form a metabolic pool of components
indistinguishable as to origin . . . This idea can scarcely be reconciled with the classical
comparison of a living being to a combustion engine nor with the theory of independent
exogenous and endogenous types of metabolism . . . The classical picture must thus be replaced
by one which takes account of the dynamic state of body structure.”
In 1941, Schoenheimer was invited to give the prestigious Dunham Lectures at the Harvard
Medical School. The materials and notes that he prepared for the lectures, from which some
of the quotations above are taken, were later published (19) under the title “The Dynamic
State of Body Constituents.” This lucid summary of his innovative work made a deep impres-
sion on the biochemists of the generation to follow.
Schoenheimer had apparently been subject to attacks of depression and was undergoing a
period of considerable personal stress when tragically in September of 1941 he ended his own
life (12). Forty-three years of age at the time of his death, he was at the height of his powers.
Fortunately many of the projects that he had begun were carried forward by very able
collaborators, one whom took up the cholesterol problem.
Konrad Bloch and the Biosynthesis of Cholesterol

Konrad Bloch (Fig. 3) was born in 1902 in Neisse, a town in the eastern German province of
Silesia, the second child of a prosperous Jewish family (23). In his boyhood, Bloch evinced little
interest in science other than nature studies, but his attendance in a course of organic
chemistry at the Munich Technische Hochschule taught by Hans Fischer marked a turning
point for him. Fischer, later to receive a Nobel Prize, was one of the remarkable group of gifted
German chemists who then dominated the study of natural products. Although Fischer’s
lectures were delivered in a monotone, Bloch found the material fascinating and he realized
that he had found his field (23).
In 1934, the brutal Nazification of Germany prevented Bloch from continuing his studies
there. Hans Fischer came to his rescue by recommending his appointment at the Schweiz-
erisches Hoehensforschungs Institut in Davos, Switzerland, the scene where Thomas Mann
placed the tuberculosis sanitarium in his novel The Magic Mountain.
In Davos, Bloch worked for a time on the lipids of the tubercle bacillus. In 1936, however, he
was refused permission to continue to reside in Switzerland. Desperate, he applied to R. J.
Anderson at Yale, with whom he had some correspondence concerning his research. He
promptly received two letters, the first from the Dean of the Medical School of Yale University
informing him that he had been appointed assistant in Biological Chemistry and the second
from Anderson informing him that there was no salary attached to this position. He showed
the first letter, but not the second, to the United States consul in Frankfurt and received a
life-saving visa to immigrate to the United States.
Upon arrival in New York, Bloch applied to Hans Clarke’s department for admission as a
graduate student. The sole formality in those happy days was an interview with Clarke
himself. The most important question, Bloch later jested, was: “Do you play a musical
instrument?” Fortunately, Bloch could say that he played the cello, an answer agreeable to
Clarke, who loved chamber music.
Shortly after completion of his work for the Ph.D. degree under Clarke’s supervision, Bloch
joined Schoenheimer’s group. In 1940, Schoenheimer suggested that he investigate the origin
of the hydroxyl oxygen in cholesterol. Was it water or O2? The thought that it might be
molecular oxygen showed the remarkable prescience of Schoenheimer because direct oxygen-
ation was without precedent at that time. Unfortunately, Bloch found the technical problems
H50
FIG. 3. Konrad Bloch. Photo made available through the courtesy of Mrs. Konrad Bloch.
of the mass spectrometry of oxygen compounds intractable in the state of technology of 1940
and was forced to give up the project. In 1956, however, he returned to the problem and with
his student Tchen (24) showed that molecular oxygen is indeed the source of the hydroxyl
oxygen.
As Bloch (23) recalled: “Schoenheimer’s untimely death in 1941 left his associates without
the leader and the inspired leadership they so admired. We feared that we might have to look
for jobs elsewhere, but Hans Clarke encouraged us to continue as heirs to the wealth of projects
Schoenheimer had begun and developed . . . How the division of ‘spoils’ came about I do not
recall—it may have been by drawing lots. At any rate, David Shemin ’drew’ amino acid
metabolism, which led to his classic work on heme biosynthesis. David Rittenberg was to
continue his interest in protein synthesis and turnover, and lipids were to be my territory.”
Bloch now began his independent studies of the biosynthesis of cholesterol. It was a
formidable enterprise. In the era before NMR, infrared, and mass spectroscopy, the determi-
nation even of the chemical structure of cholesterol, with its 27 carbon atoms arranged in four
rings and with a branched hydrocarbon side chain, had been a challenge to the world’s greatest
chemists of natural products. The Nobel Prizes in chemistry for 1927 and 1928 had been
awarded to Heinrich Wieland and Adolf Windaus, respectively, for their work on the structure
of cholesterol and the closely related bile acids, but it was not until 1932 that the fully correct
structure was established.
In his 1928 Nobel lecture (25), Windaus stated: “This formula [of cholesterol] is very
complicated and has no similarity to the formulae of sugars, fatty acids, or the amino acids
which occur in protein. The synthesis of such a substance appears to the chemist particularly
difficult, and up to now I have not dared to attempt it, as success is extremely improbable.
Furthermore, the majority of physiologists have not been inclined to believe the animal
organism capable of such a synthesis, for it is known that other seemingly simpler syntheses—
e.g. that of tyrosine and tryptophane— have not succeeded in the animal organism.”
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Bloch of course knew that Windaus’ pessimistic view of the capabilities of the animal
organism was unfounded. Schoenheimer and Rittenberg had demonstrated the extensive
incorporation of deuterium from D2O into cholesterol in the mouse and concluded that
cholesterol must be synthesized by the joining of a number of small molecules.
The pathway for the biosynthesis of cholesterol from acetate, involving more than 30
separate enzyme-catalyzed reactions, can now be found in every textbook of biochemistry. A
detailed review is beyond the scope of this essay. Here we will consider only the principal
landmarks in its three major stages: 1) acetate to “activated isoprene”; 2) “activated isoprene”
to squalene; and 3) squalene to cholesterol.
Bloch’s studies began with investigations of the overall process of formation of cholesterol in
the intact organism. Stimulated by a report from the German workers Sonderhoff and Thomas
(26), indicating that acetate is efficiently converted into the sterols of yeast, Bloch began a
series of studies demonstrating the incorporation of specifically labeled acetate into cholesterol
in the intact animal. These studies were continued and expanded after his move in 1946 to the
Department of Biochemistry at the University of Chicago, where his good friend Earl Evans,
also a product of Hans Clarke’s department, had become chairman.
I was a graduate student in the Department at this time, and so I came to know Konrad
Bloch, first as a teacher and later as a colleague and friend. He was a man of personal qualities
commensurate with his great abilities. His manner with students was friendly and easy. He
was painstakingly generous in acknowledging the research contributions of his colleagues and
of other laboratories. He was widely cultured, devoted to music, literature, and art.
In the mid-1940s, Bloch (23) was completely convinced of the truth of Lipmann’s dictum that
energy-requiring biosynthetic reactions are driven by ATP, directly or indirectly. Before this
period the synthesis of peptide bonds had been observed only by reversal of the reactions
catalyzed by proteases. In a project quite unrelated to the cholesterol problem, he and his
students began to investigate the synthesis of the tripeptide glutathione as a possible model
of protein synthesis. They were indeed able to show that the assembly of glutathione requires
the successive activation of glutamate and glutamylcysteine by ATP, but unfortunately the
mechanism proved to shed little light on the ribosomal synthesis of proteins.
Bloch was also very much aware of the potential power of microbial genetics for the analysis
of metabolic pathways, and he enrolled as a student in the famous course in microbiology
taught by C. B. Van Niel at the Hopkins Marine Station in Pacific Grove, CA. When a mutant
of the mold Neurospora crassa was isolated in Tatum’s laboratory that grew only when acetate
was added to the medium, Bloch was eager to follow this lead. He and his collaborators found
that isotopically labeled acetate was converted to ergosterol in this mutant essentially without
dilution of the isotope. Clearly the sterol could be built up entirely from acetate.
In the conversion of acetate to cholesterol, which of the carbon atoms of cholesterol were
derived from the carboxyl group and which from the methyl group? Studies carried out over a
number of years in the laboratories of Cornforth and of Popjak, as well as of Bloch, achieved
the ambitious goal of defining the origin of each of the 27 carbon atoms of cholesterol as either
the methyl or the carboxyl carbon of acetate. This work placed important constraints on
possible structures of intermediates in the scheme.
It had been known for some time that squalene (a branched, acyclic hydrocarbon found in
abundance in the livers of sharks) when fed to animals increases the levels of cholesterol in
their tissues. To test the idea that squalene might be a precursor of cholesterol, Bloch went to
the Biological Research Station in Bermuda to attempt the preparation of isotopically labeled
squalene in shark liver, but the shark proved to be an intractable subject for study (23). “All
I was able to learn was that sharks of manageable length are very difficult to catch and their
oily livers impossible to slice.” Back at the University of Chicago, however, his student Robert
Langdon was able to prepare labeled squalene by feeding rats labeled acetate along with
unlabeled squalene as an isotopic trap. Labeled squalene so obtained was then fed to rats and
found to be converted to cholesterol (27). This was an important result. In the dissection of
every biosynthetic pathway, it is particularly helpful to identify an intermediate in the middle
of the chain of reactions; the researcher can then trace the pathway both backwards and
forwards. At this stage in his work, in 1954 Bloch moved to the Department of Chemistry at
Harvard, where he was to remain for the rest of his career.
Squalene, containing 30 carbon atoms, could plausibly be considered to be built up from 6
units of isoprene, a branched, unsaturated compound containing five carbon atoms. Isoprene
H52
was already known to be a building block of other naturally occurring hydrocarbons such as
rubber, although the nature of the biologically active “isoprene donor” remained unknown.
Robinson (28) had suggested that squalene might be folded to form the basic structure of
cholesterol directly. Bloch, however, after illuminating discussions with his Harvard colleague
Robert Woodward considered that lanosterol, with a structure closely similar to cholesterol but
with three “extra” methyl groups, was likely to be an intermediate in this transformation.
Up to this point, Bloch’s experimental approach to the cholesterol problem had been largely
confined to isotopic tracer studies with intact animals or with tissue slices in which cellular
structure was preserved intact, but now he turned increasingly to the study of cell-free enzyme
systems. Rat liver homogenates, prepared by the methods developed by Nancy Bucher, were
found to catalyze the transformation of labeled squalene to lanosterol and of lanosterol to
cholesterol. Although much work remained to be done, Bloch had established the landmarks
for the final stages of the biosynthesis of cholesterol (29).
The focus now was turned to the first stages of the pathway, the conversion of acetate to the
“active isoprene donor.” A mutant strain of Lactobacillus acidophilus had been found to grow
only when acetate was added to the medium. A substance that very efficiently replaced the
acetate requirement was identified by workers at Merck, Sharpe and Dohme (30) as mevalonic
acid (isolated as the lactone). Mevalonic acid was then shown to be a very efficient precursor
of squalene and of cholesterol in homogenates of liver (31). These findings opened the way for
the elucidation of the reactions leading to the formation of the “active isoprene unit” of which
mevalonate was clearly the precursor. Progress in this area now became fast and furious with
important contributions from the laboratories of Rudney, Lynen, Cornforth, and Popjak among
others.
Bloch and his collaborators showed that the overall conversion of labeled mevalonic acid to
squalene in extracts of bakers’ yeast required ATP as well as reduced pyridine nucleotide and
manganese ions. His colleague Chen then discovered the phosphorylation of mevalonate to a
monophosphate. The further conversion of this monophosphate to the important intermediates
isopentenylpyrophosphate and dimethylallylpyrophosphate was elucidated largely by work in
Lynen’s laboratory.
The synthesis of squalene via geranyl pyrophosphate and farnesyl pyrophosphate was next
documented. As shown by the early studies of Bloch, squalene is converted in a series of steps
to lanosterol, which after several further transformations gives rise to cholesterol.
It is impossible, of course, in this highly condensed account to do justice to the vast amount
of work, still ongoing in laboratories over the world, that has led to our present knowledge of
the biosynthesis of cholesterol. It was Bloch, however, who was a prime mover in all three
phases of the problem. For this work he was awarded a Nobel Prize, with Feodor Lynen, in
1964.
Working out the pathway for the assembly of the complex structure of cholesterol was an
exemplary achievement of the era of biosynthesis, important not only because of the intrinsic
interest of its enzymology but also because of its significance for medicine. High levels of blood
cholesterol, characteristic of populations in developed countries, strongly increase the danger
of heart disease and stroke. An understanding of the detailed route of biosynthesis made it
possible to determine that the synthesis of mevalonate from HMG-CoA is a rate-making step
in the production of cholesterol. This advance made possible the development of drugs, the
family of statins, that reduce levels of blood cholesterol with a minimum of toxic side effects.
These drugs are among the most useful in modern medicine.
Konrad Bloch made outstanding contributions to fields other than the biosynthesis of
cholesterol, including the enzymic synthesis of fatty acids and the mechanism of enzyme action
(23). He died on October 15, 2000 at the age of eighty-eight.
The development of any field of science is inevitably the work of many hands. Obviously,
Lipmann, Schoenheimer, and Bloch cannot be regarded as single handedly transforming
American biochemistry. Their work was nonetheless a great gift to their adopted country and
a shining manifestation of the international character of science.
REFERENCES
1. Medawar, J., and Pyke, D. (2001) Hitler’s Gift, Arcade Publishing, New York
2. Lipmann, F. (1953) Annu. Rev. Biochem. 54, 1–32
3. Lipmann, F. (1971) Wanderings of a Biochemist, Wiley-Interscience, New York
4. Warburg, O. (1949) Wasserstoffuebertragende Fermente, Editio Cantor, Freiburg, Germany
H53
5. Lipmann, F. (1939) Nature 144, 33–34
6. Lipmann, F. (1941) Adv. Enzymol. 1, 99 –162
7. Lipmann, F. (1945) J. Biol. Chem. 160, 173–190
8. Lipmann, F., and Kaplan, N. O. (1946) J. Biol. Chem. 162, 743–744
9. Lipmann, F., Kaplan, N. O., Novelli, G., Tuttle, L. G., and Guirard, B. M. (1947) J. Biol. Chem. 167, 869 – 870
10. Lynen, F., and Reichert, E. (1951) Angew. Chem. 63, 47– 48
11. Hevesy, G. (1923) Biochem. J. 17, 439 – 445
12. Kohler, R. E., Jr. (1977) Hist. Studies Phys. Sci. 8, 257–298
13. Young, V. R., and Ajami, A. (1999) Proc. Nutr. Soc. 58, 15–32
14. Hevesy, G. (1948) Cold Spring Harbor Symp. Quant. Biol. 13, 129 –150
15. Clarke, H. T. (1958) Annu. Rev. Biochem. 27, 1–14
16. Palmer, W. (1966) in Nobel Lectures Chemistry 1922–1941, pp.333–338, Elsevier Science Publishing Co., Inc., New
York
17. Lewis, G. N. (1934) Science 79, 151–153
18. Schoenheimer, R., and Rittenberg, D. (1935) Science 82, 156 –157
19. Schoenheimer, R. (1949) The Dynamic State of Body Constituents, Harvard University Press, Cambridge, MA
20. Bernhard, K., and Schoenheimer, R. (1940) J. Biol. Chem. 133, 707–712
21. Rittenberg, D., and Schoenheimer, R. (1937) J. Biol. Chem. 121, 235–253
22. Schoenheimer, R., Ratner, S., and Rittenberg, D. (1939) J. Biol. Chem. 127, 333–344
23. Bloch, K. (1987) Annu. Rev. Biochem. 56, 1–19
24. Tchen, T. T., and Bloch, K. (1956) J. Am. Chem. Soc. 78, 1516 –1517
25. Windaus, H. O. (1996) in Nobel Lectures Chemistry 1922–1941, pp. 105–121, Elsevier Science Publishing Co., Inc.,
New York
26. Sonderhoff, R., and Thomas, H. (1937) Ann. Chem. 530, 195–213
27. Langdon, R. G., and Bloch, K. (1952) J. Biol. Chem. 200, 129 –144
28. Robinson, R. J. (1934) J. Chem. Soc. Ind. 53, 1062–1063
29. Bloch, K. (1965) Science 150, 19 –28
30. Wolf, D. E., Hoffman, C. H., Aldrich, P. E., Skeggs, H. R., Wright, L. D., and Folkers, K. (1956) J. Am. Chem. Soc.
78, 4499
31. Tavormina, P. A., Gibbs, M. H., and Huff, J. W. (1956) J. Am. Chem. Soc. 78, 4498 – 4499
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 38, Issue of September 17, pp. 39187–39194, 2004
Reflections
A PAPER IN A SERIES COMMISSIONED TO CELEBRATE THE CENTENARY OF THE JBC IN 2005
JBC Centennial
1905–2005
The Biotin Connection: Severo Ochoa, Harland Wood,

and Feodor Lynen
Published, JBC Papers in Press, May 27, 2004, DOI 10.1074/jbc.X400005200
M. Daniel Lane
From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205
Unique circumstances sometimes bring us into contact with individuals who will profoundly
influence us, particularly in our formative years. In this article I would like to reflect on the
circumstances that brought me into contact with three great biochemists, Severo Ochoa (1),
Harland Wood (2), and Feodor Lynen (3). Each entered the field by a different route: Ochoa as
a physician with an interest in physiology, Wood as a bacteriologist trained at Iowa State
University, and Lynen as an organic chemist trained in the German tradition with Nobel Prize
winner, Heinrich Wieland.
They entered the field of biochemistry in the late 1930s when the race was on to discover new
enzymes, cofactors, and metabolic cycles. Hans Krebs had formulated the tricarboxylic acid
cycle in 1937 and ornithine cycle (now known as the urea cycle) in 1932, some B vitamins had
been found to function as cofactors or prosthetic groups of enzymes, and Rudolf Schoenheimer
(Columbia University College of Physicians and Surgeons) had demonstrated the dynamic
state of tissue proteins using heavy isotopes of hydrogen and carbon (mid-1930s). This was
where the action was and it attracted many of the brightest young minds into the field. This
was the arena in which Ochoa, Wood, and Lynen were early participants. Excited by discovery,
they transmitted this excitement to their younger colleagues.
I was fortunate to have scientific associations and enduring friendships with each of them.
My connection developed through the B vitamin, biotin, and its role in the reactions catalyzed
by a family of biotin-dependent enzymes, notably carboxylases. The B vitamin, biotin, has an
interesting history not familiar to most scientists who now make use of it. Today, this vitamin
is widely used along with avidin (or its cousin, strepavidin), the specific biotin-binding protein
from egg white, to probe biochemical phenomena. Biotinylation of proteins and nucleotides and
the use of avidin to “fish out” or detect these molecules from/in complex mixtures has found
great utility.
It is a curiosity that nature has brought together within the hen’s egg the richest source of
biotin in the yolk and in the white, a “toxic” factor, avidin, which when fed to animals causes
biotin deficiency. In 1936, Kögl and Tönnis isolated 1.1 mg of biotin from more than 500
pounds of egg yolk. Paul György recognized that the distribution, fractionation behavior, and
chemical properties of Kögl’s yeast growth factor and the anti-egg white injury factor in egg
yolk (then called vitamin H) were similar. When Kögl’s pure biotin methyl ester became
available it was found to be extremely potent in protecting rats against “egg white (i.e. avidin)
injury.” Within a few years Vincent Du Vigneaud and colleagues determined the structure of
biotin, which cleared the way for an attack on the role of biotin at the molecular level.
By 1950 biotin had been implicated in a number of seemingly unrelated enzymatic processes
including the decarboxylation of oxaloacetate and succinate; the “Wood-Werkman reaction”
(discovered by Harland Wood (2)), i.e. the carboxylation of pyruvate; the biosynthesis of
aspartate; and the biosynthesis of unsaturated fatty acids. Of course, we now know that biotin
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Reflections: The Biotin Connection
functions in each of these processes as a mobile “CO2 carrier” bound covalently to a carbox-
ylase. The long sought after link between biotin and enzymatic function was provided by
Henry Lardy at the University of Wisconsin. Lardy showed that liver mitochondrial extracts
catalyzed the ATP- and divalent cation-dependent carboxylation of propionate (subsequently
shown to be propionyl-CoA) to form succinate (4). Later work in the laboratory of Severo Ochoa
found that the initial carboxylation product was methylmalonyl-CoA, an intermediate en route
to succinyl-CoA. The connection to biotin was made by Lardy with the finding that the
propionate-carboxylating activity was lacking in liver mitochondria from rats made biotin-
deficient by being fed egg white, which of course contained avidin (KD(biotin) ⬃10⫺15). Moreover,
the failure of mitochondrial extract to catalyze the carboxylation of propionate was quickly
cured by injecting the rats with biotin.
Upon joining the faculty at Virginia Polytechnic Institute in Blacksburg, Virginia in 1956,
I decided to try to determine how propionate is metabolized in the liver. Because of its unique
features, I settled on bovine liver as the tissue source of the enzyme system to address this
question, propionate being a major hepatic carbon source in ruminants. Unlike carbohydrate
digestion by monogastric animals, ruminants digest carbohydrates in the rumen, the large
anaerobic fore compartment of their multi-compartmented “stomach.” Virtually all carbohy-
drate is fermented in the rumen to short chain fatty acids, primarily acetate and propionate.
Thus glucose, the major digestion product of carbohydrates in monogastric animals, is not
available for absorption in ruminants. Propionate, produced in abundance by fermentation in
the rumen, is absorbed directly into the portal system and transported to the liver where it is
the major carbon source for gluconeogenesis, the pathway leading to glucose production.
My entry into this area coincided with Lardy’s report that propionate was somehow carbox-
ylated to form succinate. I recall writing to Henry Lardy, and he referred me to Severo Ochoa
at New York University School of Medicine. He knew that Ochoa was working on propionate
metabolism and had found that propionyl-CoA first became carboxylated to form methylma-
lonyl-CoA and then was converted to succinyl-CoA. With some trepidation about competing
with the Ochoa laboratory, I decided to forge ahead and purify propionyl-CoA carboxylase from
bovine liver mitochondria. For the reasons mentioned above bovine liver turned out to be an
excellent source of the enzyme. At that point I wrote to Severo Ochoa, and he generously gave
me a status report on their progress and put me in contact with the people in his laboratory
(Alisa Tietz, Martin Flavin, and later, Yoshito Kaziro) who were working on the enzyme. This
initiated what was to be a long relationship with Severo Ochoa and also his colleague, Yoshito
Kaziro (now in Tokyo).
About that time I applied to the National Science Foundation for a research grant to support
my work on propionate metabolism. The grant proposal was rejected because the reviewers felt
that I was really “in over my head” competing with the Ochoa laboratory and also because it
had been rumored that his laboratory had already crystallized the enzyme from muscle. I knew
that this was not true because in my correspondence with Ochoa he had indicated that the
crystals turned out to be pyruvate kinase, not propionyl-CoA carboxylase. After much anguish
I wrote to the Head of the National Science Foundation Review Committee, Louis Levin,
indicating that the Committee was mistaken: “the carboxylase had not been crystallized” and
that I thought it was inappropriate for the National Science Foundation to take a position on
a grant application based on the size of the laboratory, rather than the merit of the proposal.
A few weeks later I received a letter from Lou Levin indicating that the Study Section had
reversed its decision and that the grant would be funded. I doubt seriously if that could happen
today. Thus began my independent career in research and a developing relationship with
Severo Ochoa.
In 1959, a paper by Lynen and Knappe appeared in Angewandte Chemie (5) (later published
in full in Biochemische Zeitschrift (6)) that created tremendous excitement in my laboratory.
The paper described the rather remarkable finding that ␤-methylcrotonyl-CoA carboxylase, a
biotin-dependent carboxylase (involved in leucine catabolism in certain bacteria), catalyzed
the ATP-dependent carboxylation of “free” biotin in the absence of its acyl-CoA substrate. The
product was shown to be a labile carboxylated biotin derivative, later identified as 1⬘-N-
carboxybiotin. Because biotin was believed to be a prosthetic group covalently bound to the
enzyme and because free biotin exhibited an extremely high Km, Lynen proposed that the free
biotin had accessed the active site of the carboxylase and by mimicking the biotinyl prosthetic
group had gotten carboxylated.
H56
FIG. 1. Harland Wood, circa 1991. (Reprinted with permission of the Cleveland Plain Dealer newspaper.)
Shortly thereafter Don Halenz and I succeeded in purifying a related enzyme, propionyl-CoA
carboxylase, from bovine liver mitochondria. After convincing ourselves that it too was a
biotin-dependent enzyme, we turned our attention to how the biotinyl group was attached to
the carboxylase and what enzymatic reactions were involved in its becoming attached to the
carboxylase. Dave Kosow, also in my laboratory at Virginia Tech, had just found that extracts
of liver from biotin-deficient rats contained catalytically inactive propionyl-CoA apocarboxy-
lase. Moreover, he demonstrated that a soluble ATP-dependent enzyme system in these
extracts from the livers of the biotin-deficient animals catalyzed the covalent attachment of
[14C]biotin to the apoenzyme, thereby restoring its ability to carboxylate propionyl-CoA (7).
Moreover, Dave Kosow showed (7) that upon treating the 14C-biotinylated carboxylase with
Streptomyces griseus protease, biocytin (i.e. ⑀-N-biotinyl-L-lysine) was released. This meant, of
course, that the biotin prosthetic group had been linked to propionyl-CoA carboxylase through
an amide linkage to a lysyl ⑀-amino group. A few years later it became evident that this long
(⬃14 Å) side arm facilitates oscillation of the 1⬘-N-carboxybiotinyl prosthetic group between
catalytic centers on the enzyme (7).
After completing those experiments I invited Severo Ochoa to visit Virginia Polytechnic
Institute and to present two lectures, which he graciously agreed to do. One of these talks dealt
with propionyl-CoA carboxylase and the other with the genetic code, the two major projects
under way in the laboratory of Ochoa at the time. While he was in Blacksburg Dave and I
showed him our results on the site of attachment of biotin to the enzyme. We gave him some
of the protease and within a month of his return to New York City he confirmed our findings
with the heart propionyl-CoA carboxylase.
It was at this point in 1962 that I decided to take a sabbatical leave in Munich with Feodor
Lynen (known to his colleagues as “Fitzi”) at the Max-Planck Institüt Für Zellchemie where I
could continue the work on the enzymatic mechanism by which biotin became attached to
propionyl-CoA carboxylase. Before leaving for Munich Dave Kosow and I developed another
more potent apoenzyme system with which to investigate the “biotin loading” reaction. This
H57
system made use of Propionibacterium shermanii that expressed huge amounts of methylma-
lonyl-CoA:pyruvate transcarboxylase, another biotin-dependent enzyme studied extensively
by Harland Wood. Moreover, this organism had an absolute requirement for biotin in the
growth medium, which when grown at very low levels of biotin produced large amounts of the
apotranscarboxylase. The choice of the P. shermanii system turned out to be a good one. It so
happened that my stay in Munich coincided with Harland Wood’s sabbatical leave in Lynen’s
Institute. This was a two-fold bonus for me, first because Harland was the world’s expert on
this enzyme and second because it began a lasting personal relationship with him. He has been
a role model for me ever since that period in Munich.
Harland (1907–1991) grew up on a farm near Mankato, Minnesota. He entered Macalester
College in Minnesota where he majored in chemistry and worked his way through college.
While a student at Macalester, he met Milly Davis and in their third year of college they
married (in 1929, the year of the stock market crash and beginning of the great depression of
the 1930s). In those days this required a meeting (for approval I presume) with the President
of the college, who needn’t have been concerned as she was at his side for the next 62 years.
They were an amazing couple, a cooperative inseparable team. My wife and I shared their
friendship for more than 30 years. In 1931, Harland became a graduate student in bacteriology
in the laboratory of C. H. Werkman at Iowa State University in Ames, Iowa, where he made
a discovery that was so controversial, although correct, that it was questioned by his thesis
adviser Werkman as well as by leaders in the field of microbial metabolism including C. B. van
Niel. Harland had discovered (2) that heterotrophic organisms, such as the Propionibacteria,
were able to fix CO2. Prior to this it was believed that only auxotrophs, i.e. chemosynthetic or
photosynthetic auxotrophs, could carry out the net synthesis of organic compounds from CO2.
His discovery truly opened the area of enzymatic carboxylation in higher organisms. After
completing his Ph.D. degree Harland (Fig. 1) did postdoctoral work at the University of
Wisconsin with W. H. Peterson and then returned to Iowa State as a faculty member. Harland
was an innovator and an improviser. While at Iowa State he decided to conduct CO2 fixation
experiments using 13CO2, but because of World War II restrictions he could not gain access to
a mass spectrometer nor could he obtain “heavy” 13CO2. In true Woodsian style, he built his
own mass spectrometer and constructed a thermal diffusion column in the Science building at
Iowa State College (2). In 1946, Harland became Professor and Director of the Biochemistry
Department at Western Reserve (now Case-Western Reserve) University. He ran the most
democratic department on record in which faculty salaries were determined by the faculty at
a meeting where members voted on one another’s salary for the upcoming year!
Upon arriving in Munich in August of 1962, I indicated to Lynen that I would like to
investigate the P. shermanii “biotin loading” enzyme system, and he agreed with my proposal.
Because Harland Wood was already at the Institute, I got his advice on growth conditions and
for large scale preparations of the transcarboxylase (actually, the apotranscarboxylase). Both
Lynen and Wood were quite enthusiastic about the project. It turned out that by growing
P. shermanii in biotin-deficient medium the bacteria produced as much of the apotranscar-
boxylase as the holotranscarboxylase when the organism was grown on normal/biotin-contain-
ing medium. Within a short time I was able to resolve and purify both the apotranscarboxylase
and the synthetase that catalyzed loading biotin onto the apoenzyme (7). Dave Young, a
postdoctoral fellow who had recently completed his medical training at Duke University, and
Karl Rominger, a Ph.D. candidate under Lynen’s direction, collaborated with me on these
studies. Finally, we proved that the synthetase catalyzed a two-step reaction in which the first
step involved the ATP-dependent formation of biotinyl-5⬘-AMP and pyrophosphate after which
the biotinyl group was transferred from the AMP derivative to the appropriate lysyl ⑀-amino
group of the apotranscarboxylase.
While in the midst of these studies, a controversy developed regarding the site at which
biotin became carboxylated during catalysis. It was suggested that HCO3⫺ became incorpo-
rated into the 2⬘-position of the ureido ring of the covalently bound biotinyl prosthetic group
of biotin-dependent enzymes and that the 2⬘-carbon was then transferred to the acceptor
substrate. It was suggested that Lynen’s experiments (referred to above) had been done with
free biotin and not the biotinyl prosthetic group covalently linked to the carboxylase. Such a
mechanism would have necessitated opening and then closing the ureido ring of biotin during
the course of the reaction, which to a chemist like Lynen didn’t make chemical sense.
Moreover, this proposal was inconsistent with the known lability of free 1-N-[14C]carboxy-
H58
FIG. 2. Feodor (“Fitzi”) Lynen, circa 1980. (Reprinted with permission of the Max-Planck Gesellschaft.)
biotin. We knew from my earlier studies that enzyme-14CO2⫺, presumably enzyme-biotin-

14
CO2⫺ (prepared by incubating propionyl-CoA carboxylase with H14CO3⫺ and ATP-Mg2⫹), was
even less stable than free 1-N-carboxy-[14CO2⫺]biotin. so we set out to address the issue head
on using propionyl-CoA carboxylase as the source of enzyme-biotin-14CO2⫺. The previous spring
before going to Munich, I had found that enzyme-14CO2⫺ (derived from propionyl-CoA carbox-
ylase) could be stabilized by methylation with diazomethane, i.e. enzyme-14CO2⫺ was labile to
acid before but was stable after methylation. Moreover, digestion of methylated enzyme-14CO2⫺
(enzyme-14CO2-CH3) with S. griseus protease produced a single radioactive derivative, pre-
sumably methoxy-[14C]carbonyl-⑀-N-biotinyl lysine. This product had chromatographic prop-
erties similar, but not identical, to ⑀-N-biotinyl lysine. Because I did not have the authentic
compound for comparison, these experiments could not be completed at the time. Fortunately,
Joachim Knappe, a former member of Lynen’s research group now at the University of
Heidelberg, had synthesized the derivative and provided Lynen with a sample. Thus, we were
able to verify the presumptive identification. This proved that the covalently bound biotinyl
prosthetic group, like free biotin, was carboxylated at the 1⬘-N position (8). Shortly thereafter,
Knappe in Heidelberg and Harland Wood on sabbatical in Lynen’s laboratory in Munich
showed using a similar approach that the carboxybiotin prosthetic groups of ␤-methylcrotonyl-
CoA and transcarboxylase, respectively, had identical structures (9). Taken together these
studies proved unequivocally that the site of carboxylation of biotin was on the 1⬘-N of the
biotinyl prosthetic group.
By this point in my sabbatical in Lynen’s Institute, I began to recognize certain habits of “the
Chief.” For example, he had the habit of working in his office until late in the afternoon. Then,
around dusk, i.e. 6:00 – 6:30 p.m., he would emerge to make “rounds” in the Institute, moving
from one bench to the next to survey the day’s progress or lack of it. Of course not one of the
⬃30 investigators would consider leaving until after he had passed through. He ran a “tight
H59
FIG. 3. Severo Ochoa with colleagues viewing an enlargement of an electron micrograph of acetyl-CoA car-
boxylase (1966). From right to left: Albrecht Kleinschmidt, Erwin Stoll, Severo Ochoa, and Dan Lane.
ship”! Fitzi had an uncanny memory and could recall details of experiments done weeks
earlier.
Lynen (1911–1974) (3) (Fig. 2) was born and spent his entire life in Munich and environs. He
received his doctoral training in organic chemistry at the University of Munich with Heinrich
Wieland (Nobel Prize in Chemistry in 1927), graduating in 1937. He then married Wieland’s
daughter, Eva. He was spared the ravages of World War II because of a serious skiing accident,
which left him with a persistent limp. Perhaps Lynen’s most important contribution was the
discovery of acetyl-CoA, the elusive molecule “active acetate,” sought after by many investi-
gators including Fritz Lipmann, David Nachmansohn, and Severo Ochoa. Ochoa had discov-
ered “condensing enzyme,” now known as citrate synthase, which catalyzed the formation of
citrate from “active acetate” and oxaloacetate. These discoveries led to an important collabo-
ration between Lynen and Ochoa in which they proved that citrate synthase used acetyl-CoA,
along with oxaloacetate, to form citrate. These findings finally answered the question of how
“active acetate” entered the citric acid cycle. In 1964 Lynen received the Nobel Prize (with
Konrad Bloch) in Physiology or Medicine for his work on “the mechanism and regulation of
cholesterol and fatty acid metabolism.”
Lynen had strong connections to the United States. Many Americans came to his Institute
to do postdoctoral work or sabbaticals. During the period that Harland Wood and I spent in
Munich, the other Americans in the group included Esmond Snell, on sabbatical leave from
Berkeley, David Young, Walter Bortz, Dick Himes, Paul Kindel, Martin Stiles, and Ed
Wawskiewicz. Although Fitzi Lynen was a hard driving biochemist, he did like to socialize over
a beer or a martini. On Friday afternoons Harland would often bring a half-gallon bottle of
Gilbey’s gin to the Institute and prepare martinis in the second floor laboratory.
Shortly after returning from Munich in the Summer of 1963, I received a phone call from
Severo Ochoa, who asked if I might be interested in joining the faculty of his department at
New York University School of Medicine in New York City. My wife, Pat, and I had some
concern about moving from the bucolic setting of Blacksburg, Virginia (where we could see 20
miles from our living room window) to the big city. Nevertheless, we relished the new
challenges ahead and were ready for a change in lifestyle. We loved New York City and never
regretted having made the decision. Severo Ochoa helped make it worthwhile.
Severo Ochoa (1905–1993) (1, 10) (Fig. 3) was born in Luarca, Spain, the youngest of seven
children. His father was a lawyer and businessman. He completed his M.D. degree (with
honors) at the University of Madrid. Though never having studied with him, he was inspired
by Ramón y Cajal, the Spanish neuroanatomist and Nobel Prize winner (1906). Following
H60
medical school (1929 –1931) Ochoa joined Otto Meyerhof’s laboratory in Heidelberg where he
worked on muscle glycolysis. His early days in science were marked by the upheavals in
Europe leading up to World War II. At the time of the Spanish civil war in 1936, he left Spain
for Heidelberg for the second and final time. Then in 1938, because of the turmoil in Germany,
he moved to Oxford University in England to work in Professor Rudolph Peter’s unit. In 1941
he came to the United States where he joined Carl and Gerty Cori at Washington University
in St. Louis.
In his comments at the Nobel Prize banquet in 1964, Ochoa spoke of those who had
influenced him most.
I was deeply influenced by my great predecessor Santiago Ramón y Cajal. I entered Medical School too late
to receive his teachings directly but, through his writings and his example he did much to arouse my
enthusiasm for biology and crystallize my vocation. Among the great names that adorn the roll of Nobel
prize-winners in Medicine is that of Otto Meyerhof, my admired teacher and friend, to whose inspiration,
guidance and encouragement I owe so very much. I was very fortunate to have worked also under the
guidance of other great scientists and I wish to acknowledge my indebtedness to Sir Rudolph Peters and
to Nobel prize winners Carl and Gerty Cori who did so much to add new dimensions to my scientific
outlook and enlarge my intellectual experience.
The seven years (1964 –1970) I spent in Ochoa’s department were among the most exciting
of my scientific career. It was a small department with only a handful of faculty, which at that
time included Charles Weissman, Bob Warner, Bob Chambers, Albrecht Kleinschmidt, and
Severo. Upon arriving at New York University Medical School in August of 1962, Severo asked
me to give 15 lectures in the first year medical student biochemistry course the next month.
This course was Ochoa’s pride and joy and he and the faculty attended every lecture. (In
retrospect, I feel that this is an excellent way to ensure quality control in teaching.) At the
time, however, I hadn’t relished the idea of having a Nobel prize winner (1964, with Arthur
Kornberg, Ochoa’s first postdoctoral fellow) in the audience for the first 15 lectures in my new
scientific home. Despite knowing that my first few lectures at New York University were not
particularly good, after the lecture Severo put his hand on my shoulder and said, “That was an
excellent lecture, Dan.” I knew that it hadn’t been, but I did appreciate the encouragement.
This was typical of Severo’s behavior toward young scientists in whom he had confidence. I
suspect that his response reflected the encouragement he had received from his mentors
during his development.
Every afternoon at 3:00 p.m. we took a break for coffee in the department library where we
discussed the latest results of our experiments or a hot new paper. Because the faculty was
small, these were informal gatherings, which created a sense of camaraderie. Severo never
failed to show up for these sessions. We could always count on Charles Weissman for a good,
often slightly “off color” joke. “Have you heard the one about the ——?” Because of his innate
ability at story telling, Charles was a favorite lecturer of the medical students. His timing was
impeccable.
Severo had a princely presence in part because of his carriage, tall stature, and silver hair.
At national/international meetings, when he walked into a room he attracted hushed atten-
tion. Despite this, he had a warm personality and showed genuine concern for his colleagues,
associates, and students.
It is natural that we feel a closeness to those to whom we are related through research
interests. In Hans Krebs book, Reminiscences and Reflections (11), he illustrates the scientific
genealogy leading to Ochoa.
We talk rather loosely these days about “impact factor” (and citation index) in evaluating the
worth of one’s publications, but it is the excitement and joy of doing science, rather than the
recognition itself, that motivates us.
Research today moves at great speed. Communication is rapid, publication is rapid, and one
is left with the impression that everything of importance was done in the past 10 years.
However, science is built stepwise on the shoulders of those who came before us. Little is
taught today as to how each of our particular areas of the biological sciences developed. For
many students the “important stuff” now goes back into the past for only 7– 8 years. Most
online scientific journals go back only 7– 8 years. Fortunately, the Journal of Biological
Chemistry is the exception and is to be commended, because it is online all the way back to the
point of its origin in 1905. These Reflections may be a sign of recognition that the history of
discovery still has importance.
H61
Acknowledgment—I thank my wife, Pat Lane, who assisted with this article and shared these friendships and experiences
with me.
Address correspondence to: dlane@jhmi.edu.
REFERENCES
1. Ochoa, S. (1980) Annu. Rev. Biochem. 49, 1–30
2. Wood, H. G. (1985) Annu. Rev. Biochem. 54, 1– 41
3. Lynen, F. B. (1964) Information available on the Nobel Museum Web Site: www.nobel.se/
4. Lardy, H. A., and Adler, J. (1956) J. Biol. Chem. 219, 933–942
5. Lynen, F., Knappe, J., Lorch, E., Jutting, G., and Ringelmann, E. (1959) Angew. Chem. 71, 481– 486
6. Knappe, J., Ringelmann, E., and Lynen, F. (1961) Biochem. Z. 335, 168 –176
7. Moss, J., and Lane, M. D. (1971) Adv. Enzymol. Relat. Areas Mol. Biol. 35, 321– 442 (a review article)
8. Lane, M. D., and Lynen, F. (1963) Proc. Natl. Acad. Sci. U. S. A. 49, 379 –385
9. Lynen, F. (1967) Biochem. J. 102, 381– 400
10. Kornberg, A., Horecker, B. L., Cornudella, L., and Oró, J. (eds) (1975) Reflections on Biochemistry, pp. 1–14,
Pergamon Press, New York
11. Krebs, H. A. (1981) Reminiscences and Reflections, Oxford University Press, Oxford, UK
H62
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PROLOGUE H20 The Prostaglandins, Sune Bergström and Bengt Samuelsson

JOURNAL OF BIOLOGICAL CHEMISTRY i

JBC Historical Perspectives: Lipid Biochemistry*

JOURNAL OF BIOLOGICAL CHEMISTRY

JOURNAL OF BIOLOGICAL CHEMISTRY

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This paper is available on line at http://www.jbc.org

Konrad Bloch. Photo courtesy of the National Library of Medicine.

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Albert L. Lehninger. Photo courtesy of the National Library of Medicine.

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Photo courtesy of the Office of NIH History, National Institutes of Health.

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Photo courtesy of the Office of NIH History, National Institutes of Health.

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‡ To whom correspondence may be addressed. E-mail: eugene_kennedy@hms.harvard.edu.

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