HE JOURNAL or BroLogicaL CHEMISTRY Vol. 235, No. 3, March 1960 Printed in U.S.A. The Polymerization of Guanosine Diphosphate by Polynucleotide Phosphorylase Maxine F. Sineer,* Russet J. Hitmor, anp Leon A. Hepprrer lrom the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service, Bethesda, Maryland (Received for publication, September 9, 1959) Polynucleotide phosphorylase catalyzes the reversible poly- erization of nucleoside diphosphates (1). With enzyme prep- rations from Escherichia colt (2), Azotobacter agile (3), and Mi- ococcus lysodeikticus (4, 5) a reaction has been observed with ngle additions of adenosine, uridine, cytosine, or inosine diphos- hate to form the corresponding homopolymer. However, the blymerization of guanosine diphosphate represents a special ise. Littauer and Kornberg (2) observed no polymerization faction upon incubating GDP with fractions from EF. coli. In kperiments with polynucleotide phosphorylase from A. agile, runberg-Manago et al. (3) noted a very slow release of P; from DP, but this reaction stopped far short of the equilibrium point und for ADP and UDP. In contrast, when mixed with other ucleoside diphosphates, GDP is well utilized and polymers ich as poly AGUC! are formed (8). The experiments described below show that GDP, when pres- ht alone, is not polymerized even after many hours by enzyme ctions from A. agile or E. coli. However, polymerization of DP does take place in the presence of oligonucleotides such as pApA and ApApU, and guanosine monophosphate units are ded to the primer. No reaction occurs with GDP and oligo- heleotides, such as ApApUp, that do not contain an unsubsti- tted hydroxyl group at carbon 3’ of the terminal nucleoside tsidue. Highly purified fractions of A. agile polynucleotide hosphorylase, kindly supplied by Drs. Mii and Ochoa (6), cata- ze the polymerization of ADP, UDP, CDP, and IDP only af- fr a lag period. This lag can be overcome by ribonucleic acid hd certain other polymers (6), as well as by oligoribonucleotides various types, including pApApA, ApApU and ApApUp (see receding paper (7)). Accordingly, GDP differs from other nu- poside diphosphates in that its polymerization requires the esence of oligonucleotides of the type that can be incorporated }to the polymer. MATERIALS AND METHODS Most of the experimental procedures were the same as those the preceding paper (7) and only brief reference will be made them here. * Research Fellow of the National Institute of Arthritis and letabolic Diseases of the National Institutes of Health, United ates Public Health Service, 1956-1958. 1The abbreviations used are: EDTA, ethylenediaminetetra- etate. The biosynthetic polymers synthesized by the action polynucleotide phosphorylase are: poly A, polyadenylic acid; ly U, polyuridylic acid; poly C, polycytidylic acid; poly G, lyguanylic acid; poly AU, copolymer of adenylic and uridylic ids; poly AGUC, copolymer of adenylic, uridylic, guanylic, and tidylic acids. Rgpp is the ratio of the Rr of a given compound that of GDP. Materials—GDP was obtained from the Sigma Chemical Com- pany and samples were usually stored at 3° for several months before use. The preparations were found to contain up to 15% of 5’-GMP and up to 4% of GTP by quantitative paper chro- matography in Solvent 3 (see below) and the values stated for concentrations of GDP have been corrected. A sample of a chemically synthesized adenylic acid polymer (8) was kindly provided by Dr. A. M. Michelson, Arthur Guinness Son and Com- pany, Ltd., Chemist’s Laboratory, Dublin; it consists of short polynucleotide chains containing mixed 3’-5’ and 2’-5’ phospho- diester bridges and terminated by a cyclic 2’ ,3’-phosphoryl group. A dinucleoside monophosphate with a 2’-5’ phosphodiester bridge, namely adenylyl-(2’-5’)-uridine (9) was also a gift from Dr. Michelson. The preparation of polynucleotide phosphorylase from F. colt was a fraction carried through the first ethanol step in the pro- cedure of Littauer and Kornberg (2); its specific activity in the “exchange” assay (3) was 15. Three highly purified prepara- tions from A. agile (6) were given to us through the kindness of Drs. Mii and Ochoa; all of them were found to display a lag period in the polymerization of ADP and UDP which could be overcome by addition of a suitable primer. The fractions are designated by their specific activity in the exchange assay (3) as S.A. 70, S.A. 60, and 8.A. 350. Two fractions (S.A. 40 and 8.A. 160) that did not show well defined lag periods were also obtained from Drs. Mii and Ochoa. Snake venom phosphodiesterase was prepared by a modification of the procedure of Koerner and Sinsheimer (10). Phosphomonoesterase was fractionated from human seminal plasma (11). Methods—The solvent systems used for descending chroma- tography were: Solvent 1, isopropanol-water (70:30, volume for volume) with NH; in the vapor phase (12); Solvent 2, saturated ammonium sulfate-isopropanol-l m sodium acetate (80:2:18, volume for volume for volume) (13); Solvent 3, isobutyric acid-1 mM NH,OH-0.2 w EDTA! (100:60:0.8, volume for volume for volume) (14); Solvent 4, isopropanol, 170 ml, concentrated HCl, 44 ml, water up to 250 ml (15); Solvent 5, n-propanol-concen- trated NH,OH-water (60:30:10, volume for volume for volume) (16). Whatman No. 3MM paper was used with Solvents 1, 3, and 5, and Whatman No. 1 paper with Solvents 2 and 4. Inorganic phosphate was determined by the method of Fiske and SubbaRow (17), with the use of the Klett colorimeter with a No. 66 filter. The higher concentrations of oligonucleotide used in this study caused an interfering turbidity upon addition of am- monium molybdate. This difficulty was overcome and satis- factory assay of P; was obtained by the following procedure. An aliquot of the incubation mixture containing from 0.015 to 0.060 751 752 > TTT = ler 6 | _ 5 i 10- 4 Sg ‘ 2 OQ 6b 3°54 2 i) a 4h } - — lid A me 2F 4 a. Po dd % 2 4 6 TIME IN HOURS Fre. 1. Polymerization of GDP as measured by formation of Pi; effect of primers. The incubation mixture (0.05 ml) contained 328 ug per ml of enzyme for Curve $ and 164 wg per ml for all of the other curves. The concentration of GDP was 6.8 mm for Curves 1, 2, 8, and 13.7 mm for Curves 4, 6, and 6. Primer addi- tions: Curve 1, 4.6 mm pApApA; Curve 2, 3.8 mm pApA; Curve 3, 1.3 mm pApA; Curve 4, 1.8 mm pApApA; Curve 5, 3.8 mm pApA; Curve 6,7.1 mm pApA. Aliquots of 0.01 ml were removed for P; analysis. In control experiments, without primer, the concen- tration of P; was <0.1 mm after as long as 20 hours. I | 0.006 M GDP PRIMER =0.004 M pApA > . | I Ss 164 oa + 7 bu ao -_ oF 4 oO nN ul o-L. t oO = Moye = 82 (no primer) 0 | 2 TIME (HOURS) Fig. 2. Polymerization of GDP with different amounts of en- zyme. The incubation mixtures contained the amounts of enzyme shown in the figure (ug per ml), and the following in mm: GDP, 6; pApA, 4.0. umole of P; is made up to 0.5 ml with cold perchloric acid whose strength is such that the final concentration is 2.5%. After 5 minutes at 0° the mixture is centrifuged and 0.3 ml of the super- natant fluid is mixed with 0.8 ml of 1 n H.SO,, 0.26 ml of water, 0.16 ml of 2.5% ammonium molybdate, and 0.08 ml of reducing reagent. The mixture is centrifuged for 5 minutes at 1,500 x g in the International No. 1 centrifuge. The clear, blue, super- Polymerization of GDP Vol. 235, No. 3 natant fluid is decanted carefully into a Klett tube and read in the instrument, together with appropriate phosphate standards and blanks, 10 minutes after addition of the reducing agent. EXPERIMENTAL AND RESULTS Effect of pApA and tts Homologues on Polymerization of GDP— There is no polymerization reaction when GDP alone is incubated with polynucleotide phosphorylase from A. agile. Thus, no re- lease of P; (<0.02 pmole) from GDP occurs in 4 hours, with amounts of enzyme which would form 10 to 20 umoles of P; from ADP per hour.2? Over 30 experiments were carried out with 6 different lots of enzyme, with 0.005 to 0.05 m GDP and incubation times that varied from 4 to 24 hours. In addition, study of the reaction mixture by paper chromatography in Sol- vents 1, 2, and 3 affords no evidence for the formation of poly- nucleotide material. However, in the presence of pApA, pA- pApA, or pApApApA one observes formation of P; at a rate that is nearly linear with time until equilibrium is approached. The equilibrium point is not well defined by the data but it ap- pears to correspond to the conversion of from 70 to 80% of GDP to P; and polynucleotide (Fig. 1). It can be seen from Fig. 2 that the initial rate of formation of P; is proportional to the concentration of enzyme, in the pres- ence of 6 mm GDP and 4 mm pApA. A similar rate was ob- tained at 13.7 and 30 mm GDP, suggesting that the enzyme is saturated with GDP at a concentration of less than 6 mm. Be- cause of insufficient material it was not possible to determine the concentrations of the various primers required to saturate the enzyme. Thus the rate of P; formation is 0.9 and 2.0 umoles per hour per ml of reaction mixture in the presence of 4.3 and 7.0 mm pApA, respectively (GDP, 13.7 mm; enzyme, S.A. 70, 82 wg per ml). The trinucleotide is effective at lower concen- trations; under the same conditions mentioned above 2.8 mm pApApA gives a rate of 3 wmoles of P; per hour per mJ. It should be noted that concentrations of-pApA and pApApA that do not saturate the GDP system are more than enough to give maximal stimulation of the rate of polymerization of ADP and UDP (7). The tetranucleotide, pApApApA, was tested at con- centrations of 2,1 mm, 0.74 mM, and 0.44 mm; these levels were found to be approximately equivalent to 2.9 mm, 0.97 mu, and 0.58 mm pApApA, respectively. Thus, the tetranucleotide is effective at somewhat lower concentrations than required for the trinucleotide. There is no detectable formation of P; when any of the enzyme fractions are incubated with pApA, pApApA, or pApApApA in the absence of GDP. Also, there is no reaction in the absence of enzyme. Similar results were obtained with the E. coli polynucleotide phosphorylase. Again, in the absence of pApA or pApApA, no material with an Rr of zero was detected on paper chroma- tograms nor were any oligonucleotides containing guanosine formed. The curve of P; formation plotted against time was also quite flat except for a small initial burst. A minor con- taminant of GDP preparations appears to be rapidly dephos- phorylated by the Z. colt enzyme but not by A. agile fractions. It might also be mentioned that a primer requirement for GDP polymerization could be demonstrated with cruder enzyme prep- * In the case of enzyme fractions that showed a lag phase in the polymerization of ADP this comparison is based on the rate achieved after the lag phase, or on the initial rate in the presence of saturating amounts of poly A. March 1960 hrations from A. agile, preparations that showed no primer re- juirement for the polymerization of ADP and UDP. Effect of Other Materials That Were Tested as Primers—Table i gives a list of nucleotides that did not induce polymerization of GDP. In addition, no effect was noted with an unknown ma- terial that cecurs in filter paper and can be eluted with water, lalthough in experiments with ADP or UDP a small but signifi- cant stimulation was obtained in this way (7). Three oligonucleotides containing no phosphomonoester end yme per ml. The results were as follows: (a) ApA, at 3.6 mm, resulted in a stimulation of P; release equivalent to that ob- tained with 4.2 mm pApA, (6) ApApU (2 mn) had exactly the same effect as pApApA (2 mm). ApApA was less effective, but the significance of this is doubtful, since only a single preparation was tested. Several lots of the other oligonucleotides were used. Two thymidine oligonucleotides, namely, pT pT and pTpTpT, were inactive as primers. Products Formed from Polymerization of GDP in Presence of pApApA—When GDP is incubated with polynucleotide phos- phorylase and pApApA for a brief interval the major reaction product is pApApApG. Oligonucleotides corresponding to the jaddition of 2 and 3 guanosine monophosphate residues also ap- pear, but in smaller amounts. More highly polymerized ma- terial, with an Rp of zero in various solvent systems, cannot be detected until later in the course of incubation. These observa- tions are illustrated in the ultraviolet photograph of a chromato- jgram showing components of a typical reaction mixture at dif- ferent time intervals (Fig. 3). : Evidence for the structure of pApApApG includes the follow- ing: The material moves as a single band in Solvents 1 and 3, and its 2» is consistent with the structure assigned. Hydrolysis Taste [ Compounds that failed to stimulate polymerization of guanosine diphosphate The incubation mixtures contained the following, in smoles per ml: Tris buffer, pH 8.2, 150; MgCle, 10; EDTA, 0.4; GDP 10 or 14; and other additions as indicated below. Incubation tempera- ture, 37°. Formation of P; was determined at three time inter- vals over a period of 4 hours, and the values were corrected for P; present at zero time. Purified A. agile polynucleotide phos- phorylase (8.A. 70) was used. Except where specifically indicated, the same incubation con- ditions and the same enzyme preparation were used in the other experiments reported. Compound Concentration iM ApUp.. 0.006000 cee cee eee 5.8 ApApUp 20.00.00. cere 0.8 Adenosine. 00.0006. cee ce es 5.0 AMP 2000 cette ees 6.6 Adenosine 5’-benzyl phosphate.............. . 5.0 Adenylyl-(2’-5’)-uridine. 2.0 ..000.0000000.00.4. 6.3 mefmt 0) gs 1.2 Poly AGUC. 0 eee eee 1.0 Poly Co.cc cn cee cee 3.0 Poly Uo eee eee 3.0 Polyadenylic acid, chemically prepared (8)... . 1.2 M. F. Singer, R. J. Hilmoe, and L. A. Heppet prow were tested with 13.7 mm GDP and 82 yg of 5.A. 70 en- 753 Fig. 3. Ultraviolet: photograph of a chromatogram run in Sol- vent 3. This is from an experiment similar to that in Fig. 4. At zero time (not shown) the only visible densities corresponded to pApApa, GMP, GDP and GTP. This photograph shows partial disappearance of pApApA as the incubation proceeds, as well as the formation of pApApApG, pApApApGpG, pApApApGpGpG, and polymer (visible at 63 minutes, near top of photograph). in 1 N HCl (18) followed by quantitative chromatography in Sol- vent 4 yielded adenine and guanine in a ratio of 3.2:1.0 (theory, 3.0:1.0). Hydrolysis in 0.8 n KOH (19), followed by chroma- a] qo ney _ fh © Th I Oo, © f 132] 8 pApApA Oo | |. pApAPAPG “ ~ 4 “Ng PAPApADGp Gp | he ode pi 0 2 4 6 8 TIME IN HOURS Fic. 4. Polymerization of GDP in the presence of pApApA. The incubation mixture (0.2 ml) contained 82 wg per ml of enzyme, and the following, in ymoles per ml: GDP, 13.7 and pApApA, 2. Aliquots of 0.03 ml were chromatographed in Solvent 3 and the various bands were quantitatively transferred and run in Solvent 1. Quantitative elution was again carried out and the concentra- tion of the various nucleotides was determined.? No polymer could be detected after 15 minutes but it was found in samples removed after 40 minutes of incubation. Thereafter the concen- tration of polymer increased progressively; it reached a value equivalent to 8 wmoles of base per ml in 6.5 hours, when it ac- counted for almost all of the GDP which disappeared. 04 CONCENTRATION IN JMOLES/ ML. oO f o> tography in Solvent 1 gave adenosine 3’ ,5’-diphosphate (mixed with 2’, 5’ isomer), 3/-AMP (mixed with 2’-AMP), and guano- sine, the expected products. No guanylic acid was formed. The results of partial hydrolysis by purified snake venom phos- phodiesterase were also informative. This fraction has been shown to cleave both oligodeoxyribonucleotides (20) and poly- ribonueleotides (21) in stepwise fashion, beginning at that end of the molecule bearing an unsubstituted hydroxyl group at car- bon 3’. From pApApApG one would expect 5’-GMP and pA- pApA to be the major early products, followed later by pApA and 5/-AMP. When enzymic hydrolysis of pApApApG was carried to the extent of 40% the following were found by quantitative elution from paper chromatograms: pApApA, 0.023 pmole; 5/- GMP, 0.020 umole; 5’-AMP, 0.010 umole; pApA, 0.002 umole. Evidence for the structure of pApApApGpG is the following: Tt moves as a single band in Solvents 1 and 3, with a lower Rr than the tetranucleotide just discussed. Acid hydrolysis yielded adenine and guanine in a ratio of 1.7:1.0 (theory, 1.5:1.0). Al- Kaline hydrolysis gave adenosine 3’,5’-diphosphate (and the 2’,5' isomer), 3’-AMP (and 2’-AMP), 3/-GMP (and 2’-GMP), and guanosine. . . The hexanucleotide, pApApApGpGpG, was obtained in small amounts. It was found to contain adenylic and guanylie acid residues but there was insufficient material for accurate quanti- * As in the preceding paper (7), the assumption is made that the molar extinction coefficient of an oligonucleotide is approxi- mated by the sum of the extinction coefficients of its constituent nucleotides. Values used for the molar extinction coefficients, at 257 mp and pH 2, for AMP and GMP are 15,100 and 12,200, re- spectively. The approximation does not account for any hypo- chromic effect. Polymerization of GDP Vol. 235, No. 3 tative analysis. signed. The following values for Rapp were obtained for pApApApG, pApApApGpG and pApApApGpGpG: in Solvent 1, 0.52, 0.26, and 0.10, respectively; in Solvent 3, 1.60, 1.17, and 0.49, re- spectively. The ratio of absorbancy (pH 2) at 280 my over that at 260 my was found to be 0.33 for pApApApG, 0.42 for pApA- pApGpG, and 0.58 for pApApApGpGpG. Structure of “poly G’’—This term is restricted to material that is precipitated by 2 volumes of ethanol, that is insoluble in cold 2.5% HC!lO,, and that remains at the origin upon chromatog- raphy in Solvents 1, 2, or 3. Alkaline hydrolysis of such ma- terial yields mostly 3‘-GMP and 2’-GMP, identified by their Rr values in Solvents 1 and 2, and by the fact that they were read- ily hydrolyzed by phosphomonoesterase purified from human seminal plasma but not by 5/-nucleotidase. Smaller amounts of adenine-containing nucleotides derived from the incorporated primer are also obtained. The following experiment illustrates the preparation of “poly G,” of short average chain length, formed by addition of guano- sine monophosphate units to pApApA. The incubation mixture contained 50 ug of EH. colt polynucleotide phosphorylase, 125 umoles of Tris buffer, pH 8.2, 10 umoles of MgCh, 0.4 umole of EDTA, 1.31 wmoles of pApApA, and 27.4 umoles of GDP, in a total volume of 1.0 ml. The mixture was incubated at 37° for 25.5 hours, with toluene added after 6 hours. The formation of P; amounted to 8 wmoles. A chromatogram run in Solvent 3 showed complete incorporation of pApApA and all of the reac- tion products had an Ry of zero. Two volumes of cold ethanol were added and after 3 hours at 2° the precipitate was collected by centrifugation and dissolved in distilled water. The solu- tion was dialyzed against 1 liter of cold 0.001 m EDTA for 24 hours and then against cold, running distilled water for 48 hours. The yield of polymer, based on measurement of optical density, was 56% of what could be expected from the amount of P; that had been formed. Paper chromatographic examination of the product after dialysis showed complete removal of GDP. Hy- drolysis of a sample of the dialysis residue in 1 n HCl (18) fol- lowed by chromatography in Solvent 4 gave a guanine-adenine ratio of 2.8:1, corresponding to an average chain length of 11.4. This indicates the addition of 8.4 guanylic acid residues, on the average, to every molecule of pApApA. The material was acid insoluble, and was nondialyzable. In later experiments, with the use of much larger amounts of enzyme, poly G that had an average chain length of 30 was syn- thesized. Further, with the use of pUpUpU as a primer and treating the resultant polymer with pancreatic ribonuclease, it was possible to remove uridylic acid from the preparation and so obtain poly G free of bases other than guanine. In a typical experiment the incubation mixture (0.5 ml) contained 27.4 uzmoles of GDP, 75 wmoles of Tris buffer, pH 8.2, 5 umoles of MgCl, 0.2 umole of EDTA, 0.9 umole of pUpUpU, and 320 yg of a gel eluate fraction from A. agile (S.A. 160). The mixture was kept at 37° for 6.5 hours. The polymer was precipitated by the addi- tion of 1.0 ml of cold 5% HClO, and collected by centrifugation. It was washed with 2 ml of 3% HClO, and then with two por- tions (2 ml each) of 0.01 n HCl. The precipitate was suspended in 1 ml of water and dissolved by addition of sufficient 1 n NH,OH to bring the pH to 7.0. An opalescent, distinctly vis- cous solution was obtained. An aliquot was hydrolyzed with 0.3 n KOH (19) and the products were separated in Solvent 5 and The structure is therefore only tentatively as- March 1960 ms w T | 6.8 X10-3M GDP + 4.1X (073M pApApA 7 PhO I Pi RELEASED (AMOLES/ML) oOo l | 0 | 2 3 4 TIME IN HOURS Fig. 5. Polymerization of GDP in the presence of pApApA. fhe incubation mixture (0.2 ml) contained 82 wg per ml of enzyme nd the following, in zmoles per ml: GDP, 6.8; pApApA, 4.1, amples of 0.03 ml were removed at intervals for chromatography h Solvent 3. In A, formation of P; plotted against time. In B, hanges in the concentration of GDP and various oligonucleotides uantitatively eluted. The products were: uridine 3’,5’- (and *,5’)-diphosphate, 0:05 umole; 3’ (and 2’)-GMP, 1.28 moles; ’ (and 2’)-UMP, 0.105 umole; guanosine, 0.045 umole. From he total amount of compounds containing guanosine isolated in his experiment the yield of poly G was calculated to be 50% of hat expected from P; release. The results are close to what one ould expect from a polymer with an average chain length of 0. This can be represented diagrammatically, with vertical otted lines showing the points of alkaline cleavage: Up: Up!Up?Gp!GpGpi ------------------ GpiG |. : : : : : _ 1 2 26 1 UDP UMP GMP guanosine ince no guanosine 3’, 5’-diphosphate was found, and guanosine yas equal to uridine diphosphate, it is evident that all of the olymer chains were built onto pUpUpU as a primer. The remainder of the preparation of poly G was shaken with volume of CHCI; and yy volume of isoamyl alcohol (22), and entrifuged. The aqueous layer was removed and re-extracted nh order to complete the removal of enzyme protein. The or- anic solvents were removed by extraction with diethyl ether, ter which the aqueous solution was aerated at 40°. The poly- her solution was then treated with 500 ug of pancreatic ribonu- lease for 4 hours, at 37°, in order to cleave off uridylic acid nits. The treatments with organic solvents and precipitation rith perchloric acid were repeated. Alkaline hydrolysis of the psultant polymer gave, as expected, only 3’ (and 2’)-GMP and tuanosine. From the ratio of GMP to guanosine the average hain length was found to be 24. The removal of 3 uridylic acid esidues from the original polymer, of chain length 30, would be xpected to result in an average chain length of 27. The dis- repancy is probably due to experimental error. , M. F. Singer, R. J, Hilmoe, and L. A. Heppel 755 n & Ww NM ao Ey | | 2. 3 4 TIME IN HOURS plotted against time (Curve A, GDP; Curve B, pApApA; Curve C, pApApApG; Curve D, pApApApGpG;; Curve E, pApApApGpGpG). Two other products were found, but only in the sample removed at 240 minutes, namely pApA (0.35 mm) and polymer (equivalent to 2.0 mm mononucleotide). CONCENTRATION. IN MOLES/ ML © © Time Course of Polymerization Reactions with Different Con- centrations of GDP and Primer—Fig. 4 shows the results of an experiment in which GDP and pApApA were incubated with the A. agile enzyme. At various time intervals, aliquots of the in- cubation mixture were removed and its components were deter- mined by quantitative chromatography. It is evident from Fig. 4 that most of the primer is utilized within 15 minutes and all of it disappears within several hours. The concentration of pApA- pApG rises to nearly 0.7 mm in 40 minutes and after 6 hours falls to 0.1 mm. The pentanucleotide, pApApApGpG, accumulates only in trace amounts (not shown), whereas the hexanucleotide reaches a level of 0.11 mm in 4.5 hours, No polymer is noted after 15 minutes but it can be found in an aliquot removed after 40 minutes and thereafter its concentration rises (see legend to Fig. 4). An experiment with 13.7 mm GDP and 1 mm pApApA gave similar results. An experiment was carried out under identical conditions ex- cept that the concentration of pApApA was increased to 4 mm. This led to a more rapid accumulation of pApApApG, so that its concentration was 1.6 mm after 20 minutes. Also there was a less rapid decline with time, compared with the experiment men- tioned above. A substantial amount of pApApApGpG was formed, as well as smaller amounts of pApApApGpGpG. No polymer was evident in a sample removed after 20 minutes but it was found after 1 hour, and thereafter increased with time of incubation. In this experiment pApApA was not completely utilized; in fact, its concentration showed a small rise between 1 and 6 hours. Possible explanations are considered below. The results of an experiment in which the concentration of GDP (6.8 mm) was brought close to that of pApApA (4.1 mm) are presented in Figs. 54 and B. The changes to be observed between 40 and 240 minutes illustrate several points. First, there is a substantial decrease in the concentration of pApA- pApG. Also, there is evidence of a continued forward reaction, 56 hamely: (a) decrease in GDP (—0.9 mm), (8) increase in P; +0.6 mm), and (c) appearance of polymer (equivalent to 2.0 mm GMP). There is also evidence for a net reaction in the re- rerse direction, occurring simultaneously. Thus, no pApA is resent after 20 and 40 minutes, but 0.35 mm* is noted after 240 minutes. This simultaneous phosphorolysis of pApApA and ontinued polymerization may explain why the decrease in GDP B greater than the net increase in P;. In similar experiments, except that more enzyme was present, t was found that the composition of the reaction mixture changed lo a considerable extent after the formation of P, and the utiliza- ion of GDP came toa halt. Disappearance of pApApApG and ApApApGpG was noted, together with an increase in the con- entration of polymer and of smaller oligonucleotides. These servations led us to incubate pApApA and pApApApA with olynucleotide phosphorylase in the absence of added nucleoside iphosphate or P;. A transnucleotidation reaction was found io occur, with the simultaneous formation of smaller and larger olynucleotides. This reaction does not appear to involve the articipation of ADP or P;, at least not in the free state. A de- failed study will be reported in a future publication. The incorporation of the dinucleotide, pApA, into poly G was pss extensively investigated, but here too it was found that oligo- ucleotides containing guanine accumulated early in the course f the reaction. At later time intervals these were observed to ecrease in concentration and material with an Rp of zero was lhe major product. DISCUSSION The present results help to explain why no polymerization eaction has been observed with GDP and polynucleotide phos- horylase in the past, even though guanosine monophosphate nits have been incorporated into polynucleotide chains when a hixture of nucleoside diphosphates was used. The data indicate mat a polynucleotide cannot be synthesized de novo from GDP; | is only possible for the enzyme to catalyze the addition of fuanosine monophosphate units to a preformed oligonucleotide rimer. These results were obtained with every sample of poly- ucleotide phosphorylase that was tested. Some of these en- lyme fractions showed no primer requirement at all with the ther nucleoside diphosphates. Thus, it should be emphasized hat these results differ from those reported in the preceding aper (7), where a lag period in the polymerization of ADP or 'TP is overcome by compounds such as pApApA or ApApUp. Vith GDP the requirement for an oligonucleotide of suitable tructure appears to be absolute; without it no reaction can be etected even after many hours. The primer must have an unsubstituted hydroxyl group at prbon 3’ of the terminal nucleoside residue in order for a 3-5’ hosphodiester bridge to be established. Thus, ApUp and Ap- .pUp are inactive. ridine as the terminal nucleoside residue were used in the pres- nt study. A suitable primer for the polymerization of GDP must have tt least one 3’,5’ phosphodiester bond. It is of considerable nterest that ApA is an effective primer, whereas adenylyl-(2’- )-uridine (which contains a 2’-5’ phosphodiester bond) is not. 4 It was not possible to obtain an accurate measure of the ADP prmed by phosphorolysis along with pApA because it appeared B a very diffuse density on the chromatogram. Polymerization of GDP Compounds that contained adenosine or - Vol. 235, No. 3 No reaction could be demonstrated with poly A, poly U, poly C, or poly AGUC. Here there may have been inhibitory interac- tions, similar to the suppression of poly A synthesis by poly U (6). It is also possible that the additions of polymer that were employed provided too low a concentration of terminal nucleoside residues with free hydroxyl groups at C-3’. An effort was made to detect some reaction in an incubation mixture containing 10 mg per ml of poly A. Relatively large aliquots were removed for estimation of P;, but the results were inconclusive. It is proba- ble that a primer must be a ribose derivative since pTpT and pT pTpT were inactive. In the presence of 4 mm pApApA the rate of P, formation from GDP with purified A. agile fractions was one-fourth of that ob- tained with ADP. It is likely that even faster rates could have been observed with higher concentrations of primer. In other studies® it was shown that under suitable conditions the rate of the GDP-P; exchange reaction is nearly equivalent to that observed with other nucleoside diphosphates. It therefore appears that GDP is an effective substrate for the enzyme. These observa- tions are consistent with the findings of Mii and Ochoa (6), with highly purified fractions from A. agile, that indicate that a single enzyme is active with all of the ribonucleoside diphosphates. At present one may only speculate as to why there is an abso- jute requirement for a primer in the polymerization of GDP by polynucleotide phosphorylase whereas the same preparations of enzyme, when tested with other nucleoside diphosphates, may show only a lag period, after which a rapid reaction occurs in the absence of added primer. SUMMARY No polymerization of guanosine diphosphate occurs when poly- nucleotide phosphorylase is incubated with this compound alone. However, a polymerization reaction does take place in the pres- ence of an oligoribonucleotide containing an unsubstituted hy- droxyl group at carbon 3’ of the terminal nucleoside residue. Such oligoribonucleotides include ApA, ApU, ApApU, pApA, and pApApA. The oligonucleotide serves as a primer and successive guano- sine monophosphate units are added to it, beginning with esteri- fication of the hydroxy] group at carbon 3’. Thus, with a trinu- cleotide containing adenosine serving as primer, polynucleotides were recovered whose structures corresponded to the addition of 1, 2, and 3 guanosine monophosphate residues. A polymer that is precipitated by acid and by 2 volumes of ethanol is also formed. It is nondialyzable and its composition indicates that, on the average, up to 27 guanosine monophosphate residues have been added to each molecule of primer. REFERENCES 1. Grunserc-Manaco, M., ano Ocuoa, S., J. Am. Chem. Soc., 77, 3165 (1955). 2. Lirraver, U. Z., anp Kornpere, A., J. Biol. Chem., 226, 1077 (1957). . 3. GrunBerG-Manago, M., Ortiz, P. J., anp Ocuoa, S., Bio- chim. et Biophys. Acta, 20, 269 (1956). . Berrs, R. F:, Jrn., Biochem. J., 66, 686 (1957). . Oumsrep, P.8., Biochim. et Biophys. Acta, 27, 222 (1958). . Mu, 8., anp Ocuoa, 8., Biochim. et Biophys. Acts, 26, 445 (1957). 7. Swncer, M. F., Heprert, L. A., ano Hitmog, R. J., J. Biol. Chem., 285, 738 (1960). 5M. F. Singer, M. 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