VOL~20 (~956)

BIOCHIMICA ET BIOPHYSICA ACTA

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E N Z Y M I C S Y N T H E S I S OF P O L Y N U C L E O T I D E S I. POLYNUCLEOTIDE PHOSPHORYLASE OF
AZOTOBACTER
by MARIANNE GRUNBERG-MANAGO**, PRISCILLA J. ORTIZ AND SEVERO OCHOA

VINELA NDII ~

Department o/ Biochemistry, New York University College o/ Medicine, New York, N.Y. (U.S.A.)

As previously reported1, *, an enzyme isolated from the microorganism Azotobacter vinelandii catalyzes the synthesis of highly polymerized polynucleotides from 5'-nucleoside diphosphates with release of orthophosphate. The reaction, which requires magnesium ions and is reversible, can be represented as in Equation (I) where R stands for ribose, P-P for pyrophosphate, P for orthophosphate, and X for one or more of the following bases: adenine, hypoxanthine, guanine, uracil or cytosine.
n X - R - P - P ~- ( X - R - P ) n + n P (1)

The reaction is analogous to the reversible synthesis and breakdown of polysaccharides catalyzed by phosphorylase and, for this reason, the name polynucleotide phosphorylase has been proposed I for the new enzyme. Chemical and enzymic degradation of the biosynthetic polynucleotides, carried out in part in collaboration with Dr. LEON A. HEPPEL of the National Institutes of Health, has shown that they are made up of 5'-nucleoside monophosphate units linked to one another through 3'-phosphoribose ester bonds as in ribonucleic acid*. It has further been shown 2 that, from a mixture of ADP'**, GDP, UDP and CDP, the Azotobacter enzyme catalyzes the synthesis of polynucleotides which, in the light of chemical and enzymic evidence, cannot be distinguished from natural RNA. Recent experiments indicate that polynucleotide phosphorylase is widely distributed and suggest that this enzyme may be essential for the synthesis and
* This work was aided by grants from the National Institute of Arthritis and Metabolic Diseases (grant A-529) of the National Institutes of Health, U.S. Public Health Service; The American Cancer Society (recommended by the Committee on Growth, National Research Council); the Rockefeller Foundation; and by a c o n t r a c t ( N 6 onr 279. T.O.6) between the Office of Naval Research and New York University College of Medicine. *~ Present address: Institut de Biologie Physico-Chimique, 13, rue Pierre Curie, Paris Ve, France. *** The following abbreviations are used: Ribonucleic acid, RNA; deoxyribonucleic acid, DNA; 5'-diphosphates (pyrophosphates) of adenosine, inosine, guanosine, uridine and cytidine, ADP, IDP, GDP, UDP and CDP; 5'-monophosphates of the same nucleosides, AMP, IMP, GMP, UMP and CMP; adenosine- and inosine-5'-triphosphate, ATP and ITP; ultraviolet, U.V.; counts per minute, c.p.m.; ethylenediaminetetraacetic acid (versene), EDTA; and tris(hydroxymethyl)aminomethane, Tris.

Re/erence~ p. 285.

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breakdown of R N A in the cell. I n this connection it is of interest that POTTER a n d his collaborators 3 have d e m o n s t r a t e d the occurrence in tissues of 5'-mono-, di-, a n d triphosphates of adenosine, guanosine, uridine a n d cytidine. POTTER4 has recently suggested the nucleoside diphosphates as the most likely precursors of RNA. The partial purification of the Azotobacter enzyme, the properties of the system, a n d the preparation a n d isolation of various polynucleotides, are described in this paper. Other papers of this series will be concerned with further studies of the structure of biosynthetic polynucleotides, their physical properties, the distribution a n d the isolation of polynucleotide phosphorylase from various sources. EXPERIMENTAL
Preliminary experiments

Polynucleotide phosphorylase was discovered in the course of a s t u d y of biological phosphorylation mechanisms 5. The aim of this work was to look for soluble enzyme systems catalyzing the incorporation of radioactive orthophosphate in ATP. On i n c u b a t i o n of dialyzed Azotobacter extracts with ATP, in the presence of Mg++, there was a rapid "exchange" or incorporation of the labeled phosphate. Since the extracts contained adenylic kinase (myokinase) 6, the labeled phosphate was incorporated both in ATP a n d ADP. The early experiments were carried out with amorphous preparations of A T P which contained A D P as a c o n t a m i n a n t . However, when crystalline A T P was used there was little or no "exchange". E v e n t u a l l y , it was found t h a t the "exchange" was promoted b y ADP, prepared from crystalline A T P through reaction with glucose in the presence of hexokinase, a n d not all b y ATP or AMP. It was TABLE I
PHOSPHATE "EXCHANGE" AND LIBERATION WITH VARIOUS NUCLEOTIDES

Nucleotide*

Purity

P incorporation in I5 rain c.p.m,

P liberation in 30 rain pmoles

AMP AT]? AT]? AD]? AD]? AD]? + boiled extract *** AT]? + AMP ADP (no Mg++) ITP ID]?

Crystalline Amorphous Crystalline Amorphous Crystalline*~ Amorphous Crystalline Amorphous Amorphous Amorphous

o 5ooo 256 41ooo 4100o 42oo0 17200 o 195 57000

o 0.07 0.35 o.35 0.20 o 0.o6 0.52

Samples contained (in/,moles), Tris buffer, ioo; MgCI~, IO; and EDTA, i. Final volume, i.o ml; incubation at 3o. In addition, the samples for phosphate incorporation (pH, 8.1) contained potassium phosphate buffer, 2.3, with s2P-labelled orthophosphate (I42,ooo c.p.m.); nucleotide as indicated, 3; and enzyme of specific activity 1.94 (assay i) with 0.83 mg of protein (1.6 units). The samples for phosphate liberation (pH, 7.4) contained nucleotide as indicated, 5; and enzyme of specific activity i. 7 (assay I) with o.35 mg of protein (0.6 unit). * All nucleotides are the 5'-mono- or polyphosphates. ** ]?repared from crystalline ATP by reaction with glucose in the presence of crystalline hexokinase, and isolation of the AD]? by ion-exchange chromatography7. *** Initial .4zotobacter extract (9-7 mg of protein per ml) heated for 3 minutes at ioo . o.2 ml of supernatant fluid used for experiment.
Re/erences p. 2,~'5.

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271

further found t h a t nucleoside diphosphates other t h a n ADP, for example I D P , promoted the "exchange" reaction a n d that, simultaneously with the incorporation of labeled phosphate, there was a liberation of orthophosphate (c/. Reaction i) which also required magnesium ions. These experiments were performed with enzyme preparations in which the activity had been partially purified employing the "exchange" with A D P as an assay. Typical d a t a with partially purified enzyme preparations are shown in Table I. With the exception of amorphous ATP, only the nucleoside diphosphates were active in giving rise to either incorporation of phosphate or phosphate liberation ; the mono- and triph0sphates had little or no activity. Positive results were also obtained with ATP plus AMP owing to the presence of adenylic kinase. This enzyme catalyzes the reaction ATP + AMP ~ 2ADP. Similar results are shown in Fig. I. These experiments were carried out with an enzyme preparation largely freed of adenylic kinase b y precipitation with acetone. After incubation of the nucleotides with the enzyme, in the presence of Mg ++, and deproteinization of the reaction mixture the nucleotides were separated by paper c h r o m a t o g r a p h y in the solvent system of KREBS AND HEMSs followed b y autoradiography to localize the radioactive spots containing 32p. It will be seen that on incubation of mixtures of A D P or I D P with other nucleotides only A D P and I D P became labeled. The fact that AMP was not labeled when A D P and AMP were incubated together indicated that the radioactive phosphate was incorporated in the terminal phosphate of ADP. On incubation of I D P and A D P together both became labeled although, in this experiment, the labeling of A D P was weak. Fig. 2 shows the time course of incorporation of 3*P-orthophosphate in A D P as well as t h a t of liberation of orthophosphate from ADP. A number of experiments were carried out with I D P as substrate to determine the stoictiiometry of the reaction. I D P , rather than ADP, was used because adenylic kinase is not active on IDP. W h e n the incubated reaction mixtures were deproteini~ed
1.25

t.O0
//
O0

.,..t

0.T5 0.50

o ~ .C() ~ ]~ =0.25

P LIBERATION I I [ I 15 30 45 60 NINUTE$

Fig. i. Enzymic incorporation of 32P-orthophosphate in ADP and IDP. Conditions as in phosphate incorporation experiments of Table I. Paper chromatography by the method of KREBS A~rDHEMS Spots located with U.V. lamp. Cors. responding autoradiograms to the right of sketch of each chromatographic strip. Re/erences p. 285.

Fig. 2. Enzymic incorporation of srP-orthophosphate in ADP (Curve I) and liberation of orthophosphate from ADP (Curve 2) as a function of time. The conditions were those of the standard assays described in the text. 20.5 pg of enzyme of specific activity lO.6 (o.2z8 unit) were used for Curve I, and 61.6/~g of enzyme of specific activity i3.o (0.8 unit) for Curve 2. The specific activities are expressed in terms of assay I.

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with trichloroacetic acid, there was a disappearance of total nucleotide (determined by measurement of U.V. absorption) from the supernatant fluid corresponding to the amount of I D P disappearing and that of orthophosphate liberated. On the other hand, there was no disappearance of total nucleotide when the reaction mixture was deproteinized by heating for I or 2 minutes at IOO (Table II). However, thereaction product could not be eluted from Dowex-I (formate) columns with formic acid at concentrations much higher than those required to elute the most acidic mononucleotides. A typical experiment is shown in Table II. In this experiment 2.0 ml aliquots of the protein-free supernatant fluid were used for the separation of nucleotides by ion-exchange chromatography. The samples were brought to 50 ml with water and made alkaline by addition of 0.5 ml of concentrated ammonia. The solution was chromatographed on a Dowex-i-(formate) column (4 I cm). The~resin used was Nalcite SDR, 200-400 mesh, 2% cross-linkage. After placing the sample on the column, it was washed with water. Then it was eluted successively with o. I M sodium formate (which removes the orthophosphate), o.15 M sodium formate with o.oi M formic acid (which elutes the IMP), 0.2-0. 3 M sodium formate with o.oi M formic acid (which elutes the IDP) and, finally, with 0. 4 M sodium formate with o.oi M formic acic~ (which elutes ITP). It may be seen in the table that, on incubation for 2o and 6o minutes, the disappearance of I D P and the liberation of orthophosphate were equimolar. It m a y also be seen from the A values of the last column that there was a deficit in the nucleotide recovered from the column of the same order of magnitude as the I D P disappearance and orthophosphate liberation. As already reported 2, it was further found that the product of the reaction of I D P or ADP, in the presence of the enzyme and magnesium, was non-dialyzable against distilled water or dilute salt solutions. It could be quantitatively precipitated with trichloroacetic acid or alcohol in the cold; either of these two methods could be used for its isolation. The product was soluble in water, yielding relatively viscous solutions with a typical nucleotide ultraviolet absorption spectrum, and it remained at the origin of paper chromatograms whichever the solvent system used. All these facts indicated that the product was a polynucleotide.
T A B L E II
REACTION W I T H I D P Incubation rain Nuc.deotid recovered lrora coluffln

Octhophosptuae

IMP

IDP

o 2o A 60 .4

1.32 3.8o + 2.48 6.4 + 5.08

o.6 o.6 o o. 6 o

lO. 7 8.6 --2.1 6.5 --42

I1. 3 9.2 --2.1 7. I --4.2

T h e reaction m i x t u r e c o n t a i n e d t h e following c o m p o n e n t s (in p m o l e s per ml), Tris buffer, p H 8 . I , 36; MgC1 a, 4.8; p o t a s s i u m p h o s p h a t e , p H 8.i, 1.3; I D P , 11.5; a n d e n z y m e of specific a c t i v i t y 4.6 (assay i) w i t h 0.54 m g of p r o t e i n (2.5 units). F i n a l v o l u m e , 8.5 ml. 2.5 m l aliquots r e m o v e d a t t i m e zero a n d after i n c u b a t i o n for 20 a n d 60 m i n u t e s a t 30 . A f t e r h e a t i n g for z m i n u t e s a t lOO , cooling, a n d r e m o v i n g t h e p r e c i p i t a t e d p r o t e i n b y c e n t r i f u g a t i o n , aliquots of t h e s u p e r n a t a n t fluid were t a k e n for t h e d e t e r m i n a t i o n of o r t h o p h o s p h a t e (o.1 m l ) a n d nucleotides (2.0 ml). Values expressed i n / , m o l e s p e r ml of reaction m i x t u r e . T h e s m a l l a m o u n t of I M P w a s p r e s e n t as a c o n t a m i n a n t of t h e I D P p r e p a r a t i o n . T h e r e was no c h a n g e in t h e a m o u n t of t o t a l nucleotide ( d e t e r m i n e d b y U.V. a b s o r p t i o n a t w a v e - l e n g t h 247 rap) d u r i n g i n c u b a t i o n .

Re/erencesp. 285.

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Isolation o[ enzyme Enzyme assays. The enzyme has been assayed in either of two ways: (I) by determining the rate of incorporation of radioactive orthophosphate into nueleoside diphosphates (phosphate "exchange" assay) and (2) by determining the rate of liberation of orthophosphate from nucleoside diphosphates. Either ADP or IDP, but mostly the former, has been used for assay purposes.
Assay I. T h e reaction m i x t u r e c o n t a i n s t h e following c o m p o n e n t s (in /,moles) : Tris buffer, p H 8.1, IOO; MgC12, 5; A D P (or I D P ) , 2.5; p o t a s s i u m p h o s p h a t e , p H 8.i, with ioo,ooo-2oo,ooo c . p . m . 32p, 3. 6 (or 2.5) ; a n d e n z y m e . F i n a l v o l u m e , i.o ml. If A D P is used, t h e m o l a r ratio A D P to o r t h o p h o s p h a t e s h o u l d be o.7; if I D P is used, t h e ratio I D P to o r t h o p h o s p h a t e s h o u l d be i.o. It is c o n v e n i e n t to a d d a s m a l l a m o u n t of E D T A for a s s a y of t h e e n z y m e d u r i n g t h e initial s t e p s of t h e purification. T h e reaction m i x t u r e is i n c u b a t e d for 15 m i n u t e s at 3 o ' . W i t h s m a l l a m o u n t s of e n z y m e t h e reaction is linear for a t least 60 m i n u t e s . O n e u n i t of e n z y m e is defined as t h e a m o u n t which c a t a l y z e s t h e incorporation of i . o / ~ m o l e of 3~PO4~ u n d e r t h e conditions of t h e a s s a y in 15 m i n u t e s at 30 . T h e r a d i o a c t i v i t y in t h e organic p h o s p h a t e fraction, d e t e r m i n e d as described in t h e n e x t section, is u s e d to calculate t h e a m o u n t of o r t h o p h o s p h a t e i n c o r p o r a t e d from t h e expression :* /,moles p h o s p h a t e incorporated = c.p.m, i n c o r p o r a t e d (/~moles p h o s p h a t e + # m o l e s ADI') c.p.m, p h o s p h a t e
T h e specific a c t i v i t y of t h e e n z y m e is expressed as u n i t s per m g of protein. P r o t e i n was d e t e r m i n e d either b y t h e b i u r e t m e t h o d 9 with crystalline o v a l b u m i n , previously dialyzed free of salt, as a s t a n d a r d , or s p e c t r o p h o t o m e t r i c a l l y b y m e a s u r i n g t h e a b s o r p t i o n of light at w a v e - l e n g t h s 280 a n d 26o m/~, with a correction for t h e nucleic acid c o n t e n t from t h e d a t a g i v e n b y XVARBURG AND CHRISTIAN 10. T h e first m e t h o d w a s used to d e t e r m i n e t h e protein c o n t e n t of t h e e x t r a c t a n d t h e first a m m o n i u m sulfate fraction, w i t h high nucleic acid content, t h e second m e t h o d was u s e d for t h e later steps in purification. Determination o] ovthophosphate incorporation. This p r o c e d u r e follows t h a t described b y R o s e AND OCHOAxl with s o m e modifications. T h e o r t h o p h o s p h a t e i n c o r p o r a t e d is given b y t h e r a d i o a c t i v i t y r e m a i n i n g in t h e protein-free filtrate after r e m o v a l of t h e o r t h o p h o s p h a t e . T h e p r o c e d u r e is b a s e d on t h e q u a n t i t a t i v e conversion of o r t h o p h o s p h a t e to a m m o n i u m p h o s p h o m o l y b d a t e a n d t h e r e m o v a l of t h e l a t t e r b y e x t r a c t i o n w i t h isobutanol, according to BERENBLUM AND CHAIN 12. T h e reaction is s t o p p e d b y addition of o.I ml of 40o trichloracetic acid per i.o ml of reaction m i x t u r e . After c e n t r i f u g a t i o n , aliquots are t a k e n for d e t e r m i n a t i o n of t h e specific r a d i o a c t i v i t y of t h e o r t h o p h o s p h a t e a n d t h e r a d i o a c t i v i t y fixed. F o r r e m o v a l of t h e orthop h o s p h a t e , u s u a l l y I.o ml of t h e protein-free filtrate is m a d e u p with w a t e r to 3.o ml. To this solution are a d d e d o.3 m l of i o . o M H~SO4, 1. 5 ml of 5 o a m m o n i u m m o l y b d a t e , a n d . 5 . o m l of isobutanol. A slow s t r e a m of air is b u b b l e d t h r o u g h t h e m i x t u r e , u s u a l l y for one m i n u t e , to o b t a i n good m i x i n g . After allowing t h e liquid p h a s e s to s e p a r a t e b y s t a n d i n g , t h e u p p e r isobutanol layer is r e m o v e d b y aspiration a n d discarded. Tile a q u e o u s phase, w h i c h m u s t be clear**, is w a s h e d w i t h 3.0 ml of ether; t h e e t h e r is r e m o v e d a n d discarded. A n a l i q u o t (usually I.O ml) of t h e a q u e o u s phase, is t a k e n for d e t e r m i n a t i o n of t h e r a d i o a c t i v i t y of t h e o r g a n i c a l l y - b o u n d p h o s p h a t e . W h e n t h e p r o c e d u r e is p e r f o r m e d on a control reaction m i x t u r e to w h i c h t h e e n z y m e is a d d e d after trichloracetic acid, it is f o u n d t h a t over 99 % of t h e r a d i o a c t i v i t y (i.e., of t h e o r t h o p h o s p h a t e ) is r e m o v e d . Such a control is included with e v e r y e x p e r i m e n t a l r u n a n d t h e r a d i o a c t i v i t y of this b l a n k s a m p l e is s u b t r a c t e d f r o m t h a t in t h e e x p e r i m e n t a l ones to correct for t h e s m a l l a m o u n t of o r t h o p h o s p h a t e n o t r e m o v e d . T h i s s h o u l d a l w a y s be v e r y small. Assay 2. T h e r a t e of liberation of o r t h o p h o s p h a t e from A D P is u s e d in t h i s assay. For e c o n o m y r e a s o n s t h e a s s a y is carried o u t w i t h c o n c e n t r a t i o n s of A D P , w h i c h a l t h o u g h higher t h a n t h o s e u s e d in A s s a y i, are insufficient to s a t u r a t e t h e e n z y m e as far as t h e rate of liberation of p h o s p h a t e is concerned. A t s a t u r a t i o n levels of A D P , t h e rate of liberation of p h o s p h a t e is s o m e t h r e e t i m e s faster t h a n o b t a i n e d in t h e s t a n d a r d assay. T h e reaction m i x t u r e c o n t a i n s t h e following c o m p o n e n t s (in/~moles), Tris buffer, p H 8.I, 7 o - I o o ; MgCl 2, 5; A D P , io; a n d e n z y m e . F i n a l v o l u m e , i.o ml. T h e a m o u n t of e n z y m e s h o u l d be s u c h t h a t t h e reaction rat0 r e m a i n s * T h i s is n o t a n a c c u r a t e c a l c u l a t i o n of t h e a b s o l u t e a m o u n t of p h o s p h a t e i n c o r p o r a t e d a n d is u s e d o n l y as a c o n v e n i e n t e x p r e s s i o n of t h e a c t i v i t y of t h e e n z y m e u n d e r t h e c o n d i t i o n s of A s s a y x. ** Occasionally t h e a q u e o u s p h a s e m a y n o t be clear after one e x t r a c t i o n w i t h isobutanol; w h e n this h a p p e n s , t h e r e m o v a l of o r t h o p h o s p h a t e is incomplete. A second e x t r a c t i o n u s u a l l y e l i m i n a t e s t h e r e m a i n i n g p h o s p h a t e satisfactorily.

Re/erences p. 285.

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essentially linear with t i m e d u r i n g t h e i n c u b a t i o n ; t h i s a m o u n t is u s u a l l y below x,5 units, as d e t e r m i n e d b y A s s a y i. Usually, two s a m p l e s are r u n for 5 a n d I5 m i n u t e s , respectively. T h e i n c u b a t i o n is carried o u t with s h a k i n g a t 3 o . O n e u n i t of e n z y m e is defined as t h e a m o u n t w h i c h c a t a l y z e s P"EXCHANGE"UNITS (CURVE I) 0.2 0.4 0.6 t h e liberation of z / , m o l e of o r t h o p h o s p h a t e per 15 m i n ' I i 1 i I a t 3 o. T h e specific a c t i v i t y is defined as u n i t s per m g 0.6 of protein. T h e r e a c t i o n is s t o p p e d w i t h trichloracetic acid as in A s s a y z a n d o r t h o p h o s p h a t e d e t e r m i n e d b y ~ 0.5 t h e m e t h o d of LOHMANN AND JENDRASSlK 18. T h e r a t e of p h o s p h a t e liberation is p r o p o r t i o n a l to t h e concent r a t i o n of e n z y m e b e t w e e n t h e limits of o a n d i. 5 ~ 0.4 (Assay z) units. W h e n t h e e n z y m e is free or a l m o s t free of adenylic kinase, t h e r a t e of p h o s p h a t e liberation r e m a i n s c o n s t a n t for at least I hour. I n t h e presence of adenylic k i n a s e it m a y be n e c e s s a r y to calculate i 0:3 112 t h e reaction r a t e f r o m t h e 5 m i n u t e i n c u b a t i o n value. As s h o w n in Fig. 3, t h e reaction rate, u n d e r t h e _ 0.1 / (~) P LIBERATION conditions of either A s s a y z or z, is p r o p o r t i o n a l to t h e c o n c e n t r a t i o n of e n z y m e over a fairly wide r a n g e I t I I I. of e n z y m e dilutions. It m a y also be seen t h a t t h e ratio 0 ' 0.5 I.O 1.5 of p h o s p h a t e i n c o r p o r a t i o n (Assay z) to t h a t of phosP "EXCHANGE'UNITS (CURVE 2) p h a t e liberation r a t e (Assay 2) is a p p r o x i m a t e l y Fig. 3. R a t e of incorporation of ~ZP-or_.5-2.8. A s s a y z h a s been t h e one r o u t i n e l y u s e d for t h o p h o s p h a t e in A D P (Curve I) a n d of purification of t h e e n z y m e . Unless otherwise s t a t e d , p h o s p h a t e liberation from A D P (Curve 2) u n i t s a n d specific activities of t h e e n z y m e p r e p a r a t i o n s as a f u n c t i o n of e n z y m e concentration. t h r o u g h o u t this p a p e r are e x p r e s s e d in t e r m s of A s s a y i. C u r v e I was o b t a i n e d with a solution of Purification o] enzyme e n z y m e (z.o2 m g of protein per ml) of Azotobacter vinelandii (strain original) * was g r o w n as specific a c t i v i t y z0.6; C u r v e 2, with a described b y HYND~AN el alA 4. T h e ceils were harsolution of e n z y m e (1.o8 m g of protein v e s t e d after x 5 h o u r s of g r o w t h ; a t t h i s t i m e a i :5 per ml) of specific a c t i v i t y 22.5. Specific dilution of t h e m e d i u m s h o u l d read a b o u t zi 5 in a activities are expressed in t e r m s of K l e t t colorimeter w i t h filter No. 54 o. T h e yield was a s s a y I. T h e o r d i n a t e r e p r e s e n t s # m o l e s a r o u n d 6 g of w e t cells p e r liter of m e d i u m . of p h o s p h a t e i n c o r p o r a t e d or liberated in Step z. Extraction. T h e e n z y m e , which is easily 15 m i n u t e s a t 3 o. soluble, c a n be e x t r a c t e d f r o m fresh, acetone-dried, or lyophilized cells. F r e s h or dried eell p r e p a r a t i o n s r e t a i n their a c t i v i t y for a long t i m e w h e n stored a t - - 1 8 . T h e fresh cells are g r o u n d in a m o r t a r with double their weight of a l u m i n a A3oz (325 mesh, A l u m i n u m C o m p a n y of America) a t o% t h e n e x t r a c t e d w i t h 4.5 v o l u m e s of o . I 5 M KC1 a n d c e n t r i f u g e d for 30 m i n u t e s a t 300o-45o0 g. T h e residue of cells a n d a l u m i n a is r e - e x t r a c t e d w i t h 2.o v o l u m e s of o.I5 M KC1 for 3 rain a n d t h e m i x t u r e is c e n t r i f u g e d as before. T h e s u p e r n a t a n t e x t r a c t s are c o m b i n e d a n d c e n t r i f u g e d for 3 o m i n u t e s at o a n d i6,ooo r.p.m, in t h e high speed h e a d of t h e I n t e r n a t i o n a l Centrifuge. T h e s u p e r n a t a n t fluid is t h e n dialyzed a g a i n s t o.ol M p h o s p h a t e buffer, p H 7.o, for 2 h o u r s w i t h stirring. I n t h e case of dried cell p r e p a r a t i o n s , t h e e x t r a c t i o n is carried o u t in t h e s a m e way, e x c e p t t h a t a n e q u a l weight of a l u m i n a a n d 22. 5 a n d io.o v o l u m e s , respectively, of o . z 5 M KCI are used. T h e e x t r a c t c o n t a i n s f r o m 8 - z 5 m g of protein p e r ml. A n a m o u n t of e x t r a c t c o n t a i n i n g o . 5 - I . o m g of p r o t e i n is used per a s s a y (Assay z). T h e specific a c t i v i t y of t h e e x t r a c t varies from 0. 3 to o. 5. Step 2. First ammonium sul/ate ]vactionation. T h e e x t r a c t is diluted, if necessary, with o.oi M p h o s p h a t e buffer, p H 7.o, to c o n t a i n io m g of p r o t e i n p e r ml a n d cooled to 0% Solid a m m o n i u m s u l f a t e is a d d e d with m e c h a n i c a l stirring to m a k e 0.35 s a t u r a t i o n . A f t e r stirring for 5 m i n u t e s , t h e m i x t u r e is c e n t r i f u g e d in t h e cold r o o m at t o p speed in t h e Servall angle centrifuge (2o, ooo g). T h e precipitate, w h i c h c o n t a i n s ~ - h y d r o x y b u t y r i c d e h y d r o g e n a s e , h a s been used for t h e purification of t h i s e n z y m e ; t h i s p r e p a r a t i o n will be described elsewhere. Solid a m m o n i u m sulfate is a d d e d to t h e s u p e r u a t a n t solution as a b o v e to m a k e its degree of s a t u r a t i o n 0.46. T h e precipitate c o n t a i n s m o s t of t h e polynucleotide p h o s p h o r y l a s e activity. T h i s fraction, w h i c h c o n t a i n s f r o m 20 to 25 % of t h e p r o t e i n of t h e extract, is dissolved in o.oi M p o t a s s i u m p h o s p h a t e buffer, p H 7.4, a n d dialyzed w i t h stirring in t h e cold for z h o u r s a g a i n s t t h e s a m e buffer. A n a m o u n t c o n t a i n i n g a b o u t o.2 m g of p r o t e i n is u s e d p e r assay, T h e specific a c t i v i t y of t h i s fraction is a b o u t 2.o. Step 3. Adsorption and elution Item calcium phosphate gel. T h e dialyzed s o l u t i o n of t h e 0 . 3 5 0.46 a m m o n i u m s u l f a t e p r e c i p i t a t e is diluted, if necessary, w i t h o,o1 M p o t a s s i u m p h o s p h a t e * W e are i n d e b t e d to Dr. P. W. WILSON, D e p a r t m e n t of Bacteriology, School of Agriculture, U n i v e r s i t y of Wisconsin, Madison, Wisconsin, for a c u l t u r e of t h e o r g a n i s m .

Re/erences p. 185.

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275

buffer, p H 7.4, to c o n t a i n 9 - ~ o m g of p r o t e i n per ml a n d cooled to o . To 95 ml of t h e e n z y m e solution is a d d e d 0.9 m l of x . o M a c e t a t e buffer, p H 5.i. T h e final p H of this solution is 5.8 a n d t h e s a l t c o n c e n t r a t i o n a b o u t o . o z M . 15 m l of c a l c i u m p h o s p h a t e gel 15 (dry weight, 20 m g per ml) are a d d e d to 96 m l of t h e a b o v e solution. A f t e r stirring for 20 m i n u t e s a t o t h e gel, which c o n t a i n s a b o u t 9 % of t h e p r o t e i n of t h e fraction, a n d 5 % of t h e a c t i v i t y , is c e n t r i f u g e d off a n d discarded. 5.0 m l of c a l c i u m p h o s p h a t e gel are n o w a d d e d to t h e s u p e r n a t a n t solution a n d t h e m i x t u r e is stirred a n d c e n t r i f u g e d as above. T h e gel is eluted first with 30-4 ml of o . t M p o t a s s i u m p h o s p h a t e buffer, p H 6.0, at o and, a f t e r c e n t r i f u g a t i o n in t h e cold, with 15 ml of o.oI M p o t a s s i u m p h o s p h a t e buffer, p H 7-3. T h e buffers used for elution are at o b u t t h e elutions are carried o u t at r o o m t e m p e r a t u r e . T h e e l u a t e s are c o m b i n e d a n d dialyzed at o a g a i n s t o.ol M p o t a s s i u m p h o s p h a t e buffer p H 7.4, for 2 h o u r s w i t h stirring. An a m o u n t of eluate c o n t a i n i n g a b o u t 0.05 m g of p r o t e i n is u s e d p e r assay. T h e specific a c t i v i t y of t h e eluate, which c o n t a i n s a b o u t i8 % of t h e p r o t e i n of t h e fraction w i t h 7 6 % of t h e activity, is 8.0 to ~o.o. T h e overall recovery of e n z y m e in t h e s u p e r n a t a n t fluid a n d gel e l u a t e s is a b o u t 90 %. Step 4. Second a m m o n i u m sul[ate #actionation. T h e dialyzed eluate, c o n t a i n i n g a b o u t 2. 5 m g o f - p r o t e i n per ml, is f r a c t i o n a t e d at o w i t h s a t u r a t e d a m m o n i u m sulfate a d j u s t e d to p H 7.4 w i t h a m m o n i u m h y d r o x i d e . T h r e e fractions are o b t a i n e d : fraction 3, b e t w e e n t h e limits of o a n d 0.45 a m m o n i u m sulfate s a t u r a t i o n , fraction z b e t w e e n 0.45 a n d 0.55 s a t u r a t i o n , a n d fraction 3 b e t w e e n 0.55 a n d 0.60 s a t u r a t i o n . T h e a m m o n i u m s u l f a t e precipitates are collected b y c e n t r i f u g a t i o n in t h e cold r o o m ( a b o u t 5 ) a t h i g h speed in t h e Servall Angle centrifuge, dissolved in a s m a l l v o l u m e of o.oi M p o t a s s i u m p h o s p h a t e buffer, p H 7.4, a n d dialyzed at o with s t i r r i n g a g a i n s t t h e s a m e buffer for several h o u r s . F r a c t i o n x, w h i c h c o n t a i n s a b o u t ~7 % of t h e p r o t e i n a n d 15 % of t h e activity, a n d fraction 3 . c o n t a i n i n g i o o of t h e p r o t e i n a n d x 1 % of t h e a c t i v i t y o f t h e eluate, are discarded. F r a c t i o n 2, w h i c h c o n t a i n s _,z% of t h e p r o t e i n a n d 73,o of t h e a c t i v i t y of t h e eluate is t h e best p r e p a r a t i o n of t h e e n z y m e so far obtained. An a m o u n t of fraction c o n t a i n i n g from o.o2 to 0.03 m g of p r o t e i n is used per a s s a y ; its specific a c t i v i t y is from I8 to -2. T h e overall recovery of a c t i v i t y in t h e t h r e e a m m o n i u m sulfate fractions a m o u n t s to 99 %. A s u m m a r y of t h e purification procedure, s t a r t i n g with acetone-dried cells, is g i v e n in T a b l e III. This r u n w a s p a r t i c u l a r l y successful b o t h as r e g a r d s purification a n d yield. In two large-scale p r e p a r a t i o n s from fresh cells, carried o u t s u b s e q u e n t l y * , t h e specific a c t i v i t y of t h e e n z y m e a t s t a g e 4 was 14.6 a n d ~.1.6, with a yield of 28 a n d 24 %, respectively. T h e a m m o n i u m sulfate s t e p s are generally reproducible b u t t h e degree of success with t h e calcium p h o s p h a t e gel step d e p e n d s on t h e p a r t i c u l a r gel p r e p a r a t i o n or b a t c h used a n d t h e procedure finally utilized at this s t a g e m a y h a v e to d e v i a t e from t h e one described here. TABLE III
PURIFICATION OF POLYNUCLEOTIDE PHOSPHORYLASE OF

Azotobacter vinelandii specif,c activity unitslmg

protein
Yield per cen~

Step

Volume ml

Units (Assay z)

Protein mg

Ratio" 28~t26

L 2. 3. 4.

Dialyzed e x t r a c t (NH4)sSOI (o.35-o.46) Cas(PO4)l gel eluate (NH4)ISO4 (o.45--.55)

376 95 58 5

x84o 175o 15oo io2o

345 8Io 142 45

0.5 0. 7 i.i

o.5 2.2 io.6 22.5

xoo 95 82 56

x5.o g of acetone-dried cells (A. vinelandii, s t r a i n original) e x t r a c t e d w i t h an e q u a l w e i g h t of a l u m i n a a n d 4o0 m l of o . x s M KCI. * R a t i o of light a b s o r p t i o n at 2280 to t h a t a t 2z6o m/~. I n t h e r u n of Table III, besides A s s a y I, on w h i c h t h e spe.cific activities g i v e n in t h e table are based, A s s a y 2 w a s also used a t each step. T h e specific activities b a s e d on this a s s a y a t s t e p s x, 2, 3, a n d 4, respectively, were o.I 7, 0.95, 3.3 o, a n d 8.30 (c[. T a b l e IIl). T h u s , t h e ratios of rate of p h o s p h a t e i n c o r p o r a t i o n to rate of p h o s p h a t e liberation, i.e., t h e ratios of specific activities d e t e r m i n e d b y A s s a y x to t h o s e d e t e r m i n e d b y A s s a y z, a t each step, were 2.9, z.3, 3.z, a n d z.7, for s t e p s t t h r o u g h 4, respectively, t h e a v e r a g e v a l u e of t h e ratio b e i n g 2.76 (O*. also Fig. 3). T h u s , t h r o u g h o u t t h e purification so far achieved of t h e Azotobacter e n z y m e , t h e ratio of t h e r a t e of p h o s p h a t e i n c o r p o r a t i o n to t h a t of p h o s p h a t e liberation r e m a i n e d c o n s t a n t .
* B y Dr. M. STAEHRI.IN a n d Mr. M. C. SCHNEIDER.

Re/erences p. a85.

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Properties o~ enzyme Stability. Preparations of the enzyme at any step of purification remain active for
periods as long as one year if kept in the frozen state at - - 18 . However, the enzyme loses activity on thawing, especially on repeated freezing and thawing. Although the enzyme is relatively stable at an acid pH (4.5 to 5.5), it cannot be stored frozen at this pH and loses about 70% of its activity on thawing unde r these conditions. Heating for 5 minutes at 5 0 and pH 5.8 destroys 50% of the enzyme activity. However, only IO to 20% is lost if this heating is carried out at pH 6. 4. The enzyme is completely destroyed by heating for 5 minutes at 60 and pH 5.6, or for I minute at IOO and pH 7.4. pH Optimum. Under the conditions of Assay I the phosphate incorporation reaction has a rather sharp optimum at pH 8.1. At pH 6.4 the activity is 28% and at pH IO.O, 50% of that at pH 8.1. Under the conditions of Assay 2, the phosphate liberation reaction has a plateau of maximum activity between pH 7.5 and pH 9.0; at pH 6. 4 the activity is 30% lower. A~nity constants. Under the conditions of Assay I the system is saturated with concentrations of ADP of the order of 2.5" Io-3M and concentrations of MgC12 between I and 2.Io-3M. The corresponding half saturation concentrations were 2.5"IO -4M and 5"Io-4M, respectively. Much higher concentrations of nucleoside diphosphates are required to saturate the system in the case of Assay 2. Thus, saturating concentrations of ADP and IDP were of the order of I o - l M and 5" Io-*M, respectively. The half saturating concentrations were 2.7.Io-2M and Io-*M, respectively. The concentrations of MgC12 required to saturate the system of Assay 2 were about the same as those in Assay I, namely between I and 2. Io-SM; the half saturation concentration for MgC1, was 4"I-*M. Whereas small variations in the ratio of the concentration of nucleoside diphosphate to that of orthophosphate do not appear to influence significantly the reaction rate in the case of Assay 2, in that of Assay I the rate of incorporation of orthophosphate is markedly dependent on the above concentration ratio. In this case, the optimum concentration ratio of nucleoside diphosphate to orthophosphate was o.7 for ADP and I.O for IDP. The reaction rates decrease rather sharply for values of the concentration ratio above or below the optimum.
T A B L E IV
EFFECT OF INHIBITORS

Addition to assay system

Per cent inhibition Assay z Assay 2

Test substance

Concentralio n (molar)

P o t a s s i u m arsenate Potassium arsenate P o t a s s i u m fluoride p-chloromercuribenzoate z,4-dinitrophenol

AMP

.4 o.oi 0.o 4 o.oool 5 0.00o 5

o.oi

7 44 13 27 o
IO

o o o 18
o

?~TP Sodium p y r o p h o s p h a t e

o.002 o.oi

xo 18

E n z y m e of specific activity 16 with 0.037 mg of protein (0.6 Unit) used for the first five experiments. E n z y m e of specific activity 12 with 0.048 mg of protein (0.58 unit) used for the last three experiments. Conditions of s t a n d a r d assays.

Re/erences p. 285.

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277

Inhibitors. As s h o w n in T a b l e IV, t h e e n z y m e is r e l a t i v e l y i n s e n s i t i v e to poisons b e i n g e s s e n t i a l l y u n a f f e c t e d b y fluoride a n d b y low c o n c e n t r a t i o n s of a r s e n a t e . H i g h e r c o n c e n t r a t i o n s of t h e l a t t e r (o.oi M ) i n h i b i t e d 4 4 % t h e i n c o r p o r a t i o n r e a c t i o n , a l t h o u g h t h e y h a d no effect on t h e l i b e r a t i o n of o r t h o p h o s p h a t e , p - C h l o r o m e r c u r i b e n z o a t e h a d r e l a t i v e l y l i t t l e effect; h e n c e , S H g r o u p s do n o t s e e m to be e s s e n t i a l for t h e a c t i v i t y of t h e e n z y m e . T h e i n c o r p o r a t i o n r e a c t i o n (Assay i) w a s r e l a t i v e l y l i t t l e a f f e c t e d b y t h e p r e s e n c e of A M P , A T P or p y r o p h o s p h a t e . On t h e w h o l e t h i s r e a c t i o n was m o r e s e n s i t i v e to i n h i b i t o r s t h a n t h e l i b e r a t i o n of o r t h o p h o s p h a t e . Synthesis o/polynucleotides
T h e i n c o r p o r a t i o n of o r t h o p h o s p h a t e i n t o n u c l e o s i d e d i p h o s p h a t e s a n d t h e l i b e r a t i o n of p h o s p h a t e t h e r e f r o m o c c u r n o t o n l y w i t h t h e d i p h o s p h a t e s so far c o n s i d e r e d , n a m e l y A D P a n d I D P , b u t also w i t h a n y of t h e o t h e r d i p h o s p h a t e s s u c h as G D P , U D P , a n d C D P , or w i t h m i x t u r e s of t w o or m o r e n u c l e o s i d e d i p h o s p h a t e s . T a b l e V i l l u s t r a t e s t h e i n c o r p o r a t i o n of a * P - o r t h o p h o s p h a t e , m e a s u r e d as d e s c r i b e d for A s s a y I, TABLE V
$ 2 p ' * E X C H A N G E " W I T H N U C L E O S I D E D I P H O S P H A T E S SINGLY AND MIXED

t'P incorporated Nucleosidediphosp~ pmoles Expt. r

c.p.r~,

Expt. 2

c.p.m.

ADP, 2.0 GDP, i.o UDP, 4.0 CDP, 2.o ADP + GDP + UDP + CDP + ADP + GDP + UDP + CDP**

I6,6oo 8,000 22,500 xo,2oo 1,27o

1o8,ooo 29,000 I42,3oo 79,6oo 5,80o i3,6oo

The samples contained the following components (in pmoles per ml), Tris buffer, pH 8.0, ioo; MgCls, 5 ; potassium phosphate (pH 8.o) with 95,0o0 c.p.m. 3~p in experiment i and 690,00o c.p.m. a2p in experiment 2, 2; enzyme of specific activity 18 with 0.o63 mg of protein (1.12 unffs) in experiment I and of specific activity 14 with o.o46 mg of protein (0. 7 unit) in experiment 2; and nucleoside diphosphates as indicated. Other conditions as in standard assay I. * Each in same amount as when tried singly. * * Each in half the amount as when tried singly. Fig. 4. Enzymic incorporation of 32p-orthophosphate in nucleoside diphosphates singly and in combination. Conditions as in phosphate incorporation experiments of Table V. Paper chromatography by the method of KREBS AND HEMSs. (A) Strip to the left, U.V. print of markers of GDP (G), UDP (U), CDP (C), and ADP (A). GDP and UDP are not separated by the isobutyric acid-ammonia solvent system ; strip to the right, autoradiograms of separate experiments with single nucleoside diphosphates. (B) Strip to the right, autoradiogram of experiment with simultaneous incubation of all four nucleoside diphosphates ; strip to the left, U.V. print of simultaneous incubation experiment (center) and of markers of GDP and ADP or UDP and CDP, respectively, on either side.

PP

tPt e

Re/erences p. 285.

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VOL. 2 0

(1956)

in the presence of ADP, GDP, UDP, and CDP, or a mixture of the four nucleoside diphosphates. The autoradiograms of Fig. 4 show that the radioactive phosphate is incorporated into each of the diphosphates whether incubated singly or in combination. These results suggested that the enzyme is able to catalyze the synthesis of either "single" polynucleotides, i.e., polynucleotides containing a single basic unit, or " m i x e d " polynucleotides, i.e., polynucleotides containing two or more basic nucleotide units. As already reported ~ this was t~ confirmed b y subsequent work. 80 + GMP On incubation of purified polynucleotide phosphorylase with nucleo70 ~..~,~r +_.~ ~ _ . . - - - - - - - - = AGUC side diphosphates singly or in combiuMP nation, in the presence of Mg ++, there is a liberation of orthophosphate. The - 50 reaction reaches equilibrium and comes 40 to a standstill when 60 to 80% of the acid labile phosphate has been released 30 as orthophosphate. The time course of the reaction with ADP, IDP, GDP, ~0 U D P and CDP, or with mixtures of I0 GMP either A D P and UDP, or ADP, GDP, UDP, and CDP, is shown in Fig. 5. 0 r i l l I 'II I t 0 2 4 6 8 I0 25 26 Addition of more nucleoside diphosHOURS phate when the liberation of orthoFig, 5. Orthophosphate liberation during synthesis phosphate has stopped disturbs the of polymers. The reaction mixtures contained Tris buffer, pH 8.z, 5o, and MgC12, zo/+moles per ml. equilibrium and causes further libeFor "single" polymers 50 mg per ml were used ration of phosphate until a new equiof each of the following nucleoside diphosphates, ADP, IDP, UDP and CDP, and 25 mg per ml of librium position is reached. The polyGDP. The sample for A-U polymer contained z 5 mg nucleotides which accumulate in tile of each ADP and UDP per ml; that for A-G--U-C polymer contained 7 mg per ml of each ADP, UDP reaction mixtures are usually isolated and CDP, and 3.5 nag per ml of GDP. The sam- b y precipitation with cold ethanol and ples for GMP and A-G-U-C polymers contained, purified b y solution in a small volume per ml, 2.5 mg of enzyme of specific activity 9.5 (e 4 units) ; all others contained o.5 mg of the same of water, reprecipitation with ethanol, enzyme (4.75 units). A drop of toluene was added solution, and exhaustive dialysis to each sample as a bacteriostat. Final volume, against distilled water. They are then o.5 ml. Incubation with shaking at 3o. o,oz ml aliquots were removed as indicated for orthophos- recovered from the dialyzed solution phate determination. The ordinate gives the b y lyophilization, and obtained in the percentage of acid-labile phosphate released as form of white powders. orthophosphate. It m a y be seen in Fig. 5 that all nucleoside diphosphates except G D P react readily. In the case of G D P the reaction is much slower and often comes to a standstill when a small fraction of the diphosphate has reacted. A precipitate, containing both polynucleotide and protein, is frequently obtained at this point. Addition of more enzyme brings about further reaction which, however, soon stops. So far, we have only been able to obtain small amounts of the GMP polynucleotide. It is because of this behavior th~/t G D P is used at lower concentration t h a n other nucleoside diphosphates and that much more enzyme is utilized for the preparation of polymers containing GMP. Preparation and isolation o/polymers. Single polymers have been prepared from

Re/erences p. 285.

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A D P , I D P , G D P , U D P , o r C D P , a n d are r e f e r r e d to as t h e A M P , I M P , G M P , U M P , or C M P p o l y m e r s , r e s p e c t i v e l y . T o date, t w o " m i x e d " p o l y m e r s h a v e b e e n p r e p a r e d , one f r o m a p p r o x i m a t e l y e q u i m o l a r m i x t u r e s of A D P a n d U D P a n d one f r o m m i x t u r e s of A D P , G D P , U D P , a n d C D P , in a p p r o x i m a t e m o l a r p r o p o r t i o n , 1 : o . 5 : 1 : 1 ; t h e s e p o l y m e r s are r e f e r r e d t o as t h e A - U , a n d t h e A - G - U - C p o l y m e r s , r e s p e c t i v e l y . T h e c o m p o s i t i o n of t h e s a m p l e s p e r ml of t h e r e a c t i o n m i x t u r e is, in general, as i n d i c a t e d in t h e l e g e n d to Fig. 5. S i n c e t h e i n c u b a t i o n p e r i o d s are of 24 h o u r s or longer, a s m a l l a m o u n t of t o l u e n e is a d d e d to t h e r e a c t i o n m i x t u r e s to p r e v e n t b a c t e r i a l g r o w t h . T h e p o l y m e r s are i s o l a t e d as follows: Any precipitate which may be present in the samples at the end of the incubation is removed by c_entrifugation at low temperature and low speed. The clear solution is then cooled to o% two volumes of ice-cold absolute ethanol are added, and the mixture is allowed to stand at o for one hour. The precipitated polymer is centrifuged off for io minutes at I2,OOO g and o; the supernatant fluid is decanted and the precipitate is dissolved in a minimum amount of distilled water. The polymer is reprecipitated once with two volumes of ice-cold absolute ethanol and the precipitate collected and dissolved in water as above. Any insoluble material at this point is removed by centrifugation and discarded. The clear, colorless polymer solution is dialyzed against several changes of distilled water in the cold room (about 5 ~) for 48 hours and the dialyzed solution dried from the frozen state. D e t a i l e d i n f o r m a t i o n on i n d i v i d u a l p r e p a r a t i o n s , i n c l u d i n g c o m p o s i t i o n of t h e r e a c t i o n m i x t u r e s , t h e e x t e n t to w h i c h t h e r e a c t i o n p r o c e e d e d as j u d g e d f r o m t h e l i b e r a t i o n of o r t h o p h o s p h a t e , t h e yield of l y o p h i l i z e d m a t e r i a l a n d t h e p e r c e n t a g e r e c o v e r y as p o l y n u c l e o t i d e of t h e d i p h o s p h a t e s d i s a p p e a r i n g , are r e c o r d e d in T a b l e VI. I t will be n o t e d t h a t in o r d e r to a v o i d d e g r a d a t i o n of t h e p o l y m e r s b y h e a t or acid, p r o t e i n is n o t r e m o v e d p r i o r to t h e p r e c i p i t a t i o n of t h e p o l y n u c l e o t i d e s w i t h alcohol. P r o t e i n d e t e r m i n a t i o n s in t h e s o l u t i o n s of p o l y m e r s a f t e r t h e s e c o n d a l c o h o l p r e c i p i t a t i o n i n d i c a t e d t h a t a b o u t 7 0 % of t h e p r o t e i n h a d b e e n r e m o v e d . T h u s , t h e TABLE VI
DATA ON

SYNTHESIS

OF VARIOUS

POLYMERS

Reaction mixture
No. Nucleo$ide diphosphate mg Tris buffer p H 8.x I~mole* MgClt #*mole* Emyme units Total vol. ml

Extent o/reaction* per cent

Yield o/ lyophilized material mg

Recovery** as polynucleotide per cent

i 2 3 4 5 6 7 8

ADP, iooo UDP, 45 UDP, 200 ADP, i8o UDP***, I8o ADP, IOO UDP***, I O O IDP, 200 CDP, 15o ADP, 200 GDP, IOO UDP, 20o CDP, 2oo

iooo 45 zoo 360 200 200 15o 14oo

200 90 4 72 4 4 3o 280

80 4 20 3z 19 19 16 725

2o 9 4 7 4 4 3 28

60 4 59 46 44 80 7 64

356 75 43 88 62 82 4 i54

82 65 56 87 9O-lOO 80 63 64

Incubation with shaking at 30% 40 to 46 hours. * As determined from orthophosphate liberation. ** Calculated for composition of nucleoside diphosphates as follows: ADP, N a H t A D P . 2 H 2 0 GDP, Na3GDP. 3HzO; UDP, Na3UDP. 3H~O; CDP, Na3CDP. 4H~O; IDP, Na3IDP.4.4H~O. *** Correction was made for an inorganic impurity present in this UDP sample.
Re#venees p. ~8 5.

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a m o u n t of protein rem a in in g a n d c o n t a m i n a t i n g the p o l y m e r preparations is insignificant, except in the case of th e AGUC p o l y m e r in which the a m o u n t of e n z y m e used was m u c h larger t h a n in the others.
Phosphorolysis

T he reaction c a t a l y z e d by polynucleotide phosphorylase is reversible. In the presence of t h e enzyme, o r t h o p h o s p h a t e and Mg ++, t h e biosynthetic polynucleotides undergo phosphorolysis to yield the corresponding 5'-nucleoside diphosphates. There is no reaction in t h e absence of orthophosphate. Q u a n t i t a t i v e d a t a on the stoichiom e r r y of the reaction with I D P in b o t h directions h a v e been presented previously 1. We h a v e as yet no accurate d a t a on the position of the equilibrium. In the direction of p o l y n u c l e o t i d e synthesis and u n d e r our usual e x p e r i m e n t a l conditions (pH 8.1, 3o), the reaction comes to a standstill when the ratio of the c o n c e n t r a t i o n of orthop h o s p h a t e to t h a t of nucleoside d ip h o s p h a t e is from 1.5 to 2. This is t h e case with A D P or I D P . U n d e r these conditions the reaction favors polynucleotide synthesis, as would be e x p e c t e d from the fact t h a t the p y r o p h o s p h a t e bonds of nucleoside diphosphates are c o n v e r t e d to the phosphodiester bonds of the polynucleotide. TABLE VII
PHOSPHOROLYSIS OF B I O S Y N T H E T I C POLYMERS AND NUCLEIC ACIDS ~, p O rl hophosphate incorporated disappearance c.p.m, pmoles

Experiment No.

Polymer

Source ol polymer

sap added c.p.m. 1"o-a

Enzyme units*

AMP RNA AMP + RNA AMP RNA RNA + IDt'ase AMP + RNA AMP UMP A-U IMP RNA RNA followed by IDPase IMI' + RNA AMP RNA AMP + RNA RNA AM P RNA RNA RNA

Synthetic
. t. vinelandii

3.65

9.3

1,38o,ooo
I 6 , OOO

2.75
O

320,000 Synthetic
A. vinelandii

~.85 1.5
0 o

1.68

9-3

86o,ooo
25,600 I 1,600

-'76,00o Synthetic Synthetic Synthetic Synthetic

,4. vinelandii

o.40 o.8o o.7o

0.58

o.3i

4.6

7i,ooo

85,ooo
28,000

0.35

6.2

82,000

7,600 3,850 60,500 0.55

6.2 200,000

0.50
I . IO

Synthetic
E. coli Str. pyogenes

J 1,7oo
I OO, OOO I 1,7OO

o
O

Synthetic Yeast Ox liver Wheat germ

o.82

4.6

380,000 I8,8oo 9,300 5,800

Samples contained (in pmoles per ml), Tris buffer, pH 8.I in experiments I to 3, pH 7.o in experiment:s 4 to 6, ioo; MgC1v 5; potassium phosphate, 7 to 9 with a2p as iudicated; polymer, 0. 5 to 3.0 rag: and enzyme (specific activity 15 to 2o) as indicated. Final volume, i. 5 ml. Incubation oo minutes at 3o. When two different polymers were present, equal amounts of each were added. Values expressed per ml of reaction mixture.
'~ . k s s a y I .

l,'c/eremes p. 25'5.

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Phosphorolysis of "single" polymers can be readily detected and measured by the disappearance of orthophosphate. The biosynthetic A-U and A-G-U-C polymers are also phosphorolyzed although less readily than "single" polymers. Moreover, the enzyme can catalyze the phosphorolysis of natural RNA from Azotobacter v i n d a n d i i as well as from other sources. However, with the exception of turnip yellow mosaic virus RNA*, nucleic acids, including that from Azotobacter, do not seem to be phosphorolyzed easily. Under these conditions one may be unable to detect disappearance of orthophosphate. Nevertheless, the occurrence of phosphorolysis can be readily detected by the use of 3*P-orthophosphate which is incorporated into the nucleoside diphosphates, and these can be identified chromatographically and autoradiographically. Calf thymus DNA and a sample of yeast RNA "core" i.e., the fraction of RNA which following exhaustive digestion with pancreatic ribonuclease is non-dialyzable against distilled water, were not attacked ~. Thus, as far as phosphorolysis is concerned, the Azotobacter polynucleotide phosphorylase does not appear to be specific for A z o b a c t e r RNA. Table VII shows data on the phosphorolysis of biosynthetic polymers and natural ribonucleic acids by the Azotobacter enzyme. Both the incorporation of labeled phosphate (determined as described for Assay I) and the disappearance of orthophosphate were measured in most cases. Only in the case of biosynthetic polynucleotides was there a measurable disappearance of orthophosphate although incorporation of labeled phosphate occurred throughout. It may further be Fig. 6. t'hosphorolysis of RNA. Samseen (Experiments I, 2, 4 and 5) that RNA inhiples of R NAfrom A zo/obacter v i ~ elan d i i (first two autoradiograms from left), bited to a greater or lesser extent the phosphorStreptococcus pyogenes and Escherichia olysis of the AMP or the IMP polymer. Attention coli (last two autoradiograms) were used. Aliquots of the corresponding is called to the RNA phosphorolysis of Experisamples of experiments 4 and 5 of ments 2 and 4 in which a separate aliquot Table VII, following deproteinization, contained, or was subsequently incubated with, were chromatographed on paper by ~ inosine diphosphatase (IDPase). This enzymO e the method of KREBSANDHE,~tS and autoradiograms obtained. The center catalyzes the hydrolysis of IDP, GDP and UDP strip is the sketch of a chromatographic to the corresponding 5'-nucleoside monophospha- strip with markers (fromtop to bottom) of GDP, UDP, CDP and ADP. The tes but is inactive on ADP and CDP. Where second strip from left was derived from IDPase was used, the radioactivity incorporated a sample incubated with IDPase (see text) before chromatography. was halved indicating that the enzyme hydrolyzed the GDP and UDP (produced by phosphorolysis) with release of the terminal radioactive phosphate as orthophosphate. This conclusion is supported by the autoradiograms of Fig. 6. Comparison with the markers shown on the central strip suggests that the two upper spots on the first strip correspond to GDP and UDP, whereas the two lowest spots on this strip correspond to CDP * L. A. HEPPELand J. D. SMITH, unpublished experiments.
Re/erences p. 285.

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and ADP. The middle spot is probably labeled ATP formed from labeled ADP by adenylic kinase contaminating the Azotobacter enzyme preparation. It may be seen (second strip from left) that the two upper spots largely disappeared from the sample treated with IDPase following incubation with RNA. The last two strips in Fig. 5 suggest the formation of radioactive GDP, UDP, CDP and ADP by phosphorolysis of RNA from Escherichia coli or Streptococcus pyogenes. They correspond to two of the samples of Experiment 5 in Table VII.
DISCUSSION

The results of work already published1, 2 and of further work to be reported in detail in subsequent papers of this series, show that the polynucleotides synthesized by polynucleotide phosphorylase consist of 5'-nucleoside monophosphate units linked to one another through 5'-3'-phosphoribose diester bonds. The chains are terminated by a phosphate group esterified at carbon 5' of the nucleoside moiety'. The biosynthetic polynucleotides of Table VI have average chain lengths ranging from 30 for the A-G-U-C polymer to 230 for the AMP polymer'. Ultracentrifugal studies by Dr. R. C. WARNER in this laboratory suggest average molecular weights ranging from 7o,ooo to 350,000 for these polynucleotides. Higher values, namely 570,000 and 800,000 for an AMP and an IMP polymer, respectively, were obtained by the method of light scattering'*. However, during the preparation of the latter polymers the equilibrium was shifted at various time intervals by supplementary additions of nucleoside diphosphate to promote further synthesis. It is possible that polymers of larger size are obtained by multiple additions of nucleoside diphosphates. Some of the biosynthetic polymers can be stretched into fibres1~ from which Dr. ALEXANDERRICH has obtained X-ray diffraction patterns suggesting a high degree of orientation***. "Single" polymers, such as the AMP polynucleotide, give X-ray diffraction patterns which are very similar, although not identical to those given by natural RNA. On the other hand, biosynthetic polynucleotides containing both purine and pyrimidine bases, such as the A-U and the A-G-U-C polymer, give patterns virtually identical to those of natural RNA. Thus, the information at present available on structure, base ratios, size, X-ray diffraction patterns, and behavior toward different enzymes~ indicates that the "mixed" polynucleotides synthesized by polynucleotide phosphorylase from 5'-nucleoside diphosphates are closely related to RNA. Indeed, the A-G-U-C polymer, i.e., the one containing adenylic, guanylic, uridylic and cytidylic acid residues, appears to be indistinguishable from natural RNA. It cannot as yet be decided whether the reaction catalyzed by polynucleotide phosphorylase represents a general biological mechanism for the synthesis of ribonucleic acids. Wide distribution of the enzyme would favor such a possibility. Recent work indicates that the distribution of the enzyme is rather wide. It has been found in extracts of a number of bacterial cells, irrespective of whether the organisms are aerobic or anaerobic, gram positive or gram negative. The enzyme has also been found in yeast extracts and in extracts of green plants (spinach). However, no unequivocal evidence has yet been obtained for the presence of the enzyme in extracts
* J. D. SMITH a n d L. A. HEPPEL, u n p u b l i s h e d e n d - g r o u p assays. * * L. F. CAVALIERI a n d M. ROSOFF, u n p u b l i s h e d e x p e r i m e n t s . *** A. RICH, u n p u b l i s h e d e x p e r i m e n t s . D. O. BRUMMOND, M. STAEHELIN a n d M. CrRUNB)~RG-MANAGO,u n p u b l i s h e d e x p e r i m e n t s ,

Re[erencesp. 285.

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of animal tissues. It may be that polynucleotide phosphorylase is present in all cells although in widely different amounts. Larger amounts would be expected in cells which have high rates of proliferation. Polynucleotide phosphorylase may prove useful for gaining further knowledge of the chemistry and the physico-chernical properties of nucleic acids as well as in studies on the mechanism of action and specificity of nucleases. Since the enzyme can catalyze the synthesis of polynucleotide chains containing either a single rnononucleotide or two or more different nucleotides (some of which may not occur naturally in nucleic acids), a number of non-naturally occurring polynucleotides can be obtained for studies of various kinds. It has been found', for example, that "single" polynucleotides containing only adenylic or guanylic acid rapidly interact in aqueous solution to form a new polymer of much higher molecular weight than that of each of the initial reactants.This polymer migrates as a homogeneous compound in an electric field. Little is as yet known of the mechanism of action of polynucleotide phosphorylase. Studies aimed at elucidating this point may have to await further purification of the enzyme. The question of whether the enzyme, in analogy with muscle phosphorylase, can only add nucleotide units to a pre-existing polynucleotide chain, i.e., whether the enzyme requires a primer, or whether it can build a polynucleotide chain starting only from a mixture of nucleoside diphosphates is of obvious interest. No definite answer can yet be given to this question. In unpublished experiments, Dr. L. A. HEPPEL has so far failed to find evidence for accumulation of small polynucleotides on brief incubation of the enzyme with nucleoside diphosphates. The possibility that a nucleoside monophosphate, for example adenosine-5' rnonophosphate, reacts initially with a nucleoside diphosphate, such as uridine-5'-diphosphate, to give a dinucleotide has been eliminated by Dr. HEPPEL by end-group assays of the polynucleotide synthesized. Azotobac~r polynucleotide phosphorylase shows considerable lack of specificity since it can act on single nucleoside diphosphates, whether.naturally or non-naturally occurring, or on mixtures of two or more of these. It also lacks specificity in the direction of phosphorolysis as it can attack ribonucleic acids from various sources. However the possibility that, in the presence of adequate concentrations of the four naturally occurring nucleoside diphosphates, the enzyme could synthesize a polynucleotide chain with a determined sequence of nucleotides, i.e., a specific RNA, is indeed intriguing. It is conceivable that RNA bound on the enzyme might act as a prosthetic group and a template for the reproduction of its own nucleotide pattern. Answers to this question might be obtained through studies now under way of the polynucleotides synthesized by phosphorylases from different sources. Finally, an interesting corollary of the reversibility of the reaction catalyzed by polynucleotide phosphorylase is that the free energy of hydrolysis of the phosphodiester bonds in polynucleotides may not be much lower than that of the pyrophosphate bonds of nucleoside diphosphates.
METHODS AND PREPARATIONS The analytical methods used have been described or referred to in the experimental sections. For the determinations of radioactivity, aliquots of deproteinized reaction mixtures, before and R. C. WARNBR, unpublished experiments.

Rs/erences p. 285.

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after removal of orthophosphate, were counted directly as liquid samples with use of a thinwindow-Geiger-Mtiller counter. Since the radioactivity of the orthophosphate was determined in every experiment under exactly the same conditions as t h a t of other experimental samples, no corrections were applied for self-absorption or decay. Azotobacter RNA was prepared from the residue (particles) obtained by high speed centrifugation of the bacterial extract during the first step of isolation of polynucleotide phosphorylase. The material was kept frozen until used. 1oo g of residue were extracted in a Waring blendor for 15 minutes at 3-4 with IO volumes of o . I 4 M sodium chloride. The mixture was centrifuged at 3-4 at high speed in a Servall angle centrifuge (20,o00 g). The sediment was re-extracted as above with 2 volumes of o. 14 M sodium chloride and the extracts were combined. The combined extracts were heated for I hour at 85 and the protein precipitate was removed by centrifugation after cooling. To the supernatant fluid was added 4 % triehloroacetic acid to give a final concentration of 5 %- The precipitate was collected by centrifugation, stirred in o. I M Tris buffer, pH 7.35, and the insoluble residue centrifuged off and discarded. The supernatant fluid was treated once more with trichloroacetic acid as above. The precipitated nucleic acid was dissolved in Tris buffer, pH 7.35, leaving no insoluble residue, and the solution was dialyzed with stirring against distilled water at o for 48 hours. Based on U.V. absorption at wave-length 26o mp, the yield of nucleic acid at this point was 39.0 mg. The solution was adjusted to pH 7.0 and the nucleic acid was precipitated with 3 volumes of ethanol. The precipitate was collected by centrifugation, washed twice with ethanol and once with ether, and dried in vacuum over P205. An aqueous solution of tlle powder after dialysis contained 26 % DNA as determined by the DlSCHE reaction TM. Before use for the experiments of Table VII, the solution of nucleic acid was incubated for i hour at 30 with an excess of crystalline deoxyribonuclease, in the presence of MgCI 2, and dialyzed against water. The DNA content was reduced to i8 %. ATI' and ADP were obtained commercially from the Sigma Chemical Co. and the Pabst Laboratories. The crystalline disodium salt of ATP was obtained from the Sigma Chemical Laboratories. W e a r e d e e p l y i n d e b t e d t o M r DAN BROIDA, S i g m a C h e m i c a l Co., St. L o u i s , Mo., for g e n e r o u s g i f t s of l a r g e a m o u n t s of U D P , G D P , I D P a n d o t h e r n u c l e o t i d e s . W e a r e also m u c h i n d e b t e d t o D r . ALEXANDER FRIEDEN a n d Dr. SAMUEL A. MORELL, P a b s t L a b o r a t o r i e s , M i l w a u k e e , Wisc., for g e n e r o u s g i f t s of U D P a n d C D P . T h e p r e s e n t work would not have been possible without the above help. Our thanks are due to Dr. A. W. BERNHEIMER, D e p t . of M i c r o b i o l o g y , N e w Y o r k U n i v e r s i t y College of M e d i c i n e , for s a m p l e s of b a c t e r i a l , w h e a t g e r m a n d o x l i v e r R N A , t o Dr. J . R. FRESCO D e p t . of P h a r m a c o l o g y , N e w Y o r k U n i v e r s i t y College of M e d i c i n e , for h e l p w i t h t h e p r e p a r a t i o n of Azotobacter R N A a n d for s a m p l e s of y e a s t R N A a n d c a l f t h y m u s D N A , a n d t o Dr. L. A. HEPPEL, N a t i o n a l I n s t i t u t e s of H e a l t h , B e t h e s d a , Md., for a s a m p l e of y e a s t R N A " c o r e " . I D P a s e w a s k i n d l y s u p p l i e d b y Dr. G. W : E . PLAUT. W e a r e i n d e b t e d t o Dr. M. KUNITZ R o c k e f e l l e r I n s t i t u t e for M e d i c a l R e s e a r c h , N e w Y o r k , N . Y . , for a gift of c r y s t a l l i n e y e a s t h e x o k i n a s e .

SUMMARY The isolation, partial purification and some properties of polynucleotide phosphorylase of Azotobacter vinelandii are described. The enzyme catalyzes the synthesis of highly polymerized ribonucleic acidqike polynucleotides from 5'-nucleoside diphosphates with release of orthophosphate. The reaction requires magnesium ions and is reversible. Thus, the enzyme also catalyzes the phosphorolysis of polynucleotides to yield the corresponding 5'-nucleoside diphosphates. The preparation and isolation of a number of polynucleotides containing one or several kinds of mononucleotide units is described. The scope, mechanism and significance of the reaction are discussed. Re#fences p. 285.

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ENZYMIC SYNTHESIS OF POLYNUC;LEOTIDES I REFERENCES

285

x M. GRUNB,~nG-MANAGO AND S. OCHOA, J. Am. Chem. Sot., 77 (1955) 3165 s M . GRUNBERG-MANAGO, P. J. OaTxz AND S. OCHOA, Science, 122 (1955) 907. 3 H. SCHMITZ, R. B. HURLBERT AND V. R. POTTEn, J. Biol. Chem., 209 (I954) 41. 4 V. R. POTTER, Canadian Cancer Conlerence (R. W. BEGG, editor), Vol. I, New York, I955, P. 290. 5 M. GRUNBERG-MANAGO AND S. OCHOA, Federation Proc., 14 (1955) 22I. 6 H. M. KALCKAR, J. Biol. Chem., 148 (I943) I27. W. E. COHN AND C. E. CARTER, J. Am. Chem. Soc., 72 (195 o) 4273 s H. A. KREBS AND R. HEMS, Biochem. Biophys. Acta, 12 (1953) 1729 A. G. GORNALL, C. J. BARDAWlLL AND M. M. DAVID, J. Biol. Chem., I77 (I949) 751. 10 O. WARBURG AND W. CHRISTIAN, Biochem. Z., 31o (I94 I) 384 11 I. A. ROSE AND S. OCHOA, J. Biol. Chem.,.(in press). 12 I. BERENELUM AND E. CHAIN, Biochem. J., 32 (1938) 295. 1, K. LOHMANN AND L. JENDEASSlK, Biochem. Z., 178 (I926) 419 14 L. A. HYNDMAN, R. H. BURRIS AND P. W. WlLSOS, J. Bacteriol., 65 (1953) 522. 16 D. KEILIN AND E. F. HARTEEE, PrOC. Roy. Soc. (London), B, 124 (1938) 397. 16 G. W. E. PLAUT, J. Biol. Chem., 217 (1955) 235. 1~ A. RICH AND J. D. WATSON, Proc. Natl. Acad. Sci. U.S., 4 (1954) 759. is Z. DlSCHE, Mihrochemie, 8 (193 o) 4. R e c e i v e d D e c e m b e r 3 o t h , 1955