THE PURIFICATION AND CHARACTERIZATION. OF ATP:RNA ADENYLYLTRANSFERASE FROM PSEUDOMONAS PUTIDA Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY KENNETH. JOHN PAYNE 1969 Inna: LIBRARY Michigan State University This is to certify that the thesis entitled THE PURIFICATION AND CHARACTERIZATION OF ATPzRNA ADENYLYLTRANSFERASE FROM PSEUDOMONAS PUTIDA presented by Kenneth John Payne has been accepted towards fulfillment of the requirements for Ph.D. degree in Biochemistry Date JUlV 22. 1969 0-169 BIRDING BY HOME 3 SONS‘ 300K BRIBERY INC I I-nA-v mamas: THE PURIFICATION AND CHARACTERIZATION OF ATPtRNA ADENYLYLTRANSFERASE FRCM PSEUDOMONAS PUTIDA BY Kenneth John Payne AN ABSTRACT OF A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1969 ABSTRACT THE PURIFICATION AND CHARACTERIZATION OF ATP:RNA ADENYLYLTRANSFERASE FROM PSEUDOMONAS PUTIDA 3? Kenneth John Payne ATP:RNA adenylyltransferase was isolated from the 80,000 x‘g pellet of Pseudomonas putida. The enzyme, which was puri- fied from major contaminating activities and endogenous RNA, specifically catalyzed the incorporation of AMP from ATP into a polymeric product. The Km for ATP was 5 x 10'4 M. The rate of incorporation of GNP from CTP was about 7% that of AMP, while GTP, UTP, dATP, and ADP were not utilized by the enzyme. The reaction was dependent upon the cofactor, magnesium ion (Km = 3 x 10‘2 M), which could neither be replaced nor sup- plemented by manganese ion. The polymerization reaction, which was accompanied by the stoichiometric release of inorganic perphosphate, was totally dependent upon exogenous RNA. This requirement could be sat- isfied by ribosomal RNA, soluble RNA, poly C, and (Ap)3A, Kenneth John Payne while DNA, poly A, poly I, poly U, (ApleA: APA: and (AP)4 were ineffective. The Km for ribosomal RNA was 7 x 10'9 M and the ngfor soluble RNA was 1 x 10'6‘M. Ribosomal RNA functioned in the reaction by acting as a primer, providing a 5'-hydroxy1 end upon which adenylate residues were added. The polymeric product was shown to be a chain of adenylate residues, greater than 100 nucleotides in length, covalently attached to the ribosomal RNA. In addition to its role as a primer in the reaction, ribosomal RNA also appeared to act as a kinetic effector. THE PURIFICATION AND CHARACTERIZATION OF ATPzRNA ADENYLYLTRANSFERASE FROM PSEUDOMONAS PUTIDA BY Kenneth John Payne A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1969 ““1 I DEDICATED to Mom and Dad ii ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. John A. Boezi for providing the direction and freedom neces- sary to make this thesis possible. Thanks also go to Dr. F. M. Rottman, Dr. J. L. Fairley, Dr. C. J. Pollard, and Dr. R. L. Anderson for serving as members of my guidance committee. Special appreciation is expressed to Monique De Backer for her imaginative thought and technical assistance. I also wish to thank Dr. L. F. Lee, Dr. R. L. Armstrong, Gary Gerard, Kathleen Rose, and James Johnson for helpful discussions and a rewarding experience. This investigation was supported in part by a National Institutes of Health Predoctoral Fellowship (GM-31,955). iii f"““‘—‘“‘.‘fi ‘7. TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . INTRODUCTION . . . . . . . . . EXPERIMENTAL PROCEDURE . . . . . . . . RESULTS Materials and Methods . . . . . . . . Bacterial Growth . . . . . . . . . . . . Preparation of Ribosomal and Soluble RNA . Enzyme Assays . . . . . . . . . Alkaline Hydrolysis of Polymeric Product . Purification of Enzyme . . . . . . . . . . . . Preparation of Initial Extract . . . . Sedimentation . . . . . . . . . . . . Solubilization . . . . . . . . . . . . pH Fractionation . . . . . . . . . . . . . pH Concentration . . . . . . . . . . . . Sephadex G- 100 Gel Filtration . . . DEAE- -Sephadex Chromatography . . . Cellulose Phosphate Chromatography . Comments on Purification Procedure . . . . Association.with Macromolecules . Removal of Contaminating Nucleic Acid Recovery of Enzyme . . . . . . . . . . Stability of Enzyme . . . . . . . . . Contaminating Activities . . . . . . . Characteristics of Reaction . . . . . . . . General Pr0perties . . . . . Metal Ion Requirement . . . . . . . Ribonucleoside Triphosphate Requirement RNA Requirement . . . . . . . . . iv Page vi vii Characterization of Reaction Products . Stoichiometry of Reaction Products . . Chain Length Determination of Polymeric Product Nearest Neighbor Analysis of Polymeric Product. Sucrose Gradient Analysis of Polymeric Product. DISCUSSION . . . REFERENCES Page 51 51 51 55 SS 59 65 Table II. III. IV. LIST OF TABLES Page Purification of enzyme . . . . . . . . . . . . 2h Incorporation of ribonucleotides . . . . . . . 36 Stimulation of AMP incorporation by polynucleotides . . . . . . . . . . . . . Al Chain length analysis of polymeric product . . 52 Nearest neighbor analysis of polymeric product Sh vi Figure 1. NOW 10. ll. 12. LIST OF FIGURES Sephadex G-100 gel filtration of pH 3.2 precipitate enzyme fraction . . . DEAE-Sephadex column chromatography of Sephadex G-100 enzyme fraction Cellulose phosphate column chromatography of DEAR-Sephadex enzyme fraction Reaction kinetics . . . . . . . AMP incorporation versus MgCla concentration Manganese inhibition of AMP incorporation AMP incorporation versus ATP concentration . AMP incorporation versus ribosomal RNA concen- tration O O O O 0 O O O O O O 0‘ O I C O O 0 AMP incorporation versus soluble RNA concen- tration . . . . . . . . . . . . . Hill plot of AMP incorporation versus ribosomal RNA concentration . . Stimulation of AMP incorporation by oligoribonucleotides . . . . . Sucrose density gradient analysis of polymeric product . vii Page 17 50 52 55 59 to A5 A8 50 57 71—“? I4 INTRODUCTION Numerous enzymes have been reported in the past few years which catalyze the addition of the nucleoside monophosphate portion of a nucleoside triphosphate onto the 5'-end of oligo- or polynucleotides. The terminal addition of deoxyribonucleo- tides to oligo- or polydeoxyribonucleotides has been well- documented. Krakow, Coutsogeorgopoulus, and Canellakis (1) reported the isolation of a terminal deoxyribonucleotide transferase from the nuclear fraction of calf thymus gland which utilized each of the four common deoxyribonucleoside triphosphates and, in the presence of Mg2+, added the cor- responding monodeoxyribonucleotides onto the 3'-end of denatured DNA. The enzyme, which was distinct from DNA polymerase, syn- thesized short chains of deoxyribonucleotide product (less than A residues in length). Referred to as the "nuclear terminal addition enzyme" (2), it was also capable of incorpor- ating the common ribonucleoside triphosphates, but only a single ribonucleotide residue could be added to each primer molecule (2, 3). The isolation of another terminal deoxyribonucleotide transferase has been reported by Bollum (h), who utilized the soluble fraction of calf thymus gland. This enzyme, distinct from the nuclear enzyme and referred to as "polydeoxynucleotide synthetase" (5), incorporated the mononucleotide residues of each of the four common deoxyribonucleoside triphosphates onto the 3'-end of a denatured DNA or oligodeoxyribonucleotide primer. Variable chain lengths (1-600 nucleotides), random copolymeri- zation of deoxyribonucleotides, and limited incorporation of ribonucleotides could be achieved depending upon incubation conditions (5, 6, 7). Enzymes which catalyze the terminal incorporation of ribo- nucleotides from ribonucleoside triphoSphates into RNA have been demonstrated in a wide variety of systems. One of these enzymes is the well-characterized and ubiquitous ATP(CTP):tRNA nucleotidyltransferase, which synthesizes the pCpCpA sequence on the 5'-end of transfer RNA (8-12). This enzyme is specific for transfer RNA and produces a product of well-defined length and sequence, factors which distinguish it from other terminal ribonucleotide incorporating activities. The remainder of these terminal ribonucleotide incorporating activities, which have been isolated from mammalian (15-25), plant (26), avian (27-29), and bacterial (30-55) sources, have ’13 f) not been as well characterized. Nevertheless, all require a ribonucleoside triphOSphate as substrate, a divalent metal ion as cofactor, and an oligo- or polyribonucleotide as primer. In those cases in which sufficient data is available, the reactions appear to proceed, with one possible exception (see below), by the addition of monoribonucleotide residues onto i the 3'-end of the primer. The resulting products are homori- I bopolymer chains of varying lengths (1-200 nucleotides), co- valently attached to the primer molecule. Within these basic characteristics, however, a great deal of diversity exists among the various ribonucleotide incorpora- ting activities. Enzymes which are reasonably Specific for each of the four common ribonucleoside triphosphates have been prepared from Escherichia coli (ATP), calf thymus (CTP), rat liver (DTP), and spinach (CTP), while an enzyme fraction from Landschutz ascites-tumor cells will utilize any of the ribonu- cleoside triphosphates. It is not known whether the latter enzyme fraction contains one enzyme which will incorporate each ribonucleoside triphosphate or an enzyme for each one. Among the best characterized of the ribonucleotide in- corporating activities are those from E; coli. In 1962, two groups of investigators reported the isolation of specific ATP incorporating activities from this source. These enzymes were isolated by Gottesman, Canellakis, and Canellakis (50) using the soluble fraction and August, Ortiz, and Hurwitz (51) using the ribosomal pellet. In the presence of the divalent metal ions, F_g Mg2+ and Mn2+, the enzyme from the soluble fraction was reported to I catalyze the terminal addition of adenylate residues onto the 5'-end I". of a RNA primer, producing chains of 25-55 nucleotides in length. The enzyme isolated from the ribosomal pellet, referred to as "polyriboadenylate polymerase" (5h), also required ATP and a RNA primer. However, the reaction, in the presence of Mga+ alone, re- portedly produced 22.2232 synthesized chains of poly A, 100-200 nucleotides in length. Recently, Hardy and Kurland (52) also des- cribed an ATP incorporating activity from E. coli ribosomes. This enzyme, which required both Mg2+ and Mn2+ for optimal activity, did not produce 92.2222 synthesis of poly A but instead added adenylate residues onto the 5'-end of a ribosomal RNA primer. The reason for the discrepancy between the results of August, Ortiz, and Hurwitz and Hardy and Kurland is not known. However, it should be pointed out that the purification procedures used by the two groups were \,.'1 different, the specific activities of the partially purified enzymes varied, and neither enzyme was free of endogenous RNA. The purpose of this thesis is to present the purification and characterization of ATPzRNA adenylyltransferase from Pseudomonas putida. The enzyme catalyzes the incorporation of I adenylate residues from ATP into a polymeric product, with the concomitant release of inorganic pyrophosphate. The reaction I is completely dependent upon exogenous RNA and requires the co- factor, magnesium ion. The product is a homopolymer chain of adenylate residues, greater than 100 nucleotides in length, which is covalently attached to the 5'-end of the added primer. EXPERIMENTAL PROCEDURE Materials and Methods - NADP+, UDPG, glucose 6-ph03phate dehydrogenase, 2'-dATP, and all unlabeled 5'-phosphate deriva- tives of the ribonucleosides were purchased from P-L Biochemi- cals, Inc. Sephadex 6-100 and DEAE-Sephadex (A-25) were obtained from Pharmacia Fine Chemicals Inc. and Whatman cellulose phos- —‘ «car - 1421:".1“ phate (Pl-floc) from Reeve Angel. Calf thymus DNA, poly A, poly U, poly C, poly I, and phosPhoglucomutase were purchased from Sigma Chemical Co. Labeled nucleotides were obtained from Schwarz BioResearch, Inc. Pseudomonas putida bacteriOphage gh-l DNA (55, 56) was purified by the method of Thomas and Abelson (57). The gh-l and calf thymus DNA were denatured by heating at 100° for 10 minutes followed by quick-cooling. UDPG perphos- phorylase was isolated from calf liver (58) and recrystallized twice. The oligoribonucleotides, (Ap)4, (Ap)3A, (Ap)2A, and ApA, were prepared according to the procedure of Rottman and Nirenberg (59). We are indebted to Mr. Joseph Abbate and Mr. Richard Jagger of this department for gifts of Xenopus laevis ribosomal RNA and (pT)5, respectively. Protein concentrations were determined by the method of Lowry £5 al.(h0) using bovine albumen fraction V powder (Nutritional Biochemicals Corporation) as a standard. The concentrations of gh-l and calf thymus DNA and ribosomal and soluble RNA were determined spectrOphotometrically based on the extinction co- efficient E520 = 200. The concentrations of the oligoribonu- cleotides were determined spectrophotometrically using the molar extinction coefficient for AMP of lh.2 x 10'3 at 260 mp and the hyperchromicity of each oligomer (Al). Bacterial Growth - Pseudomonas putida A.5.l2 was grown with vigorous aeration at 520 in 10 liter volumes in a MicroFerm Laboratory Fermentor (New Brunswick Scientific Co.). The growth media contained, per liter, 5 g of bacto yeast extract (Difco), 6 g of (NH4)2HPO4, 5 g of KH2P04, 8 g of NaCl, 2.1 g of MgClg' 6H20, 12 g of glucose, and 5 mg of FeCla. Bacterial growth was continued until the early stationary phase at which time the culture was harvested by means of a Sharples centrifuge. Cells were stored at -20° for several months without detectable loss of activity. Preparation.g£ Ribosomal and Soluble RNA - Frozen Pseudom- onas putida cells were disrupted as described in "Results (Purification of Enzyme)" and the initial 80,000 x‘g pellet was resuspended in buffer containing 10 mM Tris-HCl (pH 8.0), 10 mM M3012, and 10 pg/ml DNase (Worthington, electrophoretically purified). After incubation at 57° for 10 minutes, the ribosomes taere collected by centrifugation at 80,000 x‘g for 120 minutes. The pellets were then resuspended in 10 mM.Mg(OAc)2-O.2% SDS (pH 5.1) by stirring at 5° for 90 minutes. An equal volume of water-saturated phenol was added, and, after stirring for 15 minutes, the suspension was centrifuged at 27,000 x.g for 10 minutes. The aqueous layer was removed and saved and the remain- ing material was re-extracted with the Mg(OAc)2-SDS buffer. The new aqueous layer was combined with the first and the combined aqueous layers were treated with an eQual volume of water-saturated phenol. The aqueous layer was separated and again treated with phenol. The final aqueous layer was made 0.5 leith reSpect to NaOAc and ribosomal RNA was precipitated by the addition of two volumes of cold ethanol. The precipitate was dissolved in 10 mM Tris-HC1 (pH 8.0). The solution was then made 0.5 M‘with respect to NaOAc and ribosomal RNA was reprecipitated with cold ethanol. This final precipitate was redissolved, dialyzed extensively against 10 mM succinate-NaOH (pH 5.5), and stored at -20°. The resulting ribosomal RNA was free of detectable small molecular weight RNA as determined by sucrose density gradient centrifu- gation. .E; coli ribosomal RNA was purified in a similar manner. For the preparation of soluble RNA, the supernatant solu- tion from the 80,000 x‘g centrifugation of the glass bead extract was utilized. The initial SDS-phenol purification procedure was similar to that for ribosomal RNA. Further purification was n“; accomplished by the slow addition of solid NaOAc to a final concentration of 5'M, with the resulting precipitate being J collected by centrifugation and discarded. Soluble RNA was then precipitated with ethanol, redissolved, and dialyzed extensively against 10 mM succinate-NaOH (pH 5.5). The resulting soluble‘ RNA.which was free of detectable high molecular weight RNA as determined by sucrose density gradient centrifugation, was stored at -20°. Enzyme Assays — The incubation temperature for all reactions was 57°. The standard assay measured the conversion of 3H-ATP into an acid-insoluble product. Unless otherwise indicated, the incubation mixture (0.5 ml) contained 50 mM glycine-NaOH (pH 9.5), 0.8 mM 3H-ATP, 20 mM MgC12, saturating amounts of P; putida ribosomal RNA, and an appropriate amount of enzyme. Samples of 100 pl were withdrawn at various times and 5 ml of cold 10% trichloroacetic acid was added to each sample. The acid-insoluble material was collected on membrane filters (Schleicher and Schuell Co., type B-6) which were then dried and monitored for radioactivity 10 in a Packard Tri-Carb liquid scintillation Spectrometer in a fluor of toluene containing A grams per liter of 2,5-bis- [2-(Sigggg-butylbenzoxazolyl)]-thi0phene. One unit of enzyme activity is defined as that amount of enzyme which catalyzes the incorporation of one nmole of AMP into an acid-insoluble product, with ribosomal RNA as primer, in 10 minutes at 57°. Specific activity is expressed as units per mg of protein. Reactions lacking either RNA, MgClg, or enzyme served as controls. For the determination of inorganic perphosphate, the reac- tion mixture (0.5 ml) contained 50 mM glycine—NaOH (pH 9.5), 0.8 mM 3H-ATP, 20 mM'Mg(OAc)2, 17 pg of P; putida ribosomal RNA, 0.2 mM UDPG, 0.2 mM NADP+, a suitable amount of enzyme and excess phosphoglucomutase, glucose 6-phosphate dehydrogenase and UDPG perphosphorylase. The assay as described by Johnson gt gl.(h2) couples the formation of inorganic pyrophosphate from ribonucleoside triphOSphate polymerization to NADP+ reduction which is measured at 5h0 mp in a spectrOphotometer. Calcula- tion of the number of nmoles of NADPH formed was made using the molar extinction coefficient of 6.22 x 103 (A5). Inorganic pyrophosphatase was measured by the disappearance of inorganic perphosphate using the coupled UDPG pyrophosphorylase ll assay system. Adenylate kinase and ATPase were measured by the conversion of 3H-ADP and 3H-ATP, respectively, to other adenylate nucleotides, which were separated by means of solvent chromatog- raphy in isobutyric acid : NH4OH : H20 (66:1:55). Polynucleo- tide phosphorylase was measured under the same conditions as described for the standard assay except that 3H-ADP was used in place of 3H-ATP. (These conditions were found to be near optimal for the ADP incorporating activity isolated from the 80,000 x‘g pellet of P. putida.) Ribonuclease was assayed by measuring the loss of acid-insoluble radioactivity and the change in the sucrose density gradient profile of enzyme-treated 14C-ribosomal RNA. Alkaline Hydrolysis 2: Polymeric Product - After incubation of the mixture for 60 minutes, the reaction was terminated by the addition of two volumes of cold 5% HC104. The resultant precipi- tate was collected by centrifugation at 12,000 x‘g for 10 minutes and the supernatant solution was discarded. The pellet was washed once with 2 ml of cold 5% HClO4 and twice with 2 ml of cold 1% HClO4. After the addition of 2.0 ml of 0.5 N KOH, the dissolved pellet was incubated at 37° for 18 hours. The solution was then neutralized with Dowex 50 (H+), which was then removed by filtration through Whatman No. A2 filter paper and washed three times with 0.5 ml 12 of 0.1 N NH40H. The combined filtrates were 1yophilized, dis- solved in H20, and spotted with the appropriate standards on Whatman No. 5MM paper for electrophoresis and Whatman No. A1 paper for solvent chromatography. Electrophoresis was carried out in 0.05 M ammonium formate (pH 5.6) at 56 volts/cm for 100 minutes. Descending solvent chromatography was performed in isobutyric acid : NH4OH : H20 (66:1:55). The radioactive lanes were cut out and counted in a liquid scintillation spectrometer. h a. jail." kil p F‘- RESULTS Purification 3f Enzyme The entire purification procedure was performed at O-AO. Preparation of Initial Extract - Frozen Pseudomonas putida, #0 g of cells which had been grown to the early stationary phase, was homogenized in a Servall Omni-Mixer with 100 g of acid- washed glass beads and 25 ml of buffer (10 mM Tris-HC1 (pH 8.0), 10 mM'MgClg, 0.1 mM EDTA). After 5 minutes of homogenization, hO ml of the same buffer was added and the entire suspension was centrifuged at 12,000 x'g for 10 minutes. The supernatant solution was decanted and recentrifuged at 12,000 x‘g for 10 min- utes. The resulting supernatant solution was carefully decanted and diluted to 100 ml with the above buffer (glass bead extract). Sedimentation - The glass bead extract enzyme fraction was centrifuged at 80,000 x'g for 120 minutes. The supernatant solution,which was devoid of detectable ATP incorporating activity, was poured off and discarded, and the pellet was stored at -20° overnight. (No difference was observed if the purification was continued immediately without freezing the high speed pellet.) Solubilization - The high speed pellet was thawed and homo- genized in 100 mM glycine-NaOH (pH 10.6) by means of a glass tube 15 1h fitted with a Teflon-tipped pestle. The resulting homogenate was centrifuged at 80,000 x.g for 120 minutes and the supernatant solution was poured off and saved. The pellet was rehomogenized with 70 ml of the same buffer followed by centrifugation at 80,000 x'g for 120 minutes. The supernatant solution was poured off and combined with the first (pH 10.6 supernatant - 15h ml). _pH Fractionation - Solid KCl was slowly added with stirring to the pH 10.6 supernatant enzyme fraction to make the solution 1 M with respect to KCl. Hydrochloric acid (1 M) was then added dropwise with stirring until a pH of 5.0 was reached. A floc- culent white precipitate formed during this procedure. After the suspension was stirred for h hours, the precipitate, which was devoid of ATP incorporating activity, was removed by centrifuga- tion at 12,000 x'g for 10 minutes and discarded. The supernatant solution was dialyzed overnight against four liters of 10 mM Tris-HCl (pH 8.0) - (pH 5 supernatant - 1&2 m1). ‘pfi Concentration - The pH 5 supernatant enzyme fraction was adjusted to pH 5.2 by the slow addition of l M hydrochloric acid. A precipitate formed which, after stirring for 15 minutes, was collected by centrifugation at 12,000 x‘g for 10 minutes. The supernatant solution, which was devoid of ATP incorporating activity, 15 was poured off and discarded. The pellet was redissolved in 22 ml of 100 mM glycine-NaOH (pH 10.1) by gentle stirring for 5 hours. The resulting solution was then dialyzed overnight against two successive four liter volumes of 10 mM Tris-HCl (pH 8.0) - (pH 5.2 precipitate - 25 ml). Sephadex Q-lOO Gel Filtration - Sephadex G-100, previously equilibrated with 10 mM Tris-HCl (pH 8.0) containing 1 M KCl, was utilized to prepare a A5 cm x 2.5 cm column. The pH 5.2 precipitate enzyme fraction was adjusted to 1 M with respect to KCl by the addition of solid KCl. A 5 ml aliquot of the enzyme solution was then applied to the gel and the column was deve10ped by the addition of 10 mM Tris-H01 (pH 8.0) containing 1 M KCl. The effluent pattern of the column is shown in Figure 1. After washing the column with at least 100 ml of the high ionic strength buffer, another 5 ml aliquot of the enzyme solution was applied and eluted in the same manner. This was repeated three more times for the remaining pH 5.2 precipitate enzyme fraction. (In order to obtain satisfactory resolution of the applied material, it was important that the protein concentra- tion of this fraction did not exceed 5 mg/ml.) The peak frac- tions from each column were pooled and dialyzed overnight against two successive four liter volumes of 10 mM Tris-HCl (pH 9.1) - (Sephadex G-100 - 92 ml). 15 was poured off and discarded. The pellet was redissolved in 22 m1 of 100 mM glycine-NaOH (pH 10.1) by gentle stirring for 5 hours. The resulting solution was then dialyzed overnight against two successive four liter volumes of 10 mM Tris-H01 (pH 8.0) - (pH 5.2 precipitate - 25 m1). Sephadex G-100 Gel Filtration - Sephadex G-100, previously equilibrated with 10 mm Tris-HCl (pH 8.0) containing 1 M KCl, was utilized to prepare a A5 cm x 2.5 cm column. The pH 5.2 precipitate enzyme fraction was adjusted to l M.with respect to KCl by the addition of solid KCl. A 5 m1 aliquot of the enzyme solution was then applied to the gel and the column was developed by the addition of 10 mM Tris-HCl (pH 8.0) containing 1 M.KC1. The effluent pattern of the column is shown in Figure 1. After washing the column with at least 100 ml of the high ionic strength buffer, another 5 m1 aliquot of the enzyme solution was applied and eluted in the same manner. This was repeated three more times for the remaining pH 5.2 precipitate enzyme fraction. (In order to obtain satisfactory resolution of the applied material, it was important that the protein concentra- tion of this fraction did not exceed 5 mg/ml.) The peak frac- tions from each column were pooled and dialyzed overnight against two successive four liter volumes of 10 mM Tris-HCl (pH 9.1) - (Sephadex c-loo - 92 m1). 16 Figure l. Sephadex G-100 gel filtration of pH 5.2 pre- cipitate enzyme fraction. A 5 m1 aliquot of the pH 5.2 preci- pitate enzyme fraction was applied to a A5 cm x 2.5 cm column of Sephadex G-100 equilibrated with 10 mM Tris-HCl (pH 8.0) containing 1 M KCl. The column was developed at a flow rate of 0.5 ml/min by the continual addition of the same buffer. Thirty ml of the 66 ml void volume was collected followed by the A0 three-ml fractions presented in the figure. A-—-A, absorb- ance at 260 mp; o---o, AMP incorporation into acid-insoluble product using ADP as the substrate; 0-—-., AMP incorporation into acid-insoluble product using ATP as the substrate. Reac- tion mixtures (0.5 ml) contained 50 mM glycine-NaOH (pH 9.5), 20 mM Mgc12, 0.8 mM 3H-ATP (1.2 x 103 cpm/nmole) or 3H-ADP (0.7 x 103 cpm/nmole), 8A pg of ribosomal RNA, and 50 pl of eluate fraction. After incubation for 10 minutes, the reac- tions were stopped by the addition of 5 ml of cold 10% trichloroacetic acid and the amount of acid-insoluble product was determined as described in "Experimental Procedure (Enzyme Assays)". Fractions 2A through 29 were pooled and dialyzed against 10 mM Tris-HCl (pH 9.1)-(Sephadex G-100 enzyme fraction). HHSWON NOIlOVHd AMP INCORPORATION (nmoles/l0 min) .- 94 :5 01 o 01 o» o I T I 9 8| 173 oc lg J I I - 04 «b 01 O U" ABSORBANCE AT 260 I 03 O ITUJ l8 DEAR-Sephadex Chromatography - A 19 cm x 1.2 cm column of DEAE-Sephadex was prepared and washed extensively with 10 mM Tris-HCl (pH 9.1). The entire Sephadex G-100 enzyme fraction was appliedtx>the column and subsequently eluted with a 100 m1 linear gradient of 0 to l M.KC1 in the same buffer. The elu- tion profile is shown in Figure 2. The peak enzyme fractions were pooled (DEAR-Sephadex - 18 ml) and stored at 2°. Cellulose Phosphate Chromatography_- A 12.5 cm x 1.2 cm column of cellulose phosphate was prepared and washed exten- sively with 10 mM phosphate buffer (pH 7.1). The DEAE- Sephadex enzyme fraction was dialyzed overnight against the same phosphate buffer (DEAE-Sephadex (dialysis) - 20 m1). As a result of this procedure, approximately two-thirds of the ATP incorporating activity was lost with the concomitant appearance of a very fine precipitate. The entire suspension was applied to the cellulose phosphate column. The enzyme was then eluted with a 100 ml linear gradient of 0 to l M KCl in 10 mM phos- phate buffer (pH 7.1). The elution profile is shown in Figure 5. The peak enzyme fractions were pooled (cellulose phosphate - 9 m1) and stored at 2°. 19 Figure 2. DEAE-Sephadex column chromatography of Sepha- dex G-lOO enzyme fraction. The entire Sephadex G-100 enzyme fraction was applied to the tOp of a 19 cm x 1.2 cm column of DEAE-Sephadex prepared in 10 mM Tris-HC1 (pH 9.1). The column was eluted at a flow rate of 0.5 mllmin by the addition of 100 ml of a 0 to l M gradient of KCl in the same buffer. Fifty three-ml fractions were collected beginning at the time of the application of the gradient to the column. No signi- ficant enzyme activity was detected in eluant previous to these fractions. Reaction conditions were the same as those described in Figure 1. O-O, AMP incorporation into acid- insoluble product using ATP as the substrate; o__o, absorbance at 280 mp. Fractions 19 through 2A were pooled and stored at 2° (DEAE-Sephadex enzyme fraction). HBSWON NOLLOVHJ AMP INCORPORATION (nmoles/IO min) .— 9‘ P- .03 OI o 01 o I T I I 0| 9| ZZ OI? I I \I .0 .0 :- O 0' O MOLARITY OF KCI I I J I .0 :— :— N m m (D in ABSORBANCE AT 280 mu Figure 2 21 Figure 5. Cellulose phosphate column chromatography of DEAE-Sephadex enzyme fraction. The entire DEAE-Sephadex enzyme fraction was applied to the top of a 12.5 cm x 1.2 cm column of cellulose phOSphate prepared in 10 mM phosphate (pH 7.1). The column was eluted at a flow rate of 0.5 ml/min by the addition of 100 m1 of a 0 to 1 M gradient of KCl in the same buffer. Thirty-six three-m1 fractions were collected beginning at the time of the application of the gradient to the column. No significant absorbance or enzyme activity was detected in eluant previous to these fractions. Reaction conditions were the same as those described in Figure 1. I .——.., AMP incorporation into acid-insoluble product using ATP as substrate. Fractions 2A through 26 were pooled and stored at 2° (cellulose phosphate enzyme fraction). HBBWHN NOLLOVEH AMP INCORPORATION (nmoles/l0 min) .- 94 .45 .03 0| 0 01 O I I I I 3| I I I I .0 .0 .- 0 GI o MOLARITY OF KCI I I I I Figure 5 ""1 an rs; 3-1 a. "I. - __' 25 Comments on Purification Procedure A summary of the purification procedure is presented in Table I. The data describes approximately a 560-fold puri- fication with an apparent 5% recovery of initial enzyme activity. Association with Macromolecules - An important aspect of I the purification is the association of the enzyme with macro- molecular cellular components. Following the preparation of the glass bead extract and removal of cellular debris, high speed centrifugation of the enzyme extract was performed. Though most cellular enzymes, including DNA-dependent RNA polymerase, remain in the supernatant solution during this procedure, the ATP incorporating activity was detected only in the pellet. Solubilization of the enzyme was accomplished by homogenization of the high speed pellet in alkaline buffer, pH 10.6. At pH 10.1 only about 50% of the enzyme was released into the supernatant fraction, while less than 10% was solu- bilized at pH 9.8. Even after solubilization from the high speed pellet, the enzyme was found to aggregate with other cellular components. The pH 5 fractionation and Sephadex G-100 gel filtration steps were ineffective unless carried out in the presence of l M salt. 2A mm: Om: oomH 00mm ooi. oon OOmHH comm OOQH an an a: 8 R R. oumcmmosa mmOasflfioo Ananmanflnv xmenanon-mauum owmqoomm coauomum Oaxaca .oBmNam mo aowumowmfiusm .H manna 25 Removal gf Contaminating Nucleic Acid - Even though the majority of the A260-absorbing material was separated from the ATP incorporating activity in the gel filtration step (Figure 1), the A250:A260 ratio of the enzyme fractions remained at 0.5. At this point in the purification, the residual nucleic acid was still in sufficient quantity to prevent any stimulation of the ATP incorporating activity by the addition of exogenous RNA. The next step, DEAE-Sephadex chromatography, was utilized to remove most of the remaining Aaeo-absorbing material (Figure 2). The A280:A260 ratio of the eluate fractions in the region of the enzyme, fractions 16 through 2A, was approximately 1.0, while the A280:A260 ratio of the later eluate fractions, 27 through 5A, was 0.5. Following this step, the ATP incorporating activity was completely dependent upon added RNA. The final purification step, cellulose phosphate chromatography (Figure 5), increased the A280:A260 ratio of the enzyme fractions to l.A. Recovery pf Enzyme - Although the data presented in Table I indicates a fairly low recovery (5%) of initial activity, much of this loss in total units can be accounted for in the puri- fication procedure. The first significant loss took place in the Sephadex G-100 gel filtration step in which two peaks of ATP incorporating activity were observed (Figure 1). Only the 26 activity peak which was separated from the bulk of the A260- absorbing material and the ADP incorporating activity was utilized for further purification. Other losses also occurred in this step, as well as in the two column chromatography steps, due to the pooling of only the peak fractions of the ATP incor- porating activity. Finally, a major loss in enzyme activity occurred, along with the concomitant appearance of a fine precipitate, during the dialysis of the DEAE—Sephadex enzyme fraction in preparation for its application to the cellulose phosphate column. Stability gf Enzyme - The stability of the enzyme fractions, up to and including the DEAE-Sephadex fraction, was such that, when stored at 2°, no loss in activity could be detected for at least a month. As mentioned above, however, dialysis of the DEAE-Sephadex enzyme fraction led to a considerable loss in activity. The cellulose phosphate enzyme fraction was also unstable, losing about 50% of its activity in one week. Contaminating Activities - The cellulose phosphate enzyme fraction was free of detectable adenylate kinase, ATPase, and inorganic pyrophosphatase activities. This enzyme fraction was also free of any primer-dependent or primer-independent (AA) polynucleotide phosphorylase. No ribonuclease activity could be detected in the DEAR-Sephadex or cellulose phOSphate enzyme fractions. Characteristics 2f Reaction General Properties - Both the rate and extent of ribo- nucleotide incorporation into an acid-insoluble product increased with temperature and were optimal at 57°. Higher temperatures, up to A50, produced an equally efficient initial reaction but the total incorporation was significantly inhibited. The optimal pH for the reaction was 9.5 using either 50 mM glycine-NaOH buffer or Tris adjusted to that pH with HCl. Ribo- nucleotide incorporation equal to approximately 50% of the optimal activity was observed with Tris-RC1 buffer at pH 8.5 and with glycine-NaOH buffer at pH 8.8 and 10.2. The rate of the reaction was directly proportional to the amount of enzyme added. The addition of 0.50, 1.5, and 5.0 pg of the cellulose phosphate enzyme fraction to the standard reaction mixture resulted in the incorporation of 0.Al, 1.8, and 5.8 nmoles of AMP, respectively, in 20 minutes. Similar proportionality was obtained with the less purified fractions. 28 The kinetics of the reaction were consistently biphasic (Figure A), independent of the purity of the enzyme or the pH of the reaction mixture. All the studies presented in this report were carried out within the initial linear range of the reaction. Metal Ion Requirement - The enzyme requires the presence of magnesium ion for activity (Figure 5). There was no detect- able activity when Mg2+wls omitted from the assay. The activity increased with increasing Mg2+ until a maximum between 15 and 50 mM, followed by considerable inhibition between 50 and A0 mM. The apparent Km for Mg2+ as determined from the Lineweaver- Burk plot shown in the insert of Figure 5 was 5 x 10'2'M. Manganese ion was ineffective as a substitute for Mg2+ in this reaction. However, whenM'n2+ replaced Mg2+ int:he standard reaction mixture in the absence or in the presence of enzyme, a radioactive material was formed, along with a visible precipitate, which was retained on a membrane filter. This non-enzymatic reaction was time-dependent, optimal at about 5 MM un2+, and dependent upon the presence of RNA. The reaction was also dependent upon pH, with the activity increasing as the pH increased from 8 to 10.5. The nature of the material formed in this non-enzymatic reaction was not determined. 29 Fi ure A. Reaction kinetics. Complete reaction mixtures (1.0 ml contained 50 mM glycine-NaOH (pH 9.5), 0.8 mM.3H-ATP (2.07 x 103 cpm/nmole), 2O mM.M@C12, 168 pg of ribosomal RNA, and 5 pg of cellulose phosphate enzyme fraction. Omissions or substitutions were as follows: 05-0, complete system; o-—o, complete system minus ribosomal RNA; A——A, complete system with 50 mM.Tris-HCl (pH 8.5) as the buffer; A-A, complete system with 3H-ADP (A.0 x 103 cpm/nmole) as the sub- strate. Samples of 100 p1 were withdrawn at the times in- dicated and product formation was determined as described in "Experimental Procedure (Enzyme Assays)". (SGWUN‘J) 3W”. 03 I 06 OQI OBI AMP INCORPORATION (nmoles/OJ ml) N (N A I I l p , _ r . .— —. y . —-I -O r - I l I I l 51 Figure 5. AMP incorporation versus MgC12 concentration. Complete reaction mixtures (0.5 m1) contained 50 mM.glycine- NaOH (pH 9.5), 0.8 mM 3H-ATP (1.26 x 103 cpm/nmole), 8A pg of ribosomal RNA, varying amounts of MgClz, and 7.5 pg of the DEAR-Sephadex enzyme fraction. After incubation for 20 minutes, the reactions were stopped by the addition of 5 ml of cold 10% trichloroacetic acid and the amount of acid- insoluble product was determined as described in "Experimental Procedure (Enzyme Assays)". O—O, complete system; 0—0, complete system minus enzyme. The insert is a Lineweaver-Burk plot of the data for the complete system up to and including 20 mM M3012. AMP INCORPORATION (nmoles/20 min) .- 9» .b .03 0| 0 01 O I I I I O a», — I §’ p "‘ O 3 —| Eco 8* ‘I “I g <- ON 28' '— '\) .£> O I‘::’- 1" .—— L—_J g; h’ '1 m 8 8h— —I q l l d.__. -I J I I I Figure 5 35 Hardy and Kurland (52) have reported an ATP incorporating activity in §;_ggli which requires both Mge+ (25 mM) and Mn2+ (2 mM) for Optimal activity. In the presence of Mg2+, Mn‘e+ produced approximately a three-fold stimulation of the activity. L4 However, as is shown in Figure 6, Mn2+, in the presence of Optimal Mg2+, did not stimulate the activity of the P; putida ‘ enzyme but instead inhibited the process. Fifty per cent inhibition was accomplished by the addition of about A mM Mug+ to the reaction mixture, while the addition of an equivi- lent amount of MgZ+, giving a final concentration of 2A mM Mge+, still produced near maximal activity (see Figure 5). A similar inhibitory effect of M112+ was observed at pH 8. Ribonucleoside Triphosphate Requirement - Of the four common ribonucleoside triphosphates, only ATP was significantly incorporated into a polymeric product by the enzyme (Table II). Under identical conditions, the rate of incorporation of CMP was about 7% that of AMP, whereas the incorporation of UMP or GMP was negligible (less than 1%). If either GTP, UTP, or CTP was added to the reaction mixture in addition to ATP, incorporation of AMP was inhibited approximately 50%. The mechanism of this inhibition is unknown. Neither ADP (see Figure A) nor d-ATP (data not shown) could replace ATP as a 5A Figure 6. Manganese inhibition of AMP incorporation. Conditions were the same as those described in Figure 5 except that 20 mM MgC12 and varying amounts of MnCle were present in each reaction mixture. A: H; complete system; o—o, complete system minus enzyme. B: H, complete system corrected for non-enzymatic reaction. ZIOUw ww AMP INCORPORATION (nmoles/20 min) :— 94 :P 8 UT 01 I T I l 36 INHI- .mo~ x m:.m .meo-mn “nod x mo.m .meo-mn mnOA x mo.m .nes-mn mn2 x ms.a .ne<-mn "Amaoat\snuv mumua>guom ugmaummm mcfisoHHOM any we onus moumnmmonawuu unamOOHosoonHu vmaopma use .:Ammmmm< mahncmv ouspoooum Hmusoaauomxm: cg confluence we pmcaaumuov was ouspoua manaaomcwupwom we uazoaw can use pace oaumomouoHsowuu Rea pHoo mo #5 m «o newugpwm ecu an nuances onus meofiuomou oeu Kmousse om new cofiumnsoca umum< .kumudccfl mm mumsmmosawuu ovdmooaosconau vmfionmaca SE m.o.v:w noumzamonawuu ovaooHosconHu coaonma 2E w.o acoHuumuw Oaxaca xovwaaomim T > O O 2 -O.8 -|.6 A8 I I I I I I O 0.8 I .6 log 8 Figure 10 1+9 Figure 11. Stimulation of AMP incor oration by oligori- bonucleotides. Reaction mixtures (0.5 ml) contained 50 mM glycine-NaOH (pH 9.5), 0.8 mM 3H-ATP (1.26 x 103 cpm/nmole), 20 mM MgC12, 25 pg of the DEAR-Sephadex enzyme fraction, and the following primers: O-—., 8A pg of ribosomal RNA; o-—o, 51 I18 of ApApApA; A—A, A7 A3 of ApApA; 0—0, A7 us of APA; and A-—A, 57 pg of ApApApAp. Samples were withdrawn at the times indicated and product formation was determined as des- cribed in "Experimental Procedure (Enzyme Assays)". (SGIOUIw) 3WIJ. 09 OOI OZI 08 AMP INCORPORATION (nmoles/OI ml) 0 01 o 01 8 I I I I O A D A9 Figure 11. Stimulation of AMP incor oration by oligori- bonucleotides. Reaction mixtures (0.5 m1) contained 50 mM glycine-NaOH (pH 9.5), 0.8 mM 3H-ATP (1.26 x 103 cpm/nmole), 20 mM.MgC12, 25 pg of the DEAE-Sephadex enzyme fraction, and the following primers: O—g, 8A pg of ribosomal RNA; 0—0, 51 us of ApApApA; A—A, A7 us of ApApA; 0-0, A7 I18 of APA; and A-—A, 57 pg of ApApApAp. Samples were withdrawn at the times indicated and product formation was determined as des- cribed in "Experimental Procedure (Enzyme Assays)". (SeInUWJ) 3WLL OOI 08 09 017 02 OZI AMP INCORPORATION (nmoles/OI ml) 0 01 I O I 01 I N ‘I’ 51 Characterization 2f Reaction Products Stoichiometry 2f Reaction Products - As measured by means of the production of NADPH in the coupled assay with UDPG pyrophosphorylase ("Experimental Procedure (Enzyme Assays)"), inorganic perphosphate was shown to be a product of the reac— tion. The amount of inorganic pyrophosphate produced was equivalent to the amount of AMP incorporated into acid- insoluble product. In two separate experiments, when ll.A and 8.2 nmoles of inorganic pyrophosphate were produced in A0 minutes, 10.8 and 7.A nmoles of AMP were incorporated into polymeric product, respectively. Chain Length Determination 2; Polymeric Product - Alkaline hydrolysis of the acid-insoluble product formed when 3H-ATP was used as substrate in the standard reaction mixture, followed by paper chromatography of the hydrolysate in isobuty- ric acid : NH40H : H20, gave the results shown in Table IV. Ninety-five to ninety-six per cent of the radioactivity co- chromatographed with 2'(5')-AMP (Rf = 0.66), while 0.5 to 0.7 % moved with adenosine (Rf = 0.85). The remaining radio- activity chromatographed as a single peak with an Rf of 0.A6. Upon electrophoresis of the alkaline hydrolysate this material migrated slightly ahead of 2'(5')-AMP and was well separated 52 .oanwu osu ca vmumOHv:H muoan ecu :a can» nonuo wouooump mm: >ua>uuomofiumu oz .muw>wuomowpmu How ponmamam was EmHmOumEouso on» was omm u moemz "pace oguhusnomfi cg msamum uOumEoueo umama kn vmumumamm mama moosvona coaumwmuwmp magfimxam one .:Auospoum owumsmaom mo mwmzfioupzm ocmexHguowoawwu mo cofiusnfiuumap .uozpoua owuoaxaoa mo mwmzfimcm summed Cameo .>H macaw 55 from the adenosine di-, tri-, and tetraphosphate regions. Since it is known that, under the conditions employed, the hydrolysis of poly A may be only 95% complete (A7), this radioactive material most probably represented unhydrolyzed oligomer(s) of adenylic acid. The length of the average polymeric product can be approxi- mated by dividing the amount of 2'(5')-AMP, which represents the internal residues of the chain, by the amount of adenosine, which represents the 5'-external residue. The results presented in Table IV indicate a length in the range of 100 to 200 adenylate residues per chain. Incomplete hydrolysis of the product and the slow conversion of 2'(5')-AMP to adenosine (A8) imply that this approximation is a minimum value. The absence of any radioactive material which might represent a 5'-externa1 residue suggests that the adenylate chain probably was not synthesized d3 novo. Nearest Neighbor Analysis 2f Polymeric Product - When a-seP-ATP was used as substrate and the resultant product was subjected to alkaline hydrolysis and analysis by paper electro- phoresis, 9A% of the radioactivity migrated with 2'(5)-AMP (Table V). A small amount of radioactivity (about 5%, data 5A .mua>«uowofiku pom ponmamcm mm: momma ecu can mfimouosmouuooam ou monounsam mes mummzaoup»: oaaamxam one no uozuaam n< .:Auu:coum uaumahaom mo mam Imaouvzm Ocaamxafiuou0gpeu vauooHozagn opfluooaosc .uozpoua oauoahaoa mo mammamam Honswam: uwmumoz .> manna 55 not shown in Table V) migrated slightly ahead of 2'(5')'AMP, corresponding to the unhydrolyzed oligomer(s) of adenylic acid observed in the chain length analysis. The remainder of the radioactivity was divided among the other three common 2'(5')- ribonucleoside monophosphates. Some of the radioactivity in the 2'(5')-CMP region undoubtedly resulted from trailing of 2'(3)'AMP which migrates just in front of 2'(5')-CMP in this system. These data, along with the results of the chain length analysis, are consistent with a polymeric product composed of long chains of adenylate residues which are attached to the 5'-end of the added ribosomal RNA. Sucrose Density_Gradient Analysis 2f Polymeric Product - Reaction products, prepared in standard assay mixtures con- taining ribosomal RNA and 3H-ATP, were analyzed by SDS- sucrose density gradient centrifugation as described in the legend to Figure 12. The data presented in this figure show that the major portion of the product sedimented in the region of the 25 and 16 S ribosomal RNA species. Chains of adenylate residues, 100 to 200 nucleotides in length, would not be suffi- cient to produce these large sedimentation values. Since noncovalent binding of poly A to ribosomal RNA does not occur 56 Figure 12. Sucrose density gradient analysis of polymeric product. Reaction mixtures (0.25 ml) contained 50 mM.Tris-HCl (pH 8.5), 0.8 mM sH-ATP (A.5 x 103 cpm/nmole), 20 mM MgClg, 1.5 pg of the cellulose phos hate enzyme fraction, and P. putida ribosomal RNA, 55.6 pg in (A) and 556 pg in (B). After incu- bation for 19 minutes, 502 pg of ribosomal RNA was added to (A) and 119 pg of soluble RNA was added to each. At 20 min~ utes the reaction waSIIOpped by cooling in ice and the addition of 50 p1 of 1% SDS. Samples of 100 p1 were withdrawn from each and treated as described for the radioactive assay in "Experimental Procedure (Enzyme Assays)". Another 100 pl was withdrawn from each and layered on separate sucrose gradients (520%) in 50 mM Tris-HCl (pH 8.1) with 0.1% SDS. The grad- ients were centrifuged at 220 for 5 1/2 hours at 59,000 rpm in a Spinco SW 59 rotor. The tubes were punctured and A6 fractions were collected. The odd-numbered fractions were measured for absorbance at 260 mp, following the addition of 0.A ml of H20 to each, and the even-numbered fractions were used to determine the acid-insoluble radioactivity. Recovery of radioactivity was 87% in (A) and 92% in (B). 57 9...... Eng $554053“. Omw ._.< moz