ACTIVE TRANSPORT* OF INORGANIC PHOSPHATE AND

MAGNESIUM IONS BY BEEF HEART MIITOCHONDRIA

BY G. P. BRIERLEY, E. BACHMANN, AND D. E. GREENt
INSTITUTE FOR ENZYME RESEARCH, UNIVERSITY OF WISCONSIN

Communicated September 28, 1962

In 19491 our laboratory described the binding of inorganic phosphate by a mito-
chondrial preparation of rabbit kidney. This binding was shown to be linked to
electron transfer since reagents that were found to inhibit this process exerted a
comparable effect on the binding Although the phosphate taken up by the mito-
chondrion estimated as inorganic phosphate, nonetheless, some of the properties
of the accumulated phosphate argued in favor of a form of phosphate other than
inorganic phosphate (hence the designation of "gel phosphate"). This binding
phenomenon was subsequently studied by Crane and Lipmann.2 The possible
relation of the phosphate binding system to the phenomenon of oxidative phos-
phorylation was the central theme of Crane and Lipmann's investigation and later
of the investigations of Pressman,3 but no simple relation could be found. Among
others, Bartley and Davies4 and Amoore5 considered phosphate binding by mito-
chondria from the standpoint of both active and passive transport. In the present
communication, we shall present evidence that the binding of inorganic phosphate
by intact beef heart mitochondria is a process coupled to electron transfer and
that this translocation of phosphate (our description of the binding phenomenon)
is a process that shares one or more intermediate steps with that of oxidative
phosphorylation but differs in respect to the final step(s). Electron transfer, in-
deed, energizes both coupled phosphorylation and coupled translocation but these
are alternative (and possibly competitive) expressions of the same underlying cou-
pling mechanism. Under the conditions and in presence of the additions specified
in the legend for Table 1, massive accumulation of inorganic phosphate by the
mitochondrion is readily demonstrable. As much as 1 ,umole of phosphate can be
bound per mg of mitochondrial protein under optimal conditions. From the esti-
mate6 of the number of mitochondria per mg of protein (9 X 109) and from the com-
puted volume of the mitochondrion of beef heart muscle (0.2 ,3) and the fluid volume
of the mitochondrion (0.1 3), it can be calculated that the final concentration of
phosphate in the mitochondrion would be about IM when 1 4mole is taken up per
mg of protein. Our own determinations of the total water content of the packed
pellets which underlie the calculations are in close agreement with those of Green
and Oda.6
The binding of phosphate is completely inhibited by the classical inhibitors of
electron transfer tested (antimycin A, cyanide, and azide), by some reagents that
uncouple phosphorylation (2,4-dinitrophenol and dicoumarol), but not by oligo-
mycin-a highly effective inhibitor of coupled phosphorylation. The concentration
levels at which these respective inhibitors achieved complete inhibition of binding
in no case exceeded the levels normally used for complete inhibition of electron trans-
fer. The binding requires: (1) a suitable substrate (there is little if any specificity
in this regard); (2) Mg++; and (3) an electron acceptor (02). These are absolute
requirements as shown by the data of Table 1. In the presence of ADP, phosphate
1928
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TABLE 1
REQUIREMENTS FOR Pi AND MG++ ACCUMULATION BY HEART MITOCHONDRIA
mntmoles/mg protein
Pi Mg++
Untreated mitochondria 30 50
Complete system 1,010 1,800
Mg++ omitted 10 35
Pi " 10 95
Substrate omitted 10 75
ADP added (10 Amoles) 130 205
ADP + hexokinase system 30 85
Complete + dinitrophenol (0.3 Mmoles) 8 75
" + antimycin (6 Mg) 40 75
" + oligomycin (12 jug) 980 1,800
Heavy beef heart mitochondria2" (5 mg protein) were incubated with shaking for 15 min at 38° in 3 ml of
0.25 M sucrose containing 50 jumoles MgC12, 35 pmoles Tris chloride of pH 7.5, 10 mnoles inorganic phosphate
(Pi), and 10 pmoles of succinate as substrate. The flasks were then rapidly cooled to 04°, and the contents were
carefully layered over 7 ml of 0.88 M sucrose in a plastic tube fitted for the No. 40 head of the Spinco ultracentri-
fuge. The pellets of sedimented mitochondria (10 min at 40,000 rpm) were suspended in 1 ml of water. Pi was de-
termined by the method of Martin and Doty2l and Mg++ by a modification of the procedure of Harvey et al.22

accumulation is drastically reduced. In a sense, the absence of added ADP is a

requirement for maximal translocation activity. At 380, the rate of accumulation
of phosphate is some 20 times as rapid as the rate at 00 (cf. Fig. 1). It should be

A.
low
0-/ 38°

a 60

D ~400 25

0 10 20 30 40 5b do 7o do
TIME-MINUTES
FIG. 1.-Accumulation of phosphate by beef heart mitochondria as a function of temperature.
The details of the experimental procedures are given in the legend for Table 1.
mentioned that the accumulation does not proceed indefinitely but levels off even
though oxidation of the substrate still proceeds.
Under the conditions of the binding experiments (high Mg++ concentration
and the absence of a phosphate acceptor (ADP) and of ADP regenerating system),
oxidative phosphorylation cannot proceed to any significant degree. Thus, coupled
translocation may be the only alternative open to the mitochondrion under these
experimental conditions. In this regard, it bears repeating that oligomycin-a
reagent that completely inhibits oxidative phosphorylation7 at relatively low
concentrations-shows no inhibition of coupled translocation at the same con-
centration levels.
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 1930 BIOCHEMISTRY: BRIERLY, BACHMANN, AND GREEN PROC. N. A. S.

Electron transfer from citric cycle substrates to oxygen leads to the highest rate
of coupled translocation. Artificial electron donors can replace the physiological
substrates (e.g., reduced silicomolybdate). The rates with these artificial electron
donors are not as high as with the physiological counterparts but they are signifi-
cant (25% or more of the maximal rates). These substitutions suggest that it is
not necessary for electrons to traverse the complete chain in order to induce coupled
translocation and hint at the possibility of more than one site for coupled trans-
location.
ATP cannot replace the oxidative process even in part. Thus, mitochondria
incubated anaerobically with ATP show no evidence of coupled translocation of
phosphate. The idea that electron transfer leads to synthesis of ATP and that it is
the ATP that energizes the translocation of phosphate has no experimental sup-
port whatsoever in the present instance. The insensitivity of coupled transloca-
tion to oligomycin provides equally powerful evidence that ATP is not involved
in the accumulation of phosphate.
When a phosphate acceptor (ADP) is added to the mitochondrial suspension
in presence of Mg++, phosphate ions, and substrate, oxidative phosphorylation
takes place, and under these conditions the rate of coupled translocation is reduced
to about 20 per cent of the maximum (cf. Table 1). When ADP is regenerated, as
in the presence of hexokinase and glucose, then the rate of coupled translocation is
almost completely inhibited. These data suggest that coupled phosphorylation
and coupled translocation can proceed simultaneously though the former process
will predominate. The possibility of competition between these two processes
for a common intermediate has to be entertained even though the relative affinities
for the common intermediate may be vastly disparate. If ADP and the hexokinase-
glucose system and some oxidizable substrate are added after maximal accumula-
tion of phosphate has taken place (about 1 ,umole per mg protein), bound phosphate
declines to a small fraction of the original level. Thus, the accumulated phosphate
can be mobilized within the mitochondrion and can provide a source of inorganic
phosphate for coupled phosphorylation.
For the routine determination of accumulated phosphate, the mitochondrial
suspension that has been incubated aerobically in presence of Mg++ and phosphate
is washed free of adhering medium by layering it atop a three-layered sucrose gra-
dient and centrifuging at 30,000 rpm (method of Pressman3). The efficiency of
this washing procedure was checked by Pressman3 and is also attested to by the
low blank for accumulated phosphate when any one of the essential components of
the system is omitted (Mg++, substrate, oxygen) or when 2,4-dinitrophenol is used
to suppress coupled translocation (cf. Table 1). When mitochondria that have
accumulated phosphate are washed again at 00 in a sucrose gradient, minimal loss
of phosphate is observed, providing adequate precautions are taken to preserve
mitochondrial integrity. t Once the mitochondrial membrane is damaged, accu-
mulated phosphate is rapidly washed out into the medium. Apparently phos-
phate in the form accumulated does not readily diffuse out of the intact mitochon-
drion at 00.
In all our experiments to date, we have found that the binding phenomenon re-
quires an intact mitochondrion. When the mitochondrial membrane is damaged
or perforated by sonication or by repeated freezing and thawing, the resulting
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 VOL. 48, 1962 BIOCHEMISTRY: BRIERLY, BACHMANN, AND GREEN 1931

particles no longer show the binding phenomenon even though the capacity for
oxidative phosphorylation may still be retained either in whole or in part. When
mitochondria that have taken up relatively large amounts of phosphate are ex-
posed to sonic irradiation, complete release of inorganic phosphate into the medium
is observed. None is found associated with the derivative particles resulting from
the fragmentation of mitochondria. The submitochondrial electron transfer par-
ticle in its phosphorylating form (ETPH)8 shows no capacity to bind phosphate.
Under optimal conditions, the Pt ratio (molecules of phosphate translocated
per two electrons transferred from substrate to oxygen) is consistently less than
1.0. Our best values to date have been in the range of 0.5-0.6. If the assumption
is made that coupled translocation takes place exclusively in the external envelope
of the mitochondrion while electron transfer can proceed both in the external en-
velope and the internal cristae, then it follows that the true ratio for coupled trans-
location (defined above) would be considerably higher than the one measured.
According to the estimate of Green and Oda,6 the external envelope represents no
more than 30 per cent of the total structured elements of the mitochondrion. If
the rate of electron transfer is uniform throughout the mitochondrion, then the cor-
rected value of the ratio for coupled translocation would be 3.3 times as high as the
observed value, i.e., about 2.0 (0.6 X 3.3). This estimate makes no allowance for
the operation of any process that would obscure the binding phenomenon-proc-
esses such as residual oxidative phosphorylation or leakage back into the medium
of some bound phosphate Thus, more than one molecule of phosphate may be
translocated in the coupled process for each pair of electrons, and there is still a
possibility that the theoretical value of the ratio for coupled translocation will be
the same as that for coupled phosphorylation, namely 3.0. In this connection, it
is of interest to point out that the initial rate of translocation of phosphate is sig-
nificantly higher with pyruvate plus malate as substrate (P/O = 3) than with suc-
cinate (P/O = 2).
Mg++ is an absolute requirement for the binding of phosphate (cf. Table 1).
Indeed, it can be demonstrated that the binding of MIg++ and of phosphate run
parallel (cf. Fig. 2), and that this binding has the same requirements and sensitivity
to inhibitors as does the binding of phosphate (cf. Table 1). A distinction may have
to be made between two kinds of Mg++ binding-one that is sensitive both to in-
hibitors of electron transfer and to dinitrophenol and that parallels the phosphate
binding-another that is insensitive both to inhibitors of electron transfer and to
dinitrophenol and is independent of phosphate binding. The former represents ac-
tive binding by coupled translocation, whereas the latter represents passive binding.
The Mg: phosphate ratio as defined above approaches a value of 1.5:1. This
ratio is approximately constant over a wide range of experimental conditions (time,
temperature, concentration of Mg++ and phosphate in the external medium).
An Ig: phosphate ratio of 1.5:1 is consistent with the formation of (Mg)3(PO4)2
within the mitochondrion--a salt with an extremely low solubility in water (ca.
0.6 ,umoles per ml at 00). Since the concentration of (Mg)3(PO4)2 within the mito-
chondrion can reach a value of 500 ,moles per ml as discussed above, it is clear that
this concentration could only be attained by precipitation of the salt.
Coincident with the coupled translocation of Mg++ and phosphate into the
mitochondrion, there is formation of acid. For each molecule of phosphate bound,
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 I932 BIOCHEMISTRY: BRIERLY, BACHAiAYN, AND GREEN PROC. N. A. S.

A. Pi B. Mg*+
SUCCINATE

g00 C _P'

* 0~~~~F
goo 1'0
0~~~~~~-WDOYrRT E

TIME - MINUTES TIME MINUTES

FIG. 2.-Accumulation of phosphate (Pi) and Mg++ as a function of time in presence of three
different substrates. P + M represents a mixture of pyruvate and malate. The concentrations
of substrates were as follows: succinate, 3.3 mM; ,B-hydroxybutyrate, 3.3 mM; pyruvate (3.3
mM) plus malate (1.67 mM). Other experimental conditions as in the legend of Table 1.

approximately one mole of acid is liberated from the mitochondrion into the external
medium. It is of interest to note that the acid formation is paralleled by a corre-
sponding increase in alkalinity of the sedimented mitochondrial pellet after resus-
pension in fresh sucrose. It may be presumed that the acid is in fact formed in-
tramitochondrially and that H+ can penetrate the membrane freely. Since the
external medium is strongly buffered, the net movement of H + from inside the mito-
chondrion to the external medium would be favored by the differential in pH that
develops during translocation.
The outstanding characteristics of phosphate and Mg binding may be summarized
as follows. (1) Phosphate can penetrate the mitochondrion rapidly only in an
active process (the passive process is relatively slow). (2) When phosphate is
actively translocated, it is accompanied by Mg. No more than one atom of Mg++
could accompany each molecule of phosphate if phosphate has to be linked to some
mitochondrial structure or system during coupled translocation. (3) Mg++ may
penetrate the mitochondrion either actively (in 1:1 relation to the phosphate) or
passively (independent of phosphate). (4) The ratio of Mg: phosphate found in the
mitochondrion as a consequence of coupled translocation conforms to the stoichi-
ometry of (Mg)3(P04)2-a salt of relatively low water solubility. (5) Coincident
with coupled translocation of Mg++ and phosphate, acid is produced in the ratio
of 1 mole per mole of phosphate bound. These observations have led us to the
following hypothesis. During coupled translocation, the ionic equivalent of Mg-
HPO4 enters the mitochondrion. Simultaneously, the ionic equivalent of MgCl2
enters by a passive process. Precipitation of (Mg)3(P04)2 then ensues with libera-
tion of acid:
MgCl2 + 2 MgHPO4 i (Mg)3(P04)2 + 2 HC1.
(Mg)3(P04id in the mitochondrion provides a power-
The deposition of the insoluble
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 VOL. 48, 1962 BI0CHEMISTRIY: BRIERLY, BAC(HMANN, AND GREEN 1933

ful pulling force for the translocatioii of Mg++ and phosphate ions. If an insoluble
precipitate was not formed, i\Ig++ ionS COUld not accumulate in the mitochondrion
since the mitochondrial membrane is probably freely permeable to Mg++. §
In the discussion thus far, stress has been laid on the active accumulation of
phosphate, and it may appear that the concomitant accumulation of 1Ig++ is a
happenstance. The available evidence strongly suggests that there is a high degree
of specificity for both the phosphate and the Mg in the active process. Phosphate
cannot be replaced by any other ion tested while MAg++ is not replaceable by any
other cation tested except MIn++. Ca++,** for example, completely inhibits the
translocation. of MIg++ and does not substitute for Mg++ in this particular process.
As we shall discuss later, there is an absolute requirement for Mg++ (or Mn++) in
the formation of the high-energy intermediates that energize the translocation phe-
nomenon. These observations suggest specific sites for Mg binding in the enzyme
systems which translocate Mg++ and make it plausible to visualize the active process
as one in which both Mlg++ and phosphate play a specific role.
The coupling of electron transfer to synthesis of ATP proceeds at three sites and
requires three factors.12-14 We consider it a reasonable probability from the avail-
able data that the same three factors are required for coupled translocation. The
uncoupling action of dinitrophenol on coupled translocation is pertinent in this re-
gard, since dinitrophenol appears to exert its uncoupling effect on both ATP syn-
thesis and translocation by decomposing the first high-energy complex prior to the
transfer of the oxidation-reduction component (perhaps DPN, coenzyme Q, and cyto-
chrome c) from this complex to the appropriate factor. The most plausible inter-
pretation is that the interaction of the high-energy factor complex with phosphate can
lead either to synthesis of ATP or to translocation. The variables that determine
the order of precedence as yet are undetermined. It is of interest to point out that
Mg++ and inorganic phosphate are both concerned in the two alternatives -coupled
phosphorylation'5' 16 and coupled translocation. The active transport process
need not necessarily involve new intermediates and separate reactions. The same
reactions and intermediates that lead to ATP synthesis may well be involved in
coupled translocation. It could be at the very last step that the switch is made.
But it is to be noted that one high-energy bond may be required to translocate one
molecule of phosphate and one atom of Mg++.
There are three independent lines of evidence that support the view that coupled
phosphorylation and coupled translocation are two alternative processes competing
for the same high-energy intermediate: (1) the equal sensitivity of both systems to
the same inhibitors of the electron transfer chain and to 2,4-dinitrophenol, dicou-
marol, and m-chlorocarbonylcyanide phenylhydrazone;23 (2) the fact that the ex-
perimentally determined P/O and the calculated and corrected Pt/O are equal in
magnitude; and (3) the insensitivity of coupled translocation to oligomycin and the
complete sensitivity of coupled phosphorylation to the same reagent under identical
experimental conditions. It is possible to visualize the transfer of a phosphoryl
group from the high-energy complex to ADP as the critical step in coupled phos-
phorylation and the transfer of a phosphate group from one position in space to
another as the critical step in coupled translocation. The former process involves
conservation of the high-energy bond; the latter process involves utilization of the
high-energy bond.
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 1934 BIOCHEMISTRY: BRIERLY, BACHMANN, AND GREEN PROC. N. A. S.

The mitochondrial membrane appears to be made up of paired arrays of ele-

mentary particles embedded in a structural protein-lipid network.'7-19 In such
paired arrays, one set of particles lies on the outside of the membrane (in contact
with the external medium), whereas the other paired set of particles lies on the in-
side of the membrane (in contact with the internal medium). If the high-energy
complex involved in transduction were poised equidistant from the paired elemen-
tary particles, simple oscillation of some functional group to which both Mg and
phosphate are attached could lead to the movement of these from outside to inside
the mitochondrion. The possibility has to be entertained of a transducer macro-
molecule in which the bond energy of the high-energy complex is used to induce
a conformational change in the protein which in effect leads to a vectorial movement
of Mg and phosphate so directed in space that the initial positions of the ions are
"outside" and the final positions "inside."
The mitochondrion is probably a model for cell membranes generally, and while
coupled translocation may appear to be a minor function of the mitochondrion,
nonetheless its mechanism may be of broad generality. If such indeed is the case,
then coupled translocation of Mg++ and phosphate in the mitochondrion provides
the first intimate glimpse of the way in which active transport goes OD in living
systems.
In this context, it is important to distinguish between elements in the mechanism
proposed above that may have general applicability to ion transport and elements
that are specific for the particular mechanism under consideration. The generalized
features are (1) a high energy compound whether generated by electron transfer or
supplied as such in the form of ATP; (2) a membrane structure with paired arrays
of repeating functional units (not necessarily the elementary particle); and (3) a
specific enzymic unit that undergoes conformational rearrangement vectorially at
the expense of some high energy bond in a donor molecule with concomitant trans-
location of one or more ions.
This work was supported in part by the Division of General Medical Sciences Graduate Train-
ing Grant 2G-88 and the National Heart Institute research grant H-458, USPH; and the Atonic
Energy Commission Contract AT( 11-1)-909 and AT(11-1)-1151. Meat by-products were
generously supplied by Oscar Mayer and Company, Madison, Wisconsin.
*
The term "active transport" is used in the sense that the movement of ions from the medium
external to the mitochondrion into the internal medium of the mitochondrion is coupled to enzymic
processes and that this movement is not determined merely by a concentration gradient.
t With the technical assistance of 1). Hadley and E. Murer; E. B. is a postdoctoral trainee of
the Institute for Enzyme Research.
t The preservation of mitochondrial integrity poses several experimental difficulties that largely
stem from the fact that the internal pH of mitochondria which have accumulated phosphate (and
which have been sedimented and washed in sucrose) is abnormally high (pH 9 or higher). None-
theless, under favorable conditions even such mitochondria will retain up to 70 per cent of ac-
cumulated phosphate when washed for a second time in the sucrose gradient.
§ The available data cannot fully exclude the possibility that either phosphate or Mg++ is ac-
tively transported and that the other partner accumulates passively and merely coprecipitates
with the ion that is actively accumulated.
** There is an entirely different system concerned with the active transport of Ca++ (cf. DeLuca
and Engstrom,9 Vasington and Murphy10 and unpublished studies in our laboratory").
'Green, D. E., W. A. Atchley, J. Nordmann, and L. J. Tepley, Arch. Biochent. Biophys., 24,
359 (1949).
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 VOL. 48, 1962 BIOCHEMISTRY: HABER AND BENNETT 1935

2 Crane, R. K., a-id F. Lipmann, J. Biol. Chem., 201, 245 (1953).

3Pressman, B. C., J. Biol. Chem., 232, 967 (1958).
4 Bartley, W., and R. E. Davies, Biochem. J., 57, 37 (1954).
5 Amoore, J. E., Biochem. J., 70, 718 (1958).
Green, D. E., and T. Oda, J. Biol. Chenm. (Japan), 49, 742 (1961).
7 Lardv, H. A., D. Johnson, and W. C. McMurray, Arch. Biochemt. Biophys., 75, 587 (1958).
8 Linnane, A. WV. and D. M. Ziegler, Biochim. Biophys. Acta, 29, 630 (1958).
9 DeLuca, H. F., and G. W. Engstrom, these PROCEEDINGS, 47, 1744 (1961).
10 Vasington, F. D., and J. V. Murphy, J. Biol. Chem., 237, 2670 (1962).
1 Brierley, G. P., E. Bachmann, E. Murer, and D. E. Green, manuscript in preparation.
12 Pinchot, G. B., these PROCEEDINGS, 46, 929 (1960).
13 Webster, G., Biochem. Biophys. R'es. Communs., 7, 245 (1962).
14 SSmith, A. L., and M. Hansen, Biochem. Biophys. Res. Commnuns., 8, 33 (1962).
15 Webster, G., unpublished studies.
16 Smith, A. L., and M. Hansen, unpublished studies.
17 Richardson, S., and D. E. Green, unpublished studies.
18 Fernandez-Moran, H., T. Oda, P. V. Blair, and D. E. Green, in preparation.
19 Fernandez-Moran, H., unpublished studies.
20 Hatefi, Y., and R. L. Lester, Biochim. Biophys. Acta, 27, 83 (1958).
21 Martin, J. B., and D. M. Doty, Anal. Chem., 21, 965 (1949).
22 Harvey, A. E., J. M. Komarmy, and G. M. Wyatt, Anal. Chem., 25, 498 (1953).
23 Heyrtler, P. G'., and W. W. Prichard, Biochem. Biophys. Res. Communs., 7, 272 (1962).

POLARIZATION OF FLUORESCENCE AS A MEASURE OF ANTIGEN-

ANTIBODY INTERACTION*
BY EDGAR HABER AND J. CLAUDE BENNETTt
HARVARD MEDICAL SCHOOL AND THE MASSACHUSETTS GENERAL HOSPITAL, BOSTON

Communicated by Herman M. Kalckar, September 17, 1962

Present techniques of measuring antigen-antibody interactions depend either
on the solubility properties of the antigen-antibody complex, as in the precipitin
method, or on specialized characteristics of the immune complex, not uniquely
dependent on specific antigen binding as hemagglutination, complement fixation,
or passive cutaneous anaphylaxis. Recent degradative studies of the antibody
molecule1 2 have demonstrated fragments which are univalent and form soluble
complexes with antigen, but may not bind to skin or fix complement.3-5 The
commonly employed method of assay of these fragments' is inhibition of the
precipitin reaction, which lacks both directness and sensitivity. In the course of
further structural investigations of antibody, it became desirable to develop a sensi-
tive and direct method of assaying the association between antigen and antibody or
its functional fragments.
The elegant work of Perrin,6 later extended by Weber,'-9 demonstrated the
facility with which polarization of fluorescence could be used to measure the rota-
tional relaxation time of macromolecules. A complex of any degree of rigidity,
such as an antigen-antibody aggregate, should have a rotational relaxation time in
solution greater than any of its components. For polarized incident light, the
following equation pertains :8
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