ENZYMES or cvcuc NUCLEOSIDE MONOPHOSPHATEJ METABOLISM IN PEA SEEDUNGS ~ " Thesis fer the Degree of Ph. D. MICHSGAN STATE UNIVERSITY PAUL P0~ CHAD UN 1971 - -....L. Aer—mm.“- the-.515 L I B R A R Y Michige 3‘1 State University This is to certify that the thesis entitled Enzymes of Cyclic Nucleoside Monophosphate Metabolism in Pea Seedlings presented by Paul Po-Chao Lin has been accepted towards fulfillment of the requirements for 912.!) degree in May MM Major professor Date April 22, 1971 0-7639 ABSTRACT ENZYMES OF CYCLIC NUCLEOSIDE MONOPHOSPHATE METABOLISM IN PEA SEEDLINGS BY Paul Po-chao Lin Two 3'-nucleotidases have been isolated and par- tially purified from germinating pea seedlings. They pro- vide useful tools for the study of pea cyclic nucleotide phosphodiesterase. The 3'-nucleotidase I with a molecular weight of 70,000 shows maximal activity at pH 5.4-5.7. It catalyzes the hydrolysis of 3'—phosphoryl linkages of 3'-AMP, 3'-GMP, 3'-UMP and 3'-CMP, with little activity toward 2'-AMP and 5'-AMP. Several lines of evidence suggest that it does not catalyze the hydrolysis of RNA, DNA, or cyclic nucleoside monophosphates. The 3'-nucleotidase II has an optimal pH at 8.0 and a molecular weight of 30,000. It catalyzes the hydrolysis of 3'-AMP, 3'-GMP, and 3'-UMP, but not 3'-CMP, 2'-AMP, and 5'-AMP. This enzyme represents about 0.2% of the total protein of homogenates of seedlings. It seems to have RNase activity associated with it. Results from a variety Paul Po-chao Lin of experiments suggest that 3'-nucleotidase II and RNase activities reside in a single protein molecule. RNase from 3'-nucleotidase II preparation catalyzes the formation of 2',3'-cyAMP from polyadenylic acid. An enzyme capable of hydrolyzing both 2',3'-cyclic nucleoside monophosphate and 3',5'-cyclic nucleoside mono- phosphate has been found and partially purified from pea seedlings. It has a molecular weight of 350,000 and an optimal pH at 5.4—6.0. It is insensitive to methylxanthines and imidazole. It catalyzes the formation of 3'-AMP ex- clusively from 2',3'-cyAMP and the formation of 3'-AMP and 5'-AMP with a ratio of 3'-AMP:5'-AMP of about 7:1 from 3',5'-cyAMP. Because there is no interconversion between 3'-AMP and 5'-AMP, both 3'-AMP and 5'—AMP are direct products from 3',5'-cyAMP. The activities toward 2',3'-cyAMP and 3',5'-cyAMP are quite similarly affected by pH, metal ions, sulfhydryl reagents, temperature, and urea. Furthermore, the two activities have similar physical prOperties. It is suggested, therefore, that a single enzyme molecule is responsible for both activities. Acti- vation energy for hydrolysis of 2',3'-cyAMP is 8.6 Kcal/mole and of 3',5'-cyAMP is 7.2 Kcal/mole. Since several lines of evidence indicate that pea cyclic nucleotide phosphodiesterase is not the enzyme which hydrolyzes RNA, a new mode of RNA degradation in higher plants, at least in pea seedlings, is proposed. This is Paul Po-chao Lin that RNase (cyclizing enzyme) may function only in cata- lyzing the formation of 2',3'—cyNMP. Further hydrolysis of 2',3'-cyclic nucleoside monophosphate is due to cyclic nucleotide phosphodiesterase. ENZYMES OF CYCLIC NUCLEOSIDE MONOPHOSPHATE METABOLISM IN PEA SEEDLINGS BY Paul Po-chao Lin A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1971 ‘ "7 ' C / DEDICATION To my mother, my wife, and my son ii ACKNOWLEDGMENTS I would like to express my sincere gratitude to Dr. J. E. Varner for his guidance and encouragement and for the freedom to learn during the course of this study. Thanks are extended to Drs. James L. Fairley, Hans Kende, and Clarence H. Suelter for serving on my guidance com- mittee. Appreciation is also expressed to the United States Atomic Energy Commission for providing the funds to support this investigation under the contract No. A-T (ll-l)-1338. iii TABLE OF CONTENTS Part Page I. THE PURIFICATION AND CHARACTERIZATION OF 3'-NUCLEOTIDASE FROM PEA SEEDLINGS . . . . 1 Introduction . . . . . . . . . . 1 Experimental Procedures . . . . . . . 2 Materials . . . . . . . . . . . 2 Methods . . . . . . . . . . . 3 Growth of Pea Seedlings . . . . . 3 Assay of 3'— Nucleotidase . . . . . 3 Assays of RNase and DNase . . . . 4 Determination of Protein Content . . 5 Preparation of DEAE- -Cellulose Ion Exchange Resin . . . . . . . . 5 Polyacrylamide Disc—Gel Electrophoresis . . . . . . . . 6 Sucrose Density Gradient Centrifugation . . . . . . . . 7 Electrofocusing Column Chromatography . . . . . . . . 8 Dowex Ion Exchange Chromatography . . 10 Experimental Results . . . . . . . . ll Purification of Enzymes . . . . . . 11 Preparation of Crude Enzyme Extract . ll Ammonium Sulfate Fractionation . . . ll Dialysis and Freezing of 50—80% Ammonium Sulfate Fraction . . . . . 12 DEAE-Cellulose Chromatography . . . 12 Chromatography of 3'-Nucleotidase I on Sephadex G- 100 Column . . . 16 Chromatography of 3'-Nucleotidase II on Sephadex G- -75 Column . . . . 21 Sucrose Density Gradient Centrifugation . . . . . . 24 Electrofocusing Column Chromatography of 3'-Nucleotidase II . . . . . . 24 iv Part II. Electrofocusing Column Chroma- tography of 3'-Nucleotidase I . . Polyacrylamide Disc-Gel Electro- phoresis of 3'—Nucleotidase II . Polyacrylamide Disc-Gel Electro- phoresis of 3'-Nucleotidase I . . Properties of the Enzyme Preparations Rate of Hydrolysis as a Function of pH and Zn++ . . . . . . . Effect of Metal Ions and Inorganic Ions on Enzyme Activity . . . . Effect of Sulfhydryl Compounds on Enzyme Activity . . . . Effect of Glycine and. Zn+ on the Inactivation of 3'-nucleotidase II at pH 5.0 . . . . Substrate Specificity, Relative Activities and Km Study . . . . Activity Toward Cyclic Nucleoside Monophosphates . . . . The Mode of the Action of 3'— Nucleotidase II on Poly A . . . Estimation of Molecular Weight, Diffusion Constant and Stokes' Radius for 3'-Nucleotidases . . Discussion . . . . . . . . . Summary . . . . . . . . . . Bibliography . . . . . . . . . THE PURIFICATION AND CHARACTERIZATION OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE FROM PEA SEEDLINGS . . . . . . . Introduction . . . . . . . . Experimental Procedures . . . . . Materials . . . . . . . . . Methods . . . . . . . . . . Thin-Layer Chromatography . . . Enzymatic and Chemical Assays Of CYNPDE I O O O O O O O Page 27 28 33 36 36 36 4O 43 43 45 47 54 56 62 64 68 68 69 69 70 71 74 Part Page Experimental Results . . . . . . . . 76 Purification of Enzyme . . . . . . 76 Preparation of Crude Extract of cyNPDE . . . . . . . . . 76 Ammonium Sulfate Fractionation . . . 77 Treatment at pH 5.0 . . . . . . . 78 Chromatography of cyNPDE on Sephadex G-200 Column . . . . . . 78 Further Attempts to Separate the Two Activities . . . . . . . . . 82 Sucrose Density Gradient Centrifugation . . . . . . . 82 Polyacrylamide Disc— —Gel Electro- phoresis . . . . . . . . . . 86 Electrofocusing Column Chroma- tography of cyNPDE . . . . . . . 86 Characterization of the Reaction PrOdUCtS O O O O O O O O O O O 89 Action on 2', 3'— —Cyclic Nucleoside Monophosphates . . . . . . . 89 Action of 3' ,5'-Cyclic Nucleoside Monophosphates . . . . . . . 93 Sucrose Density Gradient Study of the cyNPDE and Time Course of the Formation of 3'-AMP and 5'-AMP From 3',S'-cyAMP . . . . . . . . 102 Properties of the cyNPDE Enzyme . . . 108 Time Course and Enzyme Concentra— tion . . . . . . . . . . 108 Effect of pH on Enzyme Activity and Stability . . . . . . . . . 108 Influence of Various Metal Ions . . . 115 Effect of Various Concentration of Sulfhydryl Compounds and NaF . . . . 115 Effect of Urea on Enzyme Activity . . 117 Optimum Temperature and Heat Stability of Enzyme Activity . . . . 117 Effect of Organic Compounds on Enzyme Activity . . . . . 127 Rate of Hydrolysis of Cyclic Nucleoside Monophosphates . . . . . 131 vi Part Page Determination of Michaelis Constant (Km) . . . . . . . . . 133 Activity Toward Other Organic Phosphates . . . . . . . . . . 133 Discussion . . . . . . . . . . . 143 Summary . . . . . . . . . . . 147 Bibliography . . . . . . . . . . . 149 vii Table LIST OF TABLES PART I Summary of Purification of 3'-Nucleotidase I from 100 g of Pea Seedlings . . . . Summary of Purification of 3'-Nucleotidase II and RNase from 100 g of Pea Seedlings . . . . . . . . . . Effect of Zn++ on the Optimum pH for the Activities of 3'-Nucleotidase I and 3'-Nuc1eotidase II . . . . . . Effect of Inorganic Ions, Caffeine and Theophylline on 3'-Nuc1eotidase ACtiVity O O I O O O O O I 0 0 Effect of Various Sulfhydryl Compounds on 3'—Nucleotidase Activity . . . . Effect of Various Concentrations of Zn++ and Glycine on Acidic Inactivation of 3'-Nuc1eotidase II and RNase . . . . Relative 3'-Nucleotidase Activities Toward Ribonucleoside Monophosphates . Summary of Well-Characterized RNase from Higher Plants . . . . . . . Physical Properties and Elution Data (from Sephadex G-200) of Standard Proteins and Pea 3'-Nuc1eotidases . . PART II Rf Values of Bases, Nucleosides, and Nucleotides in Thin-Layer Chroma- tography O O O O O I I O O O 0 viii Page 17 18 39 41 42 44 46 53 55 73 Table Page 2. Summary of Purification of Cyclic Nucleotide Phosphodiesterase from 300 g of Pea Seedlings . . . . . . . 81 3. Enzymatic Analysis of the Hydrolysis Product Formed from 2',3'-cyNMP . . . . 92 4. Effect of Inorganic Ions on the Activity of Cyclic Nucleotide Phos— phodiesterase . . . . . . . . . . 116 5. Effect of Various Concentrations of Reducing Reagents and NaF on the Activity of Pea Cyclic Nucleotide Phosphodiesterase . . . . . . . . 118 6. Effect of Organic Compounds on the Activity of Pea cyNPDE Using 3H-3',5'—cyAMP as Substrate . . . . . 130 7. Relative cyNPDE Activities Toward Cyclic Nucleoside Monophosphates . . . 132 8. Summary of Well-Characterized 3',5'-Cyc1ic Nucleotide Phospho- diesterases . . . . . . . . . . 140 ix LIST OF FIGURES Figure PART I l. Elution Profile of Pea 3'-Nucleotidase Activities from DEAE—Cellulose Column Chromatography . . . . . . . 2. Chromatography of 3'-Nucleotidase I on Sephadex G-100 Column . . . . . . 3. Chromatography of 3'—Nucleotidase II on Sephadex G-75 Column . . . . . 4. Elution Pattern of 3'-Nucleotidase II, RNase, and DNase from Sucrose Density Gradient Centrifugation . . 5. Elution Profiles of Pea 3'-Nucleotidase from an Electrofocusing Column Chroma- tography . . . . . . . . . . . 6. The Staining Pattern of Protein and the Distribution of RNase and 3'- Nucleotidase II on a Polyacrylamide Gel after Electrophoresis . . . . . 7. The Staining Pattern of Protein and Distribution of 3'-Nuc1eotidase I on a Polyacrylamide Gel after Electrophoresis . . . . . . . . . 8. Effect of pH on the Activity of Pea 3 ' "NUCleOtidase o o o o o o o o c 9. Ion Exchange Chromatography of the Hydrolysis Product of Poly A . . . . 10. Thin-Layer Chromatography of the Hydrolysis Products Obtained from the Action of RNase (3'—Nuc1eotidase II) on Poly A . . . . . . . . . Page 13 19 22 25 29 31 34 37 49 51 Figure 2 Page 11. Calibration Curve for Molecular Weight Determination on Sephadex G-200 Column Chromatography . . . . . . . . 57 PART I I 1. Elution Profile of Pea cyNPDE from Sephadex G-200 Column Chromatography . . . 79 2. The Elution Profiles of cyNPDE Activi- ties from Sucrose Density Gradient Centrifugation . . . . . . . . . . 83 3. Staining Pattern of Protein and Dis- tribution of Enzyme Activities within a Polyacrylamide Gel after Electrophoresis . . . . . . . . . . 87 4. Elution Profile of Pea cyNPDE from an Electrofocusing Column Chromatography . . 90 5. Ion Exchange Chromatography of the Hydrolysis Product of 2',3'-cyAMP . . . . 94 6. Thin-Layer Chromatography of the Hydrolysis Products Obtained from the Action of Pea cyNPDE on 3H-3',5'-cyAMP . . . . . . . . . . 97 7. Thin-Layer Chromatography and Enzymatic Analysis of the Hydrolysis Products Obtained from the Action of Pea cyNPDE on 3H-3',5'-cyAMP . . . . . . . . . 100 8. Analysis of End Product Formation as a Function of Time from Enzymatic Hydrolysis of 3H-3',5'-cyAMP . . . . . 103 9. End Products Formed from Enzymatic Hydrolysis of 3H-3',5'-cyAMP . . . . . 106 .10. Time Course of cyNMP Breakdown by Pea cyNPDE . . . . . . . . . . . . . 109 11. Activity of Pea cyNPDE as a Function of Protein Concentration . . . . . . . 111 12. Effect of pH on the Activity of Pea cyNPDE . . . . . . . . . . . . . 113 xi Figure Page 13. Effect of the Concentration of Urea on the Activity of Pea cyNPDE . . . . . 119 14. Time Courses of the Effect of Urea on cyNPDE Activity . . . . . . . . 121 15. Temperature-Activity Profile for Pea cyNPDE . . . . . . . . . . . 123 16. Arrhenius Plot for the Determination of the Activation Energy . . . . . . 125 17. Heat Stability of Pea cyNPDE . . . . . . 128 18. Effect of Substrate (2',3'-cyNMP Concentration on the Activity of Pea Cyclic Nucleotide Phospho- diesterase . . . . . . . . . . . 134 19. Effect of Substrate (3',5'—cyNMP Concentration on the Activity of Pea Cyclic Nucleotide Phospho- diesterase . . . . . . . . . . . 136 20. The Elution Profiles of Enzyme Activities from Sephadex G-200 Column Chromatography . . . . . . . 138 21a The Elution Profiles of cyNPDE, RNase, and 3'-Nucleotidases from Sucrose Density Gradient Centrifu— gation . . . . . . . . . . . . 141 xii ADP ATP DNA DNase EDTA LFDP G-l-P G-6—P 2'-NMP 3'-NMP 5'-—NMP 2' ,3'-cyNMP 3' ,5'-cyNMP LIST OF ABBREVIATIONS 5'-diphosphate of adenosine 5'-triphosphate of adenosine deoxyribonucleic acid deoxyribonuclease ethylenediaminetetraacetate fructose-1,6-diphosphate glucose-l-phosphate g1ucose-6-phosphate 2'-nucleoside monophosphate 3'-nucleoside monophosphate 5'—nuc1eoside monOphosphate (e.q.2'-AMP, etc.) (e.q.3'-AMP, etc.) (e.q.5'—AMP, etc.) 2',3'-cyclic nucleoside monophosphate (e.q.2',3'-cyAMP, etc.) 3',5'-cyclic nucleoside monophosphate (e.q.3',5'-cyAMP, etc.) cyclic nucleotide phosphodiesterase inorganic orthophosphate p—nitrophenol phosphate polyadenylic acid inorganic pyrophosphate ribonucleic acid ribonuclease 820 w Svedberg unit (sedimentation coefficient in ' water at 20°)- One Svedberg = l x 10‘13sec. TCA trichloroacetic acid Tris tris-(hydroxymethyl) aminomethane xiv PART I THE PURIFICATION AND CHARACTERIZATION OF 3'-NUCLEOTIDASE FROM PEA SEEDLINGS Introduction Although 5'—nuc1eotidase is widely distributed in mammalian tissues (1-7) , enzymes capable of nucleotidase activity studied from various plant sources have been shown to have high specificity toward 3'—ribonuc1eotides (8-15). Furthermore, most of the 3'-nucleotidases in higher plants have been demonstrated to accompany nuclease activities (12, 14, 19). Whether the two activities were due to the same or to different enzyme molecules was not clear. The 3'-nuc1eotidases so far isolated and character- ized from higher plants in general work best on purine 3'-nucleotides (12, 15, 16). Most of the RNases studied in higher plants have been shown to be cyclizing enzymes which catalyze the formation of 2',3‘-cyNMP, with little activity toWard 2' ,3'—cyAMP and 2' ,3'-cyGMP, but not pyrimidine 2' p3'—cyNMP derivatives (12, 19, 28). However, recent Studies of rye grass (29) and sugar cane (30) RNases showed that these RNases could not hydrolyze 2' ,3'—cyNMP. Preliminary experiments indicated that germinating pea seedlings contained two 3'-nuc1eotidases. Since little work has been done on pea nucleotidase and RNase and since it was desirable to find enzymes which could serve as tools in the enzyme coupled assay of pea cyclic nucleotide phos- phodiesterase as described in the Part II, it was desirable to purify the pea 3'-nuc1eotidases and to characterize them. Part I presents the procedures for isolation, puri- fication, and characterization of the general chemical and physical properties of these 3'-nuc1eotidases. Attempts to separate the activities of 3'—nuc1eotidase II and RNase by a variety of chemical and physical means will be described. The substrate specificity of 3'-nuc1eotidase and a suggested mode of the action of RNase are also presented. Experimental Procedure Materials Early Alaska peas (Pisum satirum, Var.) were used for all enzyme preparations and were purchased from the Vaughan's Seed Co., Chicago, Illinois. The following com— pounds were commercial samples purchased from various sup- pliers as indicated: nucleotide and nucleoside derivatives, glutathione, dithiothreitol, cysteine, Coomassie blue, tris(hydroxymethyl) aminomethane, Sigma; calf thymus DNA, Worthington; DEAE-cellulose ion exchanger (0.7 meq/gm), Gallard-Schlesinger chemical; Sephadex products and blue dextran, Pharmacia; Lypogel, Gelman; enzyme protein molecu- lar weight markers, sucrose and ammonium sulfate (special enzyme grade), Mann Research Laboratory; polyadenylic acid and polyadenylic acid-8-Cl4 (0.154 uCi/mg), Miles Labora- tory; cellulose powder MN 300 (Brinkmann), Macherey and Nagel Co.; BioRad Ag-l-X 2, 400 mesh, chloride form ion exchanger, BioRad Laboratory; acrylamide, TEMED(N,N,N'-N'- tetramethylethylenediamine), BIS-acrylamide(N,N'-methylene- bisacrylamide), Eastman Kodak. A11 standard chemicals were reagent grade, and were used without further purification with the exception of acrylamide and BIS-acrylamide which were recrystallized twice from chloroform and acetone, re- spectively. High molecular weight ribosomal RNA was pre- pared from commercial yeast by the method of Crestfield et a1. (31). Methods Growth of Pea Seedlings.-—A1aska peas were surface- sterilized for 20 min in 1% sodium hypochlorite, rinsed with sterile distilled water several times, and planted in a 4-liter Erlenmeyer Flask containing moist, sterile vermiculite. After germination at 23° in the dark for about one week, seed were removed and rinsed with distilled water. Assay_of 3'-Nuc1eotidase.-—The assay measured the release of Pi from nucleoside monophosphate. The standard reaction mixture contained 0.1M K-acetate buffer, pH 5.4, or Tris-acetate buffer, pH 8.0, and 2 mM nucleoside mono- phosphate with a suitable dilution of the enzyme preparation being assayed in a total volume of 0.5 ml. The reaction mixture was incubated at 37° for 10 to 30 minutes, and the reaction terminated by the addition of 0.05 ml of cold 55% TCA. After standing in an ice bath for 15 min, the precipi— tate formed was removed by centrifugation at top speed of the International clinical centrifuge (2,000 rpm) for 10 min. The resulting supernatant was analyzed for Pi by the method of Fiske and SubbaRow (32), modified as follows: SO was added to 2 4 1.0 m1 aliquot of the supernatant solution (diluted with 0.2 ml of 2.5% ammonium molybdate in 5 N H distilled water). The color was developed by the further addition of 0.1 m1 of reducer (100 ml solution contained 0.2 g of 1-amino-2-naphthol-4-su1fonic acid, 1.2 g of sodium bisulfite and 1.2 g of sodium sulfite) and read at 660 mu on a Beckman D.U. spectrophotometer with a Gilford digital absorbance meter. A standard curve relating the absorbance to the concentration of Pi (KH2P04 as the stand- ard) was constructed for each assay. This standard curve was not affected by the presence of enzyme solution or TCA. One unit of nucleotidase activity is defined as that amount of enzyme which causes the release of 0.1 umole of Pi per 30 min under the assay conditions described above. Assays of RNase and DNase.--RNase was assayed according to the procedures of McDonald (33) and Ibuki gE_gl. (34). The reaction mixture contained 0.1 M Tris- acetate buffer, pH 6.5, 0.2 mg of yeast ribosomal RNA and an appropriately diluted enzyme preparation in a total volume of 0.5 m1. Incubation was carried out at 37° for 30 min. At the end of the incubation, 0.5 m1 of cold 3 mM uranyl acetate in 0.2 N HC1 was added. The precipitate formed after standing at 4° for 15 min was removed by centrifugation and the resulting supernatant was diluted to 3.0 ml with distilled water. The absorbance at 260 mu was then measured. One unit of RNase activity is defined as that amount of enzyme which causes an increase in the ab- sorbance at 260 mu of 0.1 unit per 30 min incubation under the assay conditions. Assay of DNase activity was essen- tially the same as that described for the RNase activity with the exception that denatured calf thymus DNA (heated 10 min at 100°, followed by quick cooling) was used instead of ribosomal RNA as substrate. One unit of DNase is de- fined as previously described for RNase. Determination of Protein Content.-—Protein concen— tration was determined according to the method of Lowry §£_§1. (35) with crystalline bovine serum albumin as a standard. Colorimetric readings were made at 660 mu. Spe— czific activity of the enzyme is defined as units per mg of Protein . Preparation of DEAE—Cellulose Ion Exchange Resin.-- DEAJE-cellulose with a capacity of 0.7 meq/g was readied for use (without acid and base treatments) by suspending 30 g in 2 liters of deionized water and pouring off the finer particles five times. The slurry was then washed with two 500 m1 of 0.01 M Tris-acetate buffer, pH 7.5 and stored at 4° in the same buffer prior to use. Polyacrylamide Disc—Gel E1ectr9phoresis.--The apparatus used in this gel electrophoresis was similar to that described by Ornstein (36) and Davis (37) with the following modifications. The glass tubes were 0.5 cm i.d.x 11.5 cm long. The height of the polyacrylamide gel columns were 8.5 cm and spacer gels were 0.5 cm. The con- centration of all the running gels were 7% (w/v). The stock solutions were prepared as follows: a. 48 m1 of 1 N HC1, 36.6 g of Tris, 0.23 ml of TEMED, and water to 100 m1. b. 28.0 g of acrylamide (2x crystallized), 0.735 g of BIS-acrylamide (2x crystallized), and water to 100 m1. c. 4 mg of riboflavin, and water to 100 m1. d. 48 m1 of l N HC1, 5.98 g of Tris, 0.46 ml of TEMED, and water to 100 m1. e. 10 g of acrylamide (2x crystallized), 2.5 g of BIS-acrylamide (2x crystallized), and water to 100 m1. f. 40 g of sucrose, and water to 100 m1. The running gel contained 0.5 part (a), 2 parts (b), 1 part (c), and 4.5 parts water. The spacer gel contained 1 part (d), 2 parts (e), 1 part (c), and 4 parts (f). Buffer for electrodes contained 0.6 g of Tris, 2.9 g of glycine and water to 1 liter, pH 8.5. Sample, 0.3 m1 of enzyme prepara- tion, was routinely layered onto the spacer gel by displace- ment of electrode buffer. Electrophoresis was performed at 4° for 45 min with a constant current of 2 mA per tube. On completion of the electrophoresis, the gels were carefully removed under water by needling and air pressure. The protein bands were located by a method similar to that described by Chrambach gE_§1. (38). The gel was stained for a minimum of 2 hr in 0.05% Coomassie blue (prepared in 12.5% TCA) and destained by diffusion in either distilled water or 5% TCA. In some cases, the gel was removed from the tube and divided into two parts along the longitudinal axis. One—half of the gel was stained for protein bands. The other half was sliced and assayed for enzyme activities under the assay conditions previously described. Sucrose Density Gradient Centrifugation.--The .linear sucrose density gradient was prepared according to true method of Martin and Ames (39) by a device which con— sists of two chambers, A and B interconnected with each otfluer when a needle valve was opened. Chamber A, loaded witdi 2.2 m1 of 20% (w/v) sucrose, was that one from which the gradient solution was delivered. Chamber B was filled with 2.4 m1 of 5% sucrose. In general the sucrose solu- tions also contained a buffer. The gradient was made in 1/2" x 2" of Beckman cellulose nitrate tube and allowed to stand at least 1 hr at 4° to smooth out before the sample (0.2-0.25 ml) was layered on the gradient. A swinging bucket rotor, SW 65 LTi (Beckman) was used for centrifugation which was routinely performed at 2° in a Beckman L-2 65B ultracentrifuge. Upon completion of the run, 10-drop fractions were collected after needle puncture of the bottom of the tube. Enzyme assays were carried out in alternate fractions with two different sub- strates for each gradient set. Electrofocusing Column Chromatography.--An LKB model 8101 electrofocusing column with a total capacity of 110 ml was used. Ampholyte carrier solution (Ampholine, LKB) and sucrose were used in order to establish a pH gradient with a density gradient. Electrofocusing was done according to the methods described in the LKB manual. The composition of gradient solution and electrode solution for the electrofocusing in the pH range from 3.0 to 6.0 were as follows: 1. Dense gradient solution: Ampholyte (40%) --------- 1.9 ml Sucrose ----------------- 28 g Distilled water --------- to 55 ml 2. Less dense gradient solution: Ampholyte (40%) --------- 0.6 ml Enzyme solution --------- varied Distilled water --------- to 55 m1 3. Dense electrode solution: NaOH -------------------- 0.3 g Sucrose ----------------- 18 g Distilled water --------- 21 m1 4. Less dense electrode solution: H2804 (conc.) ——————————— 0.1 ml Distilled water --------- to 10 m1 A potential of 350 volts (kept constant throughout the whole procedure) was applied to the column for a period of 48 hr with the aid of a Buchler model No. 3-1014 A voltage and current regulated D.C. power supply. The working temperature was maintained at 2° by circulating water and methanol solution from a thermostat water cooler, LAUDA model WB-20/R (Brinkman Instruments) through the external and internal jackets. The electric current of the electrofocusing unit gradually drOpped as proteins settled at their isoelectric ‘points along the linear pH gradient. Eventually, the cur- rrent stabilized at less than 1.0 mA indicating nearly all ifihe proteins in the electric field have been neutralized éit their isoelectric points. After completion of the run, 80~wdrop fractions were collected by draining the gradient 10 solution from the bottom of the column at a flow rate of 1 ml/min with the aid of a Gilson fraction collector. Since ampholine was found to form a precipitate with ammonium molybdate (2.5% in 5 N H 804) which was used 2 for Pi assay as previously described in the method of Fiske and SubbaRow (32), it was removed from the fractions before enzyme assays were carried out. Therefore, soon after the determination of pH, fractions within the pH range from 3.0 to 6.0 were dialyzed against 1 M NaCl solu- tion at 4° over night, then against deionized water for 6 hr. The resulting dialyzed fractions were free of ampholine and were assayed for enzyme activity. Dowex Ion Exchange Chromatography.--Although 2'-AMP, 3'-AMP, and 2',3'-cyAMP could be separated by thin-layer chromatography, the quantitative detection was not sensitive enough to tell whether 2',3'-cyAMP was the exclusive product from the action of pea RNase on synthetic polyadenylic acid. Volkin and Carter (40) have described the separation of 2'-AMP and 3'-AMP on Dowex-l-Cl-, 400 mesh column. For that procedure, a column of BioRad Ag-l-XZ, chloride form, 400 mesh, 0.5 x 5 cm, was first equilibrated with 2 mM HC1 and then calibrated by chromatographing the mix of authentic czompounds 2' and 3' isomers of adenylic acid and 2',3'- CYAMW. The same ionic strength of HC1 was used as the elliting solvent. Fractions of 60-drops (approximately 3.4 101) *were collected with a flow rate of 12 drops per min and 11 the absorbance measured at 260 mu. The column was regener- ated by washing with 50 ml of l N NaOH, 100 ml of distilled water, 50 m1 1 N HC1 and 200 ml of 2 mM HC1, in that order. Experimental Results Purification of Enzymes Preparation of Crude Enzyme Extract.--(All proce- dures in enzyme fractionation were performed at 4° or in ice bath unless otherwise specified.) Routinely, 100 g of a week-old pea seedlings, germinated in sterile vermiculite in the dark, was homogen- ized with 100 ml of deionized water for 1-2 min in a com- mercial Waring Blender. The homogenate was first squeezed through double-layered cheesecloth to remove the bulk of insoluble material, then centrifuged at 4,000 x g for 30 min. The resulting supernatant fluid was decanted through deionized water washed glass wool. The filtrate was taken as the crude enzyme extract for further purification. Ammonium Sulfate Fractionations.-—The crude extract was brought to 50% saturation by slowly adding solid ammonium sulfate (29.5 g per 100 ml of enzyme extract) (41). The solution was stirred for 30 min, and the precipitate was removed by centrifugation at 10,000 x g for 20 min and discarded. The clear supernatant fluid was decanted and brought to 80% saturation with the addition of 19.7 g of 12 solid ammonium sulfate per 100 m1 of extracted solution. After stirring and standing for at least 1 hr, the precipi- tate was collected by centrifugation at 10,000 x g for 30 min, and dissolved in 2 mM Tris—acetate buffer, pH 7.5, by vigorous mechanical stirring. The resulting enzyme solution was then taken for dialysis. Dialysis and Freezing of 50-80% Ammonium Sulfate Fraction.--The resulting enzyme solution was dialyzed in 2.3 cm diameter dialysis tubing (Union Carbide) against 20 volumes of 2 mM Tris-acetate buffer, pH 7.5, with constant agitation for 24 hr, with three changes. After dialysis, the enzyme solution was centrifuged at 10,000 x g for 10 min in order to remove a small amount of precipitate which often formed during the dialysis. Although the precipitate contained some activity, the specific activity was too low to be saved. The supernatant fluid was then fractionated further or was frozen at —20°. DEAE-cellulose Chromatography.--The dialyzed solu- tion, 25 ml (47 mg of protein/m1) was applied to a DEAE— cellulose column (2.5 x 25 cm) which had previously been equilibrated with 2 liters of 0.01 M Tris-acetate buffer (pH 7.5) at a flow rate of 1 ml per min, regulated with a polystaltic pump (Bucher Instruments). After loading the enzyme solution on the column, the adsorbent was washed with 120 ml of the same buffer with which it was equilibrated. 13 A step-gradient of NaCl solution (prepared in the same buffer as previously mentioned) was applied to the column. Fractions (8 ml) were collected and the absorbance measured at 280 mu. Enzyme assays were performed as pre- viously described. The residual material in the column was washed out with 1 M NaCl solution, and the column regener- ated by the method of Peterson and Sober (42). The elution profile of protein and enzyme activities is shown in Figure 1. The 3'-nucleotidase activity was located by assaying 0.1 m1 of each fraction with either 3'-CMP or 3'-AMP as substrate. Buffer of 0.1 M K—acetate, pH 5.4, was used for the assay of 3'-nucleotidase I activity when 3'-CMP served as substrate. A reaction mixture con- taining 0.1 M Tris-acetate buffer (pH 8.0), 2 mM ZnCl and 2: 2 mM 3'-AMP was used for the assay of 3'-nuc1eotidase II activity. Evidently, 3'-nucleotidase I activity was eluted by 0.1 M NaCl solution between fractions 52 and 70, without detectable contamination of 3'—nuc1eotidase II activity. With 0.2 M NaCl solution, 3'-nucleotidase II activity was eluted between fractions 83 and 104. Although there was a minor amount of 3'-nucleotidase I in the fraction of 3'- nucleotidase II, this impurity could be eliminated by further purification (Sephadex gel filtration). RNase and DNase activities (not shown in the figure) were located mainly between fractions 82 and 120. Fractions containing l4 =.m©ocumz= cam uxmu map cw cm>Hm mum monocmooum Hmucmsflummxm emaflmumo .Ao ..... o .o.m mm .mumuumbsm was we age-.mv HH mmmefloomaosc -.m new A.--. .e.m mm .mpmubmbsm mm ago-.mc H mmmefluomaosc-.m mo AHs\muHcs may mmHuH>Huom mahnco ucmmmummu mmcfla Umcmmo .18 omm um mocmonomom an Umuommme mm coflumuucmocoo samuonm mopmoflocfl mafia UHHOm mce .>DH>Huom memncm How UmmMmmm mcofluomum mag cam .18 owm um Umuommme mUCMQHOQO map .pmuomaaoo mum3 mcoflpomum He-ucmflm .msouum HMOHuHm> ho Umuwoflccw mm cEdHoo map on Umflammm mums .z N.o can 2 H.o .Anmmmon dawn on» Ca conmmmnmv coflumnucmocoo Homz mo mucmflcmnm mmum 039 .Hdmme mEMm may mo HE oma cows mcflcmm3 can ceoaoo map so coflumnmmmnm mamucm ecu mcflumsma umume .cfls\as H mo mums 30am m on Am.s may smudge «seamen-mane z Ho.o mo mumofia N cufl3 owumuoflaasom >Hm50H>mum A80 mm x m.mv cEsHoo wmoaoaamo-mflmo m ow omflammm mm3 AHE\chpoum me N.hvv HE mm .cofluomum monuaom EdHcoEEm cmnwamflo .wcmmumODMEouco cEdHoo mmoHDHHoo Imemo Eoum mmHuH>Huom ommcflpomaosc-.m mom mo maflmoum cofloon-I.H mudmflm 15 OBZV . HH 88:83..qu zwmiaz 20:03.“. O o --________---. o.” -. o 1 IL .82 28 I18. IT. 80. 3. _8lmNcm Op moo mmz >uH>Huom owwfloomm 30H ones 0.0 m00H 000 0.0 coflumuuaflm 000-6 xmemnmmm 0.0 00 000.0 0.00 mmoasaamo-mama 0.00 00 00H.NH 00m eommxamzv 000-00 000 00 000.00 000a ucmnmcummsm 0.x 000.0 m mE\muflcs moans me ”00% £00. CHM. .mmcflacmom mom mo m 00H Eonm H mmmcfluooaosc-.m mo coflpmowmwuom mo mumEEdm-I.H mam¢9 18 m.H mm oav mmo.oa mm moo 0mm.va 0m mnlo xmomcmmm m.H mm mm www.ma om mmH oom.ma oma mmoHoHHmolm4mo 0.0 mm 00 000.00 00 00 000.00 000 00mmx0mzc 000-00 m.H ooa mm 000.00 ooa H0 mma.0m mmma ucmumcummom m x 000.0 w mE\muHco mafia: w mE\mUHco moans m8 .060 .mm cw mmmzm UHmHM >u0>fluo< >u0>0uo¢ Gama» >u0>flpo< mufl>fluo< vaunommm .00000 camaomam Hmuoe cfimuoum coduomum HH mmmcflu Hmuoe . Iowaooz-.m mmmzm HH ommofluomaooz-.m mo 000mm .mmcflacmmm mom mo m 000 Eoum mmmzm com HH mmmcfluomaoscl.m mo cowumoHMHnom mo xumfifiomlu.m mqmfia 19 ..----. .10.m mm .eumnum Icon mm QED-.mv >u0>fluoe H emecfluoeaoozl.m .Illlll .18 0mm um eocecHOmce ecu me ceuomeee mez coflumuuceocoo cfleuoum =.mcocue2= mews: cecfluomep we erH0muem euez maemme eE>Ncm .uc\HE NH we cecfleucfiee mez econ 300m ecB .mcoflpoenm HE m CH Hemmoc eEem ecu cufiz oepoae cce Am.h may Hemmsc eumueoelmflue z Ho.o cuflz cepeucflaflove ceec cec coHcB A80 om x m.Hv GEDHoo e on ceflamme me3 cfleuoum m0 m5 om mcflcfieucoo cofluoeum eEMNce emoHoHHeo-mcmo ceueuuceocoo Hemocmwa mo HE m.m mo ucooee cc .cEdHoo OOH-u xececmem :0 H emecflooeaoocl.m mo wcmeHmOpeEouc0-.m euomflm 20 ("IWISlINfl ) AllAllDV V I Sephadex G—100 I I r 60 50 30 F RAC'I’ ION NUMBER oszv 21 Although about 90% of the applied protein was re- moved from the major enzyme fraction, the recovery of enzyme activity was only 10%. Therefore, it was only about 2-fold increased in specific activity. The reason for such low recovery is given in the "Discussion." The enzyme preparation was stored in 3 ml quantities at -20°. The summary of the purification of 3'-nucleotidase I from 100 g of pea seedlings is given in Table l, to which all values were calculated based on 3'-CMP as substrate. Chromatographyyof 3'-Nuc1eotidase II on Sephadex G-75 Column.—-The procedure for the preparation of a Sephadex G-75 column (1.5 x 60 cm) was essentially the same as described for Sephadex G-100 column with the exception that the flow rate was 14 ml per hr. The 3'-nucleotidase II preparation from the DEAE-cellulose column chromatogram, 5 ml, containing about 40 mg of protein, was applied to a column which had been equilibrated with 0.01 M Tris-acetate buffer (pH 7.5) and eluted in 4-ml fractions. Figure 3 shows the elution profile of protein and enzyme activities. This purification step routinely gave 80% recovery of 3'- nucleotidase II and RNase activities applied to the column, and an increase in specific activity of about 4-fold. The enzume activities toward 3'-AMP, RNA and de- natured DNA were eluted between fractions 15 and 30. The peaks of the three activities coincided at the same frac- tion. Further attempts to separate these three activities 22 .<-|-< .emezo “on-Ila .emezm ..--. .10.0 mm .eumuumbsm mm mz<-.mc >00>Huom HH emmeflbomHosc-.m was . .nE omm um eocecu0mce ecu we venomeee me3 coHueuuceocoo cHeuoum :.mcocuez= mecca cecHHOmec we ceEHOMHem euez mwemme eE>Ncm .uc\HE «H was even onm ecB .mcoHuomnm HE 0 GH Hemmoc eEem ecu cUH3 cepoHe one Am.n mac nemwoc euepeoe-mHHB z Ho.o cuHB ceueucHHHooe meB coHc3 AEo om x m.HV cEdHoo e on ceHHmme mes cHeooum me 00 mchHeu Icoo coHueummeHm eewuce emoHoHHeo-m¢mo ceuenuceocoo Hemocth mo HE e>Hm .cEdHoo mnlo xeoecmem :0 HH emecHuoeHooc-.m mo mcmeumoueeoncO-I.m euomHm 23 ('1W/Slan)AllAllDV O V o 52‘. 3 «32:2 ZO_._.U<~I O Q Rio xenoemmm m6 24 on Sephadex G-200 was unsuccessful. The enzyme preparation at this step was stored at -20°, over a period of 5 months without any significant loss of activity. The summary of the purification of 3'-nucleotidase II and RNase from 100 g of pea seedlings is given in Table 2. Sucrose Density Gradient Centrifugation.--Sucrose density gradients were prepared as described in "Methods." An amount of 0.2 ml of lyphogel concentrated enzyme solu- tion of 3'-nucleotidase II which had been partially puri- fied from Sephadex G-75 gel filtration (210 enzyme units toward 3'-AMP per ml) was layered on a precooled and equili- brated sucrose density gradient. The tube was centrifuged for 18 hr at 60,000 r.p.m. in a Beckman L2-65B ultracentri- nge with the temperature maintained at 2°. After centrifu- gation, lO-drop fractions were collected and assayed for enzyme activities as previously described. The enzyme activities toward 3'-AMP, RNA and de- natured DNA were eluted in an identical pattern (Figure 4). Recovery in each case was about 57%. Electrofocusing Column Chromatography of 3'- N£‘3leotidase II.--Although the activities toward 3'-AMP, RNA: and denatured DNA appeared to purify together as in the previous results, it was desirable to have additional I evidence bearing on the question of their identity. therefore attempted further purification by the technique of electrofocusing. .III .3029 u0----0 .emmzm “all .Aeueuumcom me mz¢-.mv emecHuoeHooc-.m mo wuH>Huoe eexncm .uxeu ecu cH ce>Hm eue mHHeueQ .mcoHuoeum e>HuecHepHe ecu cH meueuquSm uceHeMMHo cuH3 >0H>Huom eE>Nce MOM vexemme cam ceuoeHHoo eue3 .mcoHuoenm 00 mo Hmuou .mcoHuomum mono 10H .coHuemowHuuceo Heumc .om um UecHeucHeE mez enoueuemsee .Hc mH Mom emowHuu iceoeuuHo mmo-mq ceaxoem m CH uouon uexooc mCHmcHBm HE H mm 3m cuH3 Emu ooo.om we UeEHOMueQ me3 coHuemstuuceO .m.h mm .Hemwoc eueueoe-mHHB z H.o CH cenemenm meB uceHceum wuHmcec emouoom ecB .AHE c.0v uceHcenm wuHmceo emOHOSm mom on m e ue>o vehemeH me3 .coHueHemenm mhlw xepecmem Eoum HH emecHuoeHosc-.m mo AHE\o.m mm on mz¢-.m cue3ou muHco QHNV H8 «.0 .coHuoHom eESNce ceueuuceocoo HemocmmH eca 25 .coHuemDMHuuceo uceHceum wuHmcec emOHUSm Eonm emezo one .emezm .HH emecHuoeHooc-.m mo cueuuem coHusHMI-.0 euomHm 26 ammiaz ZO_._.U<~_“_ On ON C— \ 3.0 :0 Z0240 -. (NOIlDVUJ/Slan ) All/\llDV 27 Ten m1 of enzyme of 3'-nucleotidase II preparation (partially purified from Sephadex G-75) was dialyzed against 20 volumes of deionized water overnight in order to reduce the salt content to less than 0.5 umoles. The small amount of precipitate formed during dialysis was removed by cen- trifugation and discarded. The resulting clean supernatant enzyme solution was subjected to electrofocusing as described under "Methods." After applying a potential of 350 volts to the column for a period of 48 hr, 80-drop fractions were collected. As mentioned in "Methods," the fractions in the pH range of 3.6-6.0 were dialyzed against 1 M NaCl and deionized water in order to remove the ampholine. After dialysis, enzyme assays were performed with the standard methods. As shown in Figure 5-B, it is evident that 3'-nucleotidase II (with 3'-AMP as substrate) and RNase were eluted in an identical pattern with an iso- electric point at pH 4.8. However, the ratio of enzyme activities toward 3'-AMP and RNA was not the same as those previously observed in the earlier steps of the enzyme purification scheme. The recovery of activity was 30% for 3'-nucleotidase II and 11% for RNase. This discrepancy may be due to enzyme instability at low pH (Table 6). Electrofocusing Column Chromatography of 3'- Nucleotidase I.--Ten ml of enzyme solution of 3'-nucleotidase I (dialyzed ammonium sulfate fraction with low salt content, containing 200 units of activity toward 3'-CMP per ml) was 28 added to the column. After completion of the electro- focusing, the eluted fractions (dialyzed against 1 M NaCl overnight and against deionized water for 6 hr) were assayed for enzyme activity. With 3'-CMP as substrate, bands of activity were found at pH 4.7 and 5.3 (Figure S-A). The low recovery (7%) of enzyme activity was probably due to the absence of reducing reagents (sulfhydryl compounds) during the electrofocusing and dialysis. Polyacrylamide Disc-Gel Electrophoresis of 3'- Nucleotidase II.--With a 7% polyacrylamide gel at pH 8.5 as described under "Methods," 0.3 ml of 3'-nucleotidase II enzyme preparation from Sephadex G—75 fraction containing 54 ug of protein (with 36 units toward 3'-AMP, or 24 units toward RNA) was subjected to electrophoresis. The electro- phoresis was performed with the addition of one drop of 0.005% of bromophenol blue (Fisher Scientific Co.) as a tracking dye. When the dye band reached the end of the gel, the current was turned off, and the gels removed and cut in half horizontally and either stained with Coomassie blue or assayed for enzyme activities. For assay of enzyme activities, one of the half gels was cut into 3 mm segments and assayed for 3'-nuc1eotidase II and RNase activities in alternate segments at 37° for 10 hr. Figure 6 shows the staining pattern of protein and tflle distribution of enzyme activities within the gel after Eilectrophoresis. It is evident that 3'-nuc1eotidase II and 29 Figure 5.--Elution profiles of pea 3'-nucleotidase from an electrofocusing column chromatography. (A) Elution profile of 3'-nucleotidase I. Ten m1 of enzyme preparation (200 units toward 3'-CMP at pH 5.4/m1) as described in the text was applied to the column. Enzyme activity was assayed with 3'-CMP as substrate. Detailed experimental procedures are described under "Methods." Solid line represents the pH gradient. Enzyme activity (in units/fraction) is shown by 0-—--0. Ampholyte, pH 3.0 to 6.0, was used in this study. (B) Elution profile of 3'-nucleotidase II. Ten m1 of 3'—nucleotidase II preparation (120 units/ml toward 3'-AMP at pH 3.0, partially purified from sephadex G—75 and dialyzed against 20 volumes of deionized water over night) was applied to the column. Ampholyte, pH 3.0 to 6.0, was used in this study. pH gradient, -—————-; 3'-nucleotidase (assayed with 3'-AMP at pH 8.0) ¢———-C; RNase, 0----0. ACTIVITY (UNI‘I’SI FRACTION) 30 PH5.3 (A) 3-NUCLEOTIDASE e i I 10 o __ 5 n 'l l l _ l \ l l 1 I ' \ __ I | _‘ 5 7’ 5. I \ I \o/ P J I, \ 0 1°} 1 him I W3 '°°l— P" "8 (B)35-NUCI.EOTIDASE II F RACTION NUMBER 31 Figure 6.--The staining pattern of protein and the distribution of RNase and 3'-nucleotidase II on a poly- acrylamide gel after electrophoresis. A 0.3 ml quantity of enzyme preparation (3'- nucleotidase II) containing 54 ug of protein was subjected to electrophoresis in 7% gel as described under "Experi- mental Procedures." Electrophoresis at pH 8.5 was performed at 4° for 45 min with an applied current of 2 mA per tube. After the run, the gel was cut in half; one half was stained for protein with Coomassie blue dye, and the other half was cut into 3 mm segments. The 3'-nucleotidase and RNase activities were assayed in alternate segments with 3'-AMP and ribosomal RNA as substrate, respectively. Detailed procedures for enzyme assays are described under "Methods." The absorbance of the protein stained at 650 mu was measured as described in the text. Enzyme activity: 3'-nucleotidase, 0----0; RNase O——-—O. 32 Nucleotidase II? 3'. .1 _ _ C m. m m thZOmmmeZ: ono< T «+» Q IIIIIIII <—) I -————9 ORIGIN 33 RNase activities were found to be associated with the only major detectable protein band and their recoveries were similar, 22% and 24%, respectively. The resulting protein profile of the gel as measured at 650 mu indicated about 90% of total protein in the preparation was located in the band containing 3'-nucleotidase II and RNase activities. Because there was 1.6% recovery of protein as shown in Table 2, the 3'-nucleotidase from Sephadex G-75 fraction seems to represent 1.4% of total protein in the 4,000 x g supernatant (or 0.2% of protein of seedlings homogenate). Separation with a 10% gel gave essentially the same pattern in protein staining and enzyme distribution as that ob- served in the 7% gel. Polyacrylamide Disc-Gel Electrophoresis of 3'- Nucleotidase I.--With the same procedures as previously described for 3'-nucleotidase II, 0.3 ml of lyphogel con- centrated enzyme preparation of 3'-nucleotidase I from Sephadex G-100 fraction containing 20 ug of protein and 5.2 units toward 3'-CMP was subjected to electrOphoresis in 7% acrylamide gel. The staining pattern of protein and the distribution of enzyme activity is shown in Figure 7. Apparently, the enzyme preparation still contained a number of protein species. One major contaminating protein bands twas found. About 30% of the applied enzyme activity was recovered . 34 Figure 7.--The staining pattern of protein and dis- tribution of 3'-nucleotidase I on a polyacrylamide gel after electrophoresis. A 0.3 ml of lyphogel concentrated enzyme of 3'- nucleotidase I preparation containing 20 ug of protein was applied to a 7% polyacrylamide gel. Details were the same as described in Figure 6. Enzyme activity was assayed with 3'-CMP as substrate. 35 E °3"' “2‘ .‘3 2 a2— :2 i D 0.]!— F“ _. 0 SI < M W JULHIJOIII‘JIIITILIIILHI' (-) | TL I j (+- - - 36 Prpperties of the Enzyme Preparations Rate of Hydrolysis as a Function ofypH and Zn++.-- Figure 8 shows the enzyme activity as a function of pH using K-acetate and Tris-acetate as buffers. The pH opti- mum of 3'-nuc1eotidase I (with 3'-AMP as substrate) as shown in Figure 8-A was in the range of pH 5.4-5.7 with no significant activity at pH 7.5 or higher pH values. In contrast to 3'-nucleotidase I, 3'—nucleotidase II was shown to have an optimum pH at 8.0, with less than 50% activity at pH 6.5 (Figure 8-B). However, with RNA as substrate, 3'-nucleotidase II showed a pH Optimum around 6-7. Furthermore, as shown in Table 3, the pH optimum of 3'-nucleotidase I varied from 5.0 to 5.7 depending on the substrate used. The addition of ZnCl at a concentration 2 of 2 mM shifted the optimal pH to a lower pH value, about 4.7, with 50% inhibition on enzyme activity. However, the pH optima for 3'-nucleotidase II were the same, 8.0, on all 3'-nucleotides except 3'-CMP which the enzyme could not attack. The addition of 2 mM ZnCl2 apparently had no effect on the pH optimum of 3'-nucleotidase II, but slightly increased (20%) the enzyme activity. Effect of Metal Ions and Inorganic Ions on Enzyme Activity.--The effect of various metal ions and inorganic ions on 3'-nuc1eotidase activity was tested. All metal ions and inorganic ions were added at zero time to the 37 .o o .eueueoe-mHHB z H.o no 0 .eueueoeum z H.o "Gem: Hewmom .Amv .HH emecHuoeHUDCI.m uAHuoe ecu co mm wo uoemmm|-.m euomHm 38 #1 33:86:20 2: On 00— 0 low 100— H 33.533240 3: All/\llDV 3A|1V1 3 8 39 TABLE 3.--Effect of Zn++ on the optimum pH for the activi- ties of 3'-nucleotidase I and 3'—nucleotidase II.a Optimum pH of Optimum pH of Substrate 3'-Nuc1eotidase I 3'-Nuc1eotidase II Used -Zn++ +Zn++ -Zn++ +Zn++ 3'—AMP 5.7 4.8 8.0 8.0 3'-GMP 5.4 4.7 8.0 8.0 3'-UMP 5.0 4.6 8.1 8.0 3'-CMP 5.4 4.7 -—- --- 2'—AMP 5.6 4.7 --- --- 5'-AMP 5.6 4.7 --- --- aExperimental conditions were as described for the standard assay system except with or without addition of 2 mM ZnClz in the reaction mixture. Buffers used as given in Figure 8: 0.1 M K-acetate, pH 4.0 to 7.0; 0.1 M Tris- acetate, pH 5.5 to 10.0. Inorganic phosphate released was determined as described under "Methods." --- denotes no detectable inorganic phosphate released. 40 standard reaction mixture as described in the "Methods" except that the buffer used was Tris-acetate for the assay of both 3'-nuc1eotidase I and 3'-nucleotidase II. The final concentration of the additives were 1 mM and the re- actions were carried out at 37° for 30 min with 5 units of enzyme preparation. The variation in enzyme activity due to the presence of metal ion or inorganic ion is summarized in Table 4. It is evident that divalent cations, Mg++, Mn++, Co++, and Zn++ showed 4%, 22%, 28%, and 50% inhibition respectively on 3'-nucleotidase I activity while EDTA and monovalent cations (K+, NH4+, Na+) showed no effect. Imidazole caused a 33% inhibition. In contrast to 3'-nucleotidase II activity none of the ions lead to major increase or decrease in 3'- nucleotidase I activity except EDTA which gave an inhibition of 60%. Effect of Sulfhydryl Compounds on Enzyme Activity.-- Effect of various sulfhydryl compound such as glutathione, cysteine, and dithiothreitol on 3'-nucleotidase activity was determined with a standard reaction mixture at 37° for 30 min. Table 5 shows that there was little effect of sulfhydryl compounds on 3'-nuc1eotidase I activity except that at 4 mM there was 10-50% activation. However, sulfhy- dryl compounds at concentrations as low as 0.4 mM gave almost complete inhibition of 3'-nucleotidase II activity. 41 TABLE 4.—-Effect of inorganic ions, caffeine and theophyl- line on 3'-nucleotidase activity. Additiona 3'-Nucleotidase I 3'-Nucleotidase II Activity Remaining (%)b None 100 100 MgCl2 96 102 MnCl2 78 --- CoCl2 72 118 ZnCl2 50 109 (NH4)ZSO4 92 100 KCl 101 100 NaCl 100 100 Imidazole 67 --- EDTA 100 42 NaF 42 83 Caffeine 104 100 Theophylline 101 101 aThe final concentration of the added reagent was 1 mM. b All reaction mixtures contained 0.05 M Tris- acetate buffer, pH 5.6 for assay 3'-nucleotidase I or 0.05 M Tris-acetate buffer, pH 8.0 for assay 3'-nucleotidase II. 42 TABLE 5.--Effect of various sulfhydryl compounds on 3'- nucleotidases activity.a Concentration of Sulfhydryl Com- 3'-Nucleotidase I 3'-Nuc1eotidase II pound Added Relative Activity (%) None 100 100 Glutathione 4 x 10'4M 100 2.5 1 x 10’3M 102 1.8 2 x 10’3M 103 4 x 10’3M 111 0.9 Cysteine 2 x 10'4M 101 5.4 4 x 10'4M 100 2.0 1 x 10‘3M 126 2 x 10'3M 131 0.9 4 x 10’3M 135 Dithiothreitol 4 x 10’4M 102 2.2 1 x 10'3M 114 1.0 2 x 10'3M 126 0.8 4 x 10'3M 151 0.6 aThe standard reaction mixture (0.5 m1) contained 10 units of enzyme and assayed for 3'-nuc1eotidase activ- ity as described under "Methods." 43 Effect of Glycine and Zn++ on the Inactivation of 3'—Nuc1eotidase II at pH 5.0.--From the previous studies, 3'-nucleotidase II was shown to be unstable at pH values below 6.0. Since glycine and Zn++ have been suggested to be factors that can prevent such an inactivation of 3'- nucleotidase in mung bean sprouts (12), it was desirable to test whether such protection occurred in the pea enzyme system. The test proceeded as follows. Reaction mixture, 0.5 ml, containing 10 units of enzyme, 0.1 M K-acetate buffer and additions as shown in Table 6 was allowed to stand at 23° for 14 hr. After standing, 0.5 m1 of 0.5 M Tris-acetate buffer, pH 8.0, was added to the reaction mixture and enzyme assay was carried out with the addition of 2 mM of 3'-AMP as previously described. The result as summarized in Table 6 indicated that Zn++ protected both 3'-nucleotidase II activity and RNase activity. But, glycine showed no effect on enzyme activity. Substrate Specificity, Relative Activities and Km Sgpgy,--Since nucleotidases prepared from other plant sources have been shown to exhibit specificity toward purine 3'-nucleotides, it was desirable to know whether or not the pea nucleotidases also demonstrated such a speci- ficity. Standard assay conditions were employed for each substrate which was present at a final concentration of 2 mM. An amount of enzyme (about 5 units) which was within 44 TABLE 6.--Effect of various concentrations of Zn"-+ glycine on acidic inactivation of 3'-nucleotidase II and RNase.a Activity Remaining (%) Additions 3'-Nuc1eotidase II RNase Control (pH 7.5) 100 100 pH 7.0 treatment 98.7 94.3 pH 6.5 treatment 94.3 92.5 pH 6.0 treatment 36.2 45.2 pH 5.0 treatment 10.8 7.5 (no addition) Zn++, 2 x 10‘2M 13.6 19.2 Zn++, 2 x 10'3M 67.8 48.5 Zn++, 2 x 10‘4M 62.8 40.7 Zn++, 2 x lO-SM 17.2 25.6 Zn++, 2 x 10'6M 16.5 14.2 Zn++, 2 x 10’7M 11.6 12.7 Glycine, 2 x lO—ZM 11.2 --- Glycine, 2 x lO-BM 13.2 --- Glycine, 2 x 10’3M + Zn++, 2 x 10’5M 18.5 --- aThe solution, 0.5 ml, containing 10 units of enzyme purified from Sephadex G-75, buffer and the reagents shown in the table were allowed The solution was 0.1 M Tris—acetate to stand at room temperature for 14 hr. assayed as described under "Methods." 45 the linear range of experiment was added to each reaction mixture. The relative rates of hydrolysis, expressed as percent of maximum activity, for all nucleotides tested is given in Table 7. It shows clearly that 3'-nucleotidase I has a high specificity toward 3'-nucleotides and not toward 2'- or 5'-nucleotides. However, it did not show speci— ficity toward purine or pyrimidine 3'-nucleotides. The 3'-nucleotidase II showed speCificity for purine 3'- nucleotides. That appreciable cleavage of 3'-UMP also occurred. There was no detectable hydrolysis of 3'-CMP, 2'-AMP, and 5'-AMP. The effect of substrate concentration on the rate of hydrolysis was studied with the standard assay condition. The Km values were calculated from the Lineweaver and Burk plot of 1/S vs. l/V and found to be as follows for 3'- nucleotides with 3'-nuc1eotidase I as enzyme source: 0.6 mM for 3'-AMP or 3'-CMP, 0.75 mM for 3'-GMP, and 0.8 mM for 3'-UMP. With 3'-nuc1eotidase II, the Km value was 0.35 mM for 3'-AMP, 0.45 mM for 3'-GMP, and 0.62 mM for 3'-UMP. It is apparent that the affinity constants of the respective substrate decrease in following order: 3'-AMP, 3'-CMP > 3'-GMP > 3'-UMP for 3'-nucleotidase I and 3'-AMP > 3'-GMP > 3'-UMP for 3'-nucleotidase II. Activity Toward Cyclic Nucleoside Monophosphates.-- RNases purified from various plant sources have been shown to have a rather weak activity toward 2',3'-cyNMP, the 46 TABLE 7.--Relative 3'-nucleotidase activities toward ribo- nucleoside monophosphates. Relative Activity (%) Mono- nucleotige Pea Rye Grass Assayed 3,_ 3,_ 3,_ b Nucleotidase I Nucleotidase II Nucleotidase 3'-AMP 83 100 60 3'-GMP 95 85 100 3'-UMP 100 48 5 3'-CMP 80 0 0 2'—AMP 15 0 0 5'-AMP l2 0 0 aAssays were carried out as described under "Methods" with 4 mM substrate. bRye grass 3'-nucleotidase (purchase from Sigma Co.) was assayed at pH 7.5. 47 product from RNA degradation (12, 19, 30, 44). However, I found that 3'-nucleotidase II of pea had no activity toward either 2',3'-cyNMP or 3',5'-cyNMP (described in the second part of this thesis). The Mode of the Action of 3'-Nucleotidase II on Poly A.--Evidence suggests that the 3'-nucleotidase II from pea characterized in this study may be associated with the RNase activity as observed in the previous results. Most of the RNases so far purified and characterized from higher plants, including the enzyme from pea leaves (21, 22), are cyclizing enzymes (phosphotransferases) which catalyze the formation of 2',3'-cyNMP from RNA. The purine 2',3'-cyNMPs are hydrolyzed slowly to give nucleoside 3'-phosphates, while the pyrimidine derivatives are apparently inert to further action of the enzyme. The small amounts of hydroly- sis of purine derivatives of 2',3'-cyNMP are of doubtful significance because it is probable that small amounts of cyclic nucleotide phosphodiesterase (not RNase in this case) contamination was present and accounted for the cleavage found. With the synthetic polymer of adenylic acid as sub- strate, it seemed possible to analyze the end products formed from the action of RNase in a more precise way. In order to avoid contaminating cyclic nucleotide phosphodi- esterase, 0.2 m1 of 3'-nucleotidase II preparation from Sephadex G-75 fraction was subjected to.a 5-20% sucrose 48 .mz¢>01.m..m Use .mz<-.m .mz¢-.N mo coHueEuom eHceuoeuec o: mzocm ecoHe e >Hom no emezm .QIIII¢ mc ceucemeumeu mH euemmHoucwc chu wo cueuuem coHuoHe ece .CEdHoo Ueueuecemeu ecu ou ceHHmme mes uxeu ecu cH cecHuomec me e >H0d OHuecucwm ecu co AHH emecHuoeHUSCI.mv emezm med uo coHuoe ecu Eoum cecHeuco muoscoum mHmaHoucmc ece =.mcocuezr Mecca cecHuomec we ceueuecemeu cecu mes cEsHoo ece .IIIIII.>c c3ocm mH e>uoo coHueucHHeo ece .18 com um ceuomeea me3 eocecu0mce ecu cce .cHE\mmouc NH mo eueu onu e cuHB ceuoeHHoo euez ecoHuoeuu AHE e.m .mmev coup-om .uce>H0m coHuoHe new nounnnnuuusem men we Hum as m nuuz .Aezeso-.m..m no go 0.0 can .ez¢-.m eo me m.o .mz¢-.N m0 m8 v.ov mcCSOQEoo UHucecuoe mo coHuechEoo mooHue> e cuHs Ueueuc IHHeo umuHu mez .Eo o.m x m.o .cmeE oov .A HUVNx-H-mm cemon m0 chH00 ecB .d mHom uo uoocoum mHmmHoucwc ecu mo acmeumoueEouco emcecoxe coH-.m euomHm 49 mmmiDZ ZO. hU<¢ ”— oo. 0m 40 .V m<<H0m .mm “EemmoueEomco mo chHmo .O .emezm cuHB eeuecsocH a xHom Eomu UecHeuco uHSmem ecu m3ocm eeme cemo .Homucoo ecu me ecoHe 4 mHom mucememmem eeme ee3oeecm .nE nmm ue eemSmeeE eme3 muceuecmemsm ecu mo eocecmOmce ecu .coHueOSMHmuceo meuuc .CHE om mow cuec meuez mcHHHoc cH meuez ceHHHume uo HE m.H cuH3 ceueHe cce Ue>Heoem eme3 emoHsHHeo ecu mo mHe>meucH meueEHuceo eco .ucmHH .>.o mecca emome ecu CH czocm mccec coeHc o3u me ceuoeuec eme3 euemxHomcwc cce d mHom mo mcoHueooH eca .omm .emouememEeu Eoom ue mc v mow A>\> Humv m.m mm .moumvmz z mo.o-HocemommowH mo Eeumwm uce>H0m cH eemoHe>ec me3 EemmoueEomco ece .uxeu ecu cH cechomee eme memseeoomm ece memouxHE coHuoeem ecu mo mHHeueo .« mHom co AHH emecHuoeHoec-.mv emezm mo coHuoe ecu Eomu eecHeuco muoscomm mHmeomcmc ecu mo >cmemmoueeomco memeH-chB-I.OH emomHm 52 T - HR MATOG, W PolyA alone 2 PolyA + RNase \\\\\\ ........ :'\.\\\.\\‘- Ajnine Adenosine AMP 3’—Cy r- 4 / I I 2 \\ \. \ \ .\.‘ .5— .2 0]— 0 / {—0 53 .mmcHHeeem eem exmeHc Eomm eeHMHmsm HH emeeHuoeHooc-.m ecu mH emezm .A an cecmee mH eocemeuwHU emmeH 4 c .c3ocm mecmo e>HueHem ecu cH .uHHmm eme c3ocm memec ecu ou uceoemee mecoc ecHuoeHoozo .m ecB .eeEmOM uoscomm euecmmocm HecHu ecu meuocec .c0HuecsocH mc em meume ccoom meuemeomehc eHceuoeuee on meueouecH ecoz c .eeumHomexc mecumem meuecmmocm UHHowo echom >H3on >me> cecu .meuecmmocQOGOE eeHmoeHoo: UHHomoI.m..m ummHm uee mzzmo-.m..m acoum ch9 sum I-I ecoz ecoz emcHHeeem eem AchmHv .He ue mcee o.m III ecoz ecoz mzzmon.m..m eceo memom AvmmH~ ceEeemm ~.m III ecoz ecoz mzzmo-.m..~ mmemm emm AmmmHv .He ue meme>e0 m.m III ecoz .m.3on >me> mzzmo-.m..m me>eeH ece>< Anemmv acnmuz e-m o.5A¢.o eeoz .m.3omm >ue> mzz»0-.m..~ Aeeen .uoomv cmou lemmac oenm 0.0 8.0.: 0:62 .m.36mn snm> nzzso-.m..~ enumnemn mecEsoso AmmmHv .He ue eHomez m.m O.D.¢Ao ecoz .m.3on >me> mzzmol.m..m seen how Aeemmc .mn um umumnz m.m -- meoz .m.3omn mn0> nzzmo-.m..~ Amzc nusouen ceec mcoz commH~ .He ue e>oB mum O.D.¢Ao ecoz .m.3on >me> mzzm01.N..N me>eeH coechm 1000mc .mn um cannunmo 0-0 -- meoz .m.30mn sne> mzzao-.m..~ Assumec oumnno Ammmmvlhmmlue ceeom AmmmHv .He ue Eeccmez H.m UAD.me> mzz>0-.ms.m me>eeH eem AmmmHv .He ue meumscom H.m OAD.¢A0 ecoz .m.3on >me> m22%0|.m..m me>eeH oooecoa ecHe enamom humoam IHEHmmm . eefimom O . . m eocememem mm nHoemm muoseomm eomsom nmzzao-.m..m mcHuaHomemm .muceHm mecch 50mm emezm eeNHmeuoemecOIHHe3 mo mmeEEdmII.m mamma 54 Estimation of Molecular Weight, Diffusion Constant and Stokes' Radius for 3'-Nuc1eotidases.-—Ku1karni and Mehrotra (45) modified the equation described by Determann EE_El° (46) for the estimation of the molecular weight, diffusion constant, and Stokes' radius of a protein in relation to its reduced elution volume (Ve/Vo) using Sephadex G-150 column. I derived similar equations for a Sephadex G-200 column which was used for study of pea 3'- nucleotidases. A Sephadex G-200 column (1.5 x 120 cm) with 40-120u bead size was used (47, 48) and a bed height of 120 cm was kept throughout the study. An 0.01 M of Tris-acetate buffer, pH 7.5, was used for elution of the proteins. To assure reproducible results and to maintain the flow rate at 9 ml/hr, it was essential to keep the hydrostatic pres- sure at 40 cm. Fractions were collected at 20 min inter- vals at 4°. The column was calibrated with various com- binations of standard proteins (2 mg for each protein) as shown in Table 9. The void volume (Vo) used in the calcu- lation is the elution volume of blue dextran (Ve = 48.0 ml). With the least-squares method, the data of standard pro- teins shown in Table 9 were analyzed and gave the following three regression equations: a. log Molecular Weight = (6.2392 i 0.7428 3:. 0.0252) (Ve/V0) . 55 IHeo eme msHeem .xo>\0>0 0000.0 - 0000.0 u 000000 .000000 0o0 .Ao>\0>0 0000.0 + 0000.0 u unnuneoo eo0nsee0o 000 .Ao>\e>v mmvb.o I mmmm.m u .03 .002 000 umcoHueswe mcH3oHH0m ecu Eomm eeueHoo .mecoum ece .uceumcoo conSMMHe .ucmues meHsoeHoE mo medHe> ecee 00.0 000 0.00 0.0 000.00 010000 00 00000uo00ose-.0 00.0 0.00 0.00 0.0 000.00 010000 H meUHuOOHOSGI . m 00.0 0.00 -- -- 000.000.0 anuuxee 0:00 00.0 0.00 -- 1000 0.0 1000 000.000 20000000-600 00.0 0.00 1000 0.00 1000 0.0 1000 000.000 e00sn00u-0 00.0 0.00 1000 0.00 1000 0.0 1000 000.00 1200000 me0>onv n0snn0e 00.0 0.000 1000 0.00 1000 0.0 0000 000.00 e0esn00>o 00.0 0.000 1000 0.00 0000 0.0 1000 000.00 0 emmoe0nm0uuos0no 00.0 0.000 1000 0.00 1000 0.00 0000 000.00 e0nmmne 00.0 0.000 1000 0.00 0000 0.00 1000 000.00 c0no00o0z mm.m «.mmH III III Amvv oo0.~H o eeomcooumo 1050 100000000 0600\ so o>\0> mss0o> 000000 000 013 0mac .03 .002 e0muoum coHuon .mecoum uceumcoo conomuHa mcHeuomm emeeceum uo Aoome .memeemuoeHoscI.m eem pee xeeecmem Eomwv euee coHuoHe cce meHumemomm HeonachI.m memes 56 b. log Diffusion Constant (D° x 107 cmZ/sec.) 20W = (0.0645 : 0.0618) + (0.3769 : 0.0277) (Ve/Vo). c. log Stokes' Radius (rS x 108) = (2.2664 : 0.0587) - (0.3778 : 0.0263)(Ve/Vo). Thus, using only the experimental value of Ve for any pro- tein (assumed to have similar shape) under study and V0 of the column of Sephadex G-200 without having a calibration curve, one can determine the molecular parameters mentioned above for a protein by the above equations. For example, the value of such molecular parameters for pea 3'- nucleotidases are shown in Table 9. Apparently, 3'- nucleotidase I has a molecular weight about 70,000 and 3'- nucleotidase II has 30,000. In addition, a typical plot of the correlation between log molecular weight and reduced elution volume (Ve/V0) is shown in Figure 11. Discussion Two 3'-nucleotidase activities were found in pea seedlings and were readily separable by DEAE-cellulose column chromatography. These two enzymes were further purified by Sephadex gel filtration. The 3'-nuc1eotidase I was purified approximately 6-fold with a recovery of about 1% of that enzyme activity present in the 4,000 x g supernatant. Although the result 57 Figure ll.--Calibration curve for molecular weight determination on Sephadex G-200 column chromatography. A Sephadex G-200 column (1.5 x 120 cm) was equili- brated with 0.01 M Tris-acetate buffer, pH 7.5, at a flow rate of 9 ml/hr at 4°. The column was calibrated with various combinations of standard proteins as shown in Table 9. The elution volume (Ve) of each protein was determined by extrapolating to the center of the peak. The void volume (Vo) used in the calibration is the Ve of blue dex- tran. A typical plot of the correlation between log molecular weight and reduced elution volume (Ve/Vo) is shown in this figure. The values of log molecular weight for 3'-nuc1eotidase I and II are indicated by ----- +. Apparently, 3'-nucleotidase I has a molecular weight about 70,000 while 3'-nuc1eotidase II has 30,000. For further details, refer to the description in the text. 58 3.0 __ Ill [I \ 2.5 L'\ IIIIIIIII]T YTOCHROME C Q MYOGLOBIN TRYPSIN - SEPHADEX G-200 CHvMomPsINOGEN A ‘—“ _ ______ n ._ | -- I I— | ‘— _ ' 0 OVALBUMIN _ I 2.0— '— 5 : - _____ ____ ALBUMIN - g --- 't _. I I .. I I u— H . -" H H "" In In '7 V) In 1.5 § § —‘ I- I- _ g g U-GIOIIUIIN _. I... g g ._ z z .I .I I— M n —‘ I I : : APO-FERRITIN 1.0 | ' BLUE DEXTRAN I I -I-v-J-lv-I-I-I-l-. 4.0 4.2 44 4.6 4 8 5.0 5.2 SA 5.6 6A LOG MOLECULAR WEIGHT 59 shows that 3'-nuc1eotidase I does not need the presence of reducing reagent such as cysteine, glutathione, and dithio- threitol for maximal activity, it may need them for stabil— ization during its purification procedures as is the case for 3'-nucleotidase in wheat seedlings (14). Thus, the lack of reducing reagents during purification procedures may be the main reason for the low recovery. Furthermore, Zn++ may also stabilize the enzyme even though it inhibits enzyme activity during the assay. Unlike the enzyme puri- fied from wheat seedlings (l4) and muskmelon seeds (15), the pea 3'-nucleotidase I preparation was nearly free of con— taminating proteins. The addition of Zn++ shifted the optimum pH and decreased the specific activity. The pea 3'-nucleotidase I is quite different from other plant nucleotidases (12, 16) which have been well- characterized and shows no significant activity toward pyrimidine 3'-nucleoside monophosphates. However like other plant 3'-nucleotidases (12, 15, 16), pea 3'- nucleotidase I shows little activity toward 2'- or 2'-AMP and no activity toward cyclic nucleoside monophosphates. Based on these characterizations, 3'-nuc1eotidase I could serve as a tool for an enzyme coupled assay of cyclic nucleotide phosphodiesterase in higher plants. The molecu- lar weight 70,000 of pea 3'-nucleotidase I is higher than that of most plant nucleotidases which in general have 60 molecular weights of 15-30,000 (15). Further studies will be required to establish whether the two isoelectric points observed for 3'-nuc1eotidase I are due to enzyme dissoci— ation or the presence of two enzymes. A rather unusual combination of nuclease and 3'— nucleotidase activities purify as one species from a variety of plant sources. These include the enzyme from rye grass (16, 29), muskmelon seed (15), rice bean (17), mung bean sprouts (12), wheat seedlings (l4), and possibly soy bean (18), and germinating garlic (19). The enzyme of 3'- nucleotidase II preparation from pea seedlings in the present study was suggested to have RNase and 3'- nucleotidase combination. Although the enzyme has been purified approximately 15-fold with a recovery of 23% of the total activity present in the 4,000 x g supernatant, about 90% of the protein in the preparation is 3'-nucleotidase II. Based on this high yield and purity, further steps of purifica- tion with either DEAE-cellulose or Sephadex G-200 may pro- vide a good source for the study of the physical and chemical properties of this unusual enzyme. The ability of Zn++ to prevent both 3'-nucleotidase II and RNase inactivation at pH 5.0, and the elimination of enzyme activity by EDTA, imidazole, cysteine and other reducing reagents provide strong evidence that the enzyme concerned may be a metalloprotein, probably a zinc- . . ++ . . . contalnlng enzyme. However, added Zn has no Slgnlflcant 61 effect on enzyme activity; added Zn++ may only function to stabilize the proper tertiary or quaternary structure of the protein. A similar role of Zn++ has been observed in several enzymes such as Escherichia coli alkaline phospha- tase (58), horse liver alcohol dehydrogenase (59), Bacillus subtilis a-amylase (60), wheat seedling 3'-nucleotidase (l4), and mung bean 3'-nucleotidase (12). Pea 3'—nucleotidase II, like other plant nucleo- tidases, has high specificity for purine 3'-nuc1eoside monophosphates and also catalyzes appreciable cleavage of 3'-UMP. The enzyme shows no activity toward 3'-CMP, 2'- and 5'-AMP. Like most of the RNases so far characterized from higher plants, as described in Table 8, pea RNase (3'- nucleotidase II) catalyzes the formation of 2',3'-cyAMP from poly A, with no further formation of 2'—AMP and 3'-AMP under assay condition. Since the finding of a cyclic nucleotide phosphodiesterase in the same tissue (as de- scribed in Part II), the pea RNase (3'-nucleotidase II preparation) failure to hydrolyze 2',3'-cyclic nucleoside monophosphates is probably due to the lack of the enzyme mentioned above rather than because of the contamination of nucleotides bound to the RNase. Therefore, the mode of RNA degradation in higher plants may not follow the way which is generally accepted (20, 30, 44, 61). As mentioned earlier, the results of the present study suggest that both 3'-nucleotidase II and RNase 62 activities reside in a single protein molecule. Evidence for this suggestion is based on the following criteria. 1. The two activities maintain a constant ratio throughout the purification procedures. 2. Attempts to separate the two activities by means of gel filtration, sucrose density gradi- ent centrifugation, polyacrylamide gel electro- phoresis, and electrofocusing have been unsuc— cessful. 3. Both activities are lost during treatment at pH 5.0. Zn".+ stabilizes both activities. Both 3'-nucleotidase I and 3'-nuc1eotidase II should be useful to study the end products of cyclic NMP diesterases. Summary Two 3'-nuc1eotidases have been isolated and par- tially purified from germinating pea seedlings. The 3'-nucleotidase I shows maximal activity at pH 5.4-5.7 with no addition of Zn++. However, the optimal pH is lowered to 4.7 and enzyme activity is decreased about 50% with the addition of 2 mM Zn++. The relative rates of hydrolysis of the respective nucleotides are 3'-UMP (100%)> 3'-GMP(95%)>3'-AMP(83%) 3'-CMP(80%)>2'—AMP(15%)>5'-AMP(12%). The values of Km for the 3'-nucleotides are 0.6-0.8 mM. The enzyme does not require the presence of metal ions or sulfhydryl compounds for maximal activity. The molecular 63 weight of the enzyme is 70,000. Two isoelectric points, pH 4.7 and 5.3, were found for the enzyme which was able to hydrolyze 3'-CMP at pH 5.4. In contrast to 3'-nucleotidase I, pea 3'- nucleotidase II has maximal activity at pH 8.0. Zn++ pro- tects against inactivation of enzyme at pH 5.0. However, metal ions are not required for full activity. Sulfhydryl compounds at 0.4 mM give about 98% inhibition. The relative rates of hydrolysis of respective nucleotides is 3'-AMP (100%)>3'-GMP(85%)>3'-UMP(48%)>3'-CMP(0%), 2'-AMP(0%), 5'-AMP(0%). The Km for 3'-AMP, 3'-GMP, and 3'-UMP were 0.35 mM, 0.45 mM, and 0.62 mM, respectively. The 3'- nucleotidase II preparations showed RNase activity. At- tempts to separate RNase activity from nucleotidase activity by a variety of chemical and physical means have been un- successful. It is, therefore, suggested that 3'-nucleotidase and RNase activities reside in a single protein molecule. The enzyme has a molecular weight of 30,000 and isoelectric point of pH 4.8. It catalyzes the formation of 2',3'-cyAMP from poly A. 10. 11. 12. 13. 14. BIBLIOGRAPHY Heppel, L.A., and Hilmoe, R.J., J. Biol. 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Freeman, K.B., Can. J. Biochem., 62, 1099 (1964). Tang, W.J., and Maretzki, A., Biochim. Biophys. Acta, 212, 300 (1970). Crestfield, A.M., Smith, K.C., and Allen, F.W., J. Biol. Chem., 216, 185 (1955). Fiske, C.H., and SubbaRow, Y., J. Biol. Chem., 66, 375 (1925). McDonald, M.R., in S.P. Colowick and N.O. Kaplan (Editors), Methods in enzymology, Vol. II, Academic Press, New York, 1957, p. 427. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 66 Ibuki, F., Aoki, A., and Matsushita, S., Agr. Biol. Chem., 36, 144 (1964). Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J., J. Biol. Chem., 193, 265 (1951). Ornstein, L., Ann. N.Y. Acad. Sci., 121, 321 (1964). Davis, B.J., Ann. N.Y. Acad. Sci., 121, 404 (1964). Chrambach, A., Reisfeld, R.A., Wyckoff, M., and Zaccari, J., Anal. Biochem., 36, 150 (1967). Martin, R.G., and Ames, B.N., J. Biol. Chem., 36, 1372 (1961). Volkin, E., and Carter, C.B., J. Am. Chem. Soc., 26, 1516 (1951). Jeso, F. DI., J. Biol. Chem., 43, 2022 (1968). Peterson, E.A., and Sober, H.A., in S.P. Colowick and N.O. Kaplan (Editors), Methods in enzymology, Vol. V, Academic Press, New York, 1962, p. 3. Fischer, L., An introduction to gel chromatography, Wiley interscience, New York, 1969, p. 182. Bernard, E.A., Ann. Rev. Biochem., 38, 677 (1969). Kulkarni, A.P., and Mehrotra, K.N., Anal. Biochem., 38, 285 (1970). Determann, H., and Michel, W., J. Chromatog., 26, 303 (1966). Andrew, P., Biochem. J., 91, 222 (1964). Andrew, P., Biochem. J., 26, 595 (1965). Ackers, G., in H.C. Damm, P.K. Besch, and A.J. Goldwyn (Editors), The handbook of biochemistry and bio- physics, World, New York, 1964, p. 68. Cunningham, L.W., JR., J. Biol. Chem., 211, 13 (1954). Hartley, B.S., Nature, 201, 1284 (1964). Warner, R.C., in H. Neurath and K. Bailey (Editors), The proteins, Vol. IIA, Academic Press, New York, 1954, p. 435. 53. 54. 55. 56. 57. 58. 59. 60. 61. 67 Phelps, R.A., and Putnam, F.W., in F.W. Putnam (Editor), The plasma proteins, Vol. I, Academic Press, New York, 1960, p. 143. Yang, J.T., Advan. Protein Chem., 66, 323 (1961). Harrison, P.M., J. Mol. Biol., 6, 404 (1963). Edmundson, A.B., and Hirs, C.H.W., J. Mol. Biol., 6, 663 (1962). Edsall, J.T., in H. Neurath and K. Bailey (Editors), Vol. IB, Academic Press, New York, 1953, p. 549. Schlesinger, M.J., and Barrett, K., J. Biol. Chem., 240, 4284 (1965). Drum, D.E., Harrison, J.H., Li, T.K., Bethune, J.L., and Vallee, B.L., Proc. Natl. Acad. Sci. U.S.A., 61, 1434 (1967). Stein, E.A., and Fisher, E.H., Biochim, Biophys. Acta, 66, 287 (1960). Center, M.S., and Behar, F.J., Biochim, Biophys. Acta, 151, 698 (1968). PART II THE PURIFICATION AND CHARACTERIZATION OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE FROM PEA SEEDLINGS Introduction Since the discovery of 3',5'-cyAMP in biological tissues (1), it has been implicated as a second messenger in the action of a variety of animal hormones (2). It is also known to be a mediator of catabolite repression or the so-called "glucose-effect" in bacteria (3). Although ex- tensive studies have been made on the distribution and function of this nucleotide in animal and unicellular organisms (3-6), knowledge about this cyclic nucleotide in higher plants is meager. Preliminary attempts to detect adenyl cyclase (7) and to incorporate radioactive adenine and adenosine into 3',5'-cyNMP in pea and barley tissues were unsuccessful. However, an enzymatic system for the degradation of 3',5'- cyAMP in both pea and barley tissues has been found. It seemed necessary to study such a 3',5'-cyAMP phosphodi- esterase in more detail in order to have a better idea how to assay for adenyl cyclase or endogeneous cyclic nucleoside 68 69 monophosphates in higher plants. The partially purified phosphodiesterase hydrolyzed not only 3',5'-cyNMP but also 2',3'-cyNMP. The general accepted mode of RNA degradation in higher plants is that RNase is the enzyme which hydrolyzes both RNA and 2',3'-cyNMP (8, 9). Although a specific enzyme for the hydrolysis of 2',3'-cyNMP, but not RNA, has been found in both animal and bacterial systems (10-15), it has not yet been found in higher plants. This paper presents the detailed procedures for iso- lation and purification of cyNPDE, together with a descrip- tion of the general chemical and physical properties of the enzyme with respect to its action on the substrates 2',3'- cyAMP and 3',5'-cyAMP. The biological significance of cyNPDE and the degradation of RNA in higher plants are discussed. Experimental Procedures Materials Most of the materials used were the same as de- scribed in Part I with the following additions. Silica gel G (acc. to Stahl) was purchased from Brinkmann Instruments. The products of Packard are PPO and POPOP. The 3H-3',5'-- cyAMP with a specific activity of 16.3 Ci/umole and about 2% impurity was purchased from Schwarz BioResearch. All standard chemicals were reagent grade and were used without 70 further purification with the exception of 3H-3',5'-cyAMP which was purified according to the following procedures. With thin-layer chromatography, it was shown that most of the 2% impurity appeared to be located in the area of authentic adenosine, adenine, and 3'-AMP or 3'-AMP. The method used for purification was similar to that described for the assay of adenyl cyclase in animal system by Krishna 251213 (16). A commercial sample was first applied to a Dowex-50-H+, 200-400 mesh, column (1.5 x 8 cm) and eluted with deionized water in 2 ml frac- tions. In general, the third and fourth fractions con— tained 95% of 3',5'-cyAMP were combined and freeze-dried with the aid of VIRTIS 1yaphylizer (Model No. 10-145 MR-BA). Deionized water was used for dissolving the purified material. With thin-layer chromatography in a two- dimensional separation system as described under "Methods," purified 3H-3',5'-cyAMP was shown to be free of bases, nucleosides, and other nucleotides. About 80-85% recovery was achieved in these procedures. Methods Some of the methods used in this part of the study were the same as described in the Part I. These included the growth of pea seedlings, assay of 3'-nucleotidase, assays of RNase and DNase, determination of protein content, preparation of ion exchange resin, polyacrylamide gel 71 electrophoresis, sucrose density gradient centrifugation, and electrofocusing. Thin-Layer Chromatogrgphy.—-A1though thin-layer chromatography on ECTEOLA cellulose (17), silica gel (18, 19), cellulose powder and anion exchange cellulose (20) have been shown useful for the separation of bases, nucleo- sides and nucleotides, these methods did not completely separate either 2',3'-cyAMP or 3',5'-cyAMP from other ultra- violet light-absorbing substances, especially xanthine, xanthosine, hypoxanthine, and inosine. Mixed cellulose- silica gel thin-layer (21-23) has these properties; in aqueous solvent the silica gel is deactivated and remains inert resulting in chromatograms typical of cellulose sys- tems, in organic solvent systems the cellulose is inert and chromatograms typical of silica gel system are observed. The plates were prepared by mixing 7.5 g of silica gel G and 7.5 g of cellulose powder in 90 m1 of deionized water in a Waring Blender at top speed for 1 min. The re— sulting slurry was spread over five glass plates (20 x 20 cm) at a thickness of 250 u using a spreader in the conven— tional manner and dried overnight at room temperature. On the thin-layer plate, substances located with ultraviolet light (Mineralight UVS. 11) were scraped out and eluted with 0.5 deionized distilled water in boiling water bath for 10 min. After centrifugation to remove in- soluble material, the resulting supernatant, if radioactive 72 substrate was used, was directly poured into a scintilla- tion vial to which 15 ml of scintillation fluid was added. The radioactivity was counted in a Beckman liquid scin- tillation spectrometer. The scintillation fluid, Bray's solution (24), consisted of 60 g of naphathalene, 4 g of PFC (2,5'diphenyloxazole in scintillation grade). An 0.2 g Ixf POPOP (1,4—bis-(2-(4-methyl-5-phenyloxazolyl)))- benzene, 100 ml of absolute methanol, 20 ml of ethylene glycol and p-dioxane to make 1 liter. For other purposes, the following thin-layer, and solvent systems were used (Table l). 1. For one dimensional separation of 3',5'-cyAMP and 2',3'-cyAMP from other compounds: Thin-layer; cellulose powder MN300. Solvent system 1; isopropanol:0.03M NH4HCO3 (pH 8.6) (3:1 v/v) Solvent system 2 (l4); isopropanol:NH4OH: 0.1 M boric acid (7:1:2 v/v) Running time and temperature; 4-5 hr at 23°. 2. For two-dimensional separation of 3',5'-cyAMP and 2',3'-cyAMP for their relative compounds: Thin-layer: cellulose (MN300):silica gel G (1:1 w/w) Solvent systems and running time: First dimension: H20, 40 min. Second dimension: isopropanol:0.03 M NH4HCO3 (pH 8.6) (3:1 v/v) 4-5 hr. TABLE 1.--R 73 values of bases, nucleosides, in thin-layer chromatography. and nucleotides R A: Values { B: Cellulose Thin-layer f Cellulose-silica Gel G Thin-layer) Compound Solvent Solvent Solvent Solvent System 1a System 2 System 3C System 4 A B A Adenine 0.63 0.79 0.74 0.28 —- Adenosine 0.57 0.75 0.75 0.53 -- 5'-AMP 0.07 0.07 0.06 0.93 0.42 3'-AMP 0.08 0.10 0.20 0.93 0.23 2'-AMP 0.08 0.10 0.19 0.93 0.35 2',3'-cyAMP 0.43 0.57 0.47 0.93 0.10 3',5'-cyAMP 0.38 0.52 0.44 0.93 -- ADP 0.04 0.01 0.02 0.93 -- ATP 0.02 0.01 0.02 0.93 -- Poly A 0.00 0.00 0.00 -- -- Xanthine -- 0.42 -- 0.45 -- Hypo- xanthine -- 0.68 -- 0.58 -- Xanthosine 0.30 0.45 -- 0.93 -- Inosine —- 0.62 -- 0.93 -- 6-methyl purine 0.80 0.78 0.76 -- -- Caffeine 0.84 0.90 0.85 0.77 -- Theo- phylline 0.78 0.80 0.76 0.65 -- aSolvent system 1: pH 8.6 (3:1 v/v). b Solvent system 2: Boric acid (7:1:2 v/v). cSolvent system 3: d Solvent system 4: -0.1 M NH4Ac (1:40:10 v/v). Isopropanol--0.03 M NH4HCO3, Isopropanol-—NH4OH - 0.1 M H20. Isopropanol-(NH4)2SO4(saturated) 74 3. For the separation of 3'-AMP, 5'-AMP, and 3',5'-AMP: Thin-layer: cellulose MN 300 Solvent system: isopropanol:NH4OH:0.1 M boric acid (7:1:2 v/v) Running time and temperature: 6 h4, 23° 4. For the separation of 2'-AMP, 3'-AMP, and 2',3'-cyAMP: Thin-layer: cellulose MN 300 Solvent system: isopropanol:saturated (NH SO :0.1 M NH Ac (1:40:10 v/v). 4’2 4 4 Running time and temperature: 5 hr, 23°. Enzymatic and Chemical Assgys of cyNPDE.--Two assays were performed in the measurement of cyNPDE activity depending on the assay condition required. "Assay method- 1," an enzymatic assay, was based on the colorimetric meas- urement of Pi released from the following enzyme coupled system: Cyclic Nucleoside Monophosphate pea cyNPDE e pea '- O 3'—NMP 3 n“°1e°tldase 0 Nucleoside + Pi Unless otherwise stated, 3'-nucleotidase purified from pea seedlings as previously described was used in this coupled assay system. The standard assay was carried out in a total volume of 0.5 m1 reaction mixture containing 0.1 M 75 K-acetate buffer, pH 5.4, 2 mM substrate and a suitable amount of enzyme preparation. In general, the reaction was conducted at 37° for a total incubation time of 1 hr. An 0.1 ml of 3'-nucleotidase preparation with excess amount of activity was added at 45 min and incubated for the remaining 15 min. In certain cases, the reaction was terminated first by heating in a boiling water bath for 3 min. After cooling, the reaction mixture was then incubated with 3'- nucleotidase for 15 min. Since 3'-nuc1eotidase I has been shown to have a maximal activity at the same pH as that used for the assay of cyNPDE, the pH of the reaction mixture was kept constant at 5.4 throughout the whole procedure. However, when 3'-nucleotidase II was used, the pH of the reaction mixture had to be changed to pH 8.0 with the addi- tion of 0.1 ml of 1 M Tris-acetate buffer, pH 8.0, after 1 hr incubation at pH 5.4. The whole reaction was then terminated by the addition of 0.05 ml of cold 70% TCA solu- tion according to the method shown in the preceding part. After centrifugation, Pi in the resulting supernatant was determined by the method of Fiske and SubbaRow (25) with slight modification as described in the previous part. Heat denatured enzyme preparation was used for the control assay. In general, 3'-nucleotidase I was used in the coupled enzyme assay system for enzymatic hydrolysis of 3',5'-cyNMP. One unit of enzyme activity is defined as 0.1 umole of Pi released per hr of incubation. Specific activity of enzyme is defined as units per mg of protein. 76 "Assay method-2" measured the rate of formation of 3H-3'-AMP and 3H-5'-AMP from 3H-3',5'-cyAMP. If the enzyme preparation was contaminated with 3'-nuc1eotidase, the rate 3 of formation of 3H-3'-AMP, H-5'-AMP, and 3H-adenosine from 3H-3',5'-cyAMP was measured. The standard assay was con- ducted in 0.5 m1 of reaction mixture containing 0.1 M K— acetate buffer, pH 5.4, 2 mM 3',5'-cyAMP, suitable amount of tritium labeled 3',5'-cyAMP and enzyme preparation. After incubation at 37° for a period of time, carrier nucle- otides and nucleosides (3'-AMP, 5'-AMP, and adenosine) were added and a suitable aliquot was applied to a cellulose thin-layer plate. The chromatogram was developed in the solvent system of isopropanol:NH4OH:0.l M boric acid (7:1:2 v/v) or in isopropanol:0.03 M NH4HCO pH 8.6 3! (3:1 v/v). The remaining procedures were the same as de- scribed in the method of "Thin-layer chromatography." Experimental Results Purification of Enzyme All the procedures were carried out in ice bath or at 4°, unless otherwise stated. Preparation of Crude Extract of cyNPDE.--Routinely, 300 g of 9- to lO-day-old pea seedlings, germinated in sterile vermiculite in the dark, was homogenized with 300 m1 of deionized water for 1-2 min in a Waring Blender. The homogenate was then squeezed through a double-layer of 77 cheesecloth to remove the bulk of insoluble material. The resulting filtrate was taken as the crude extract of cyNPDE. The crude extract was centrifuged at 10,000 x g for 10 min. Ammonium Sulfate Fractionation.--The supernatant was brought to 50% saturation by slowly adding solid ammonium sulfate (29.5 g per 100 m1 of fluid) (26). The solution was stirred for 30 min and the precipitate was removed and discarded by centrifugation at 10,000 x g for 20 min. The resulting supernatant was decanted and then brought to 80% saturation with the addition of 19.7 g of solid ammonium sulfate per 100 ml of the extracted solution. After stirring for 2 hr, the precipitate was collected by centrifugation at 10,000 x g for 30 min and dissolved in 2 mM Tris-acetate buffer, pH 7.5. The resulting solution was then dialyzed in 2.3 cm diameter dialysis tubing (Union Carbide) against 20 volumes of 2 mM Tris-acetate buffer, pH 7.5, with constant agitation for 24 hr, with 3 changes. After dialysis, the solution was centrifuged at 10,000 x g for 10 min in order to remove a small amount of precipitate which formed during the dialysis. The resulting supernatant was then fractionated further or was frozen at -20°. Enzyme activity was retained at the original level after 3 months at -20°. The enzyme up to this step still contained appre- ciable amount of 3'-nucleotidase. 78 Treatment at pH 5.0.--The pH of the dialyzed enzyme preparation was adjusted to 5.0 with 0.1 M acetic acid. The precipitate formed from the pH 5.0 treatment was removed and discarded by centrifugation at 10,000 x g for 10 min. The supernatant containing 50% of the original activity with a 7- to lO-fold increase in speCific activity was adjusted to pH 7.5 with 0.1 M KOH. Chromatography of cyNPDE on Sephadex G-200 Column.-- After pH 5.0 treatment, enzyme preparation was first con- centrated with Centriflo membrane cone (Amicon) in an Inter— national portable refrigerated centrifuge (Model PR-2) and then applied to a Sephadex G-200 column (1.5 x 120 cm) which had been equilibrated with 0.01 M Tris-acetate buffer, pH 7.5. With a 40 cm hydrostatic pressure, enzyme was eluted by the same buffer and collected in 3 ml fractions at a flow rate of 9 ml per hr. Enzyme activities were determined by the standard methods. The elution profile of protein and enzyme activities is shown in Figure 1. Although only 28.2% of the total activity toward 2',3'-cyAMP or 41.5% toward 3',5'-cyAMP was recovered, about 95% of the protein originally applied to the column was removed from the major enzyme fractions. On the other hand, there was about 5-fold increase in the specific activity toward 2',3'-cyAMP and 8-fold increase for 3',5'-cyAMP as can be seen in Table 2. The summary of the purification of cyNPDE from 300 g of pea seedlings is 79 . .0......V nE owm ue eocecmomce ecu me cems ImeeE me: coHuemuceocoo cHeuomm =.meocue2= mecco eechomee me eecHEmeuee emeB SI-lec 020003030 00 000 Roll: 002003030 00 10000000 000 00000>0000 eE>0cm .eeuoeHHoo eme3 mcoHuoemu HE eemce .60 ue mc\HE 0 mo euem BoHu ecu ue meumcc eEem ecu cuHs uco eeHmmeo me3 coHuon .m.0 cm .meuusc eueueoeImHHB z H.o cu03 eeuemcHHHove ceec eec coch AEo omH x m.HV cEsHoo e ou eeHHmme me3 escho -.0..0 000300 00000 000 000 02000-.0..0 000300 00000 000 .0000000 ms 0 0000000 Icoo .HE m .coHuemememm eemuce eeueemu o.m mm ece eeuemuceocoo Hemocmmq .hcmemmoueEomco cesHoo ooqu xececmem Eomu momzmo eem mo eHHmomm coHucHMII.H emcmHm 8O ('IW/SIINn )AllAllDV I l I l 0.2 -—‘ I F RACT ION NUMBER 81 ~.N 0.mH mmm mmH 000 0.0 0Hm men men ~.~ AwthHac :oHuoemm oomIo xececaem ~.m 0.00 m.0o N.ON New 0.0m 0.00 mm 000m o.Hv eeueemu m an m.m 0.00 0.0 m.m oNvH m.mm m.m 0.0 momm NHo AweaneHcc 00000220 momIom o.m 0HH 0.0 0.0 000m 0.00 o.H m.H maH0 «new uceuecmeoee 0 x 000.00 m.m 00H o.H m.o 0000 OOH o.H 0.H mem OHmm uoemuxe ecsmo m mE\muHcc 000:: w mE\muHco muHcs me 1000 000000 000>0000 000>0000 000000 000>000< 000>000< eHeHw IHu0msm cHeH» I0m0mcm I.m..m\4%o cwom UHHHoeam Heuoe .UmOh UHMHoemw Heuoe cHeuomm coauoemm -00..00 .004 00000 . ow mo oHuem euemumccm me mz¢>oI.m..m euemumcdm me mz<>oI.m..~ .mmCHHceem ee@ 00 0 com Eomm ememeumeHeocmmoco eeHuoeHooc UHHoao mo coHueOHmumoo mo umefifiomII.~ wands 82 also shown in Table 2. Three peaks of enzyme activity toward 2',3'-cyAMP and one having activity toward 3',5'- cyAMP were consistently observed. Enzyme preparation from this purification step was further concentrated with lyphogel and stored at -20° for further use. At that temperature, the enzyme preparation was stable for at least 4 months. Although the profile of enzyme activities in Sephadex G—200 chromatogram suggested that the enzymatic hydrolysis of 2',3'-cyAMP and 3',5'- cyAMP might be due to the same protein molecule, further attempts were made to separate the two activities. Further Attempts to Separate the Two Activities Sucrose Density Gradient Centrifugation.-—A 5-20% linear sucrose density gradient was prepared according to the method of Martin and Ames (27). A swinging bucket rotor, SW 39 (Beckman), was used for the centrifugation which was performed at 34,000 r.p.m. in a Beckman L-2 65B ultracentrifuge for 12 hr. Ten-drop fractions were col- lected after centrifugation and assayed for enzyme activi- ties according to "Assay method-1" as previously described. As shown in Figure 2-A, three peaks of enzyme activity toward 2',3'—cyAMP were observed with sucrose density gradient prepared in 0.1 M Tris-acetate buffer, pH 7.5, while only one of them showed appreciable activity toward 3',5'-cyAMP. The major peak represented 50% and 80% 83 Figure 2.--The elution profiles of cyNPDE activi- ties from sucrose density gradient centrifugation. Partially purified enzyme preparation (pH 5.0 treated fraction), 0.3 ml, containing 0.39 mg protein (24 units toward 2',3'-cyAMP) was layered over a 5 to 20% sucrose density gradient. Centrifugation was performed as described in the text. Ten—drop fractions, total of 40 fractions, were collected. Enzyme assays were carried out in the alternative fractions with two different substrates for each gradient set. The 3'-nucleotidase purified from pea was used for the coupled assay system as described under "Methods." Beef liver catalase (Mol. Wt. = 247,500) was used as the marker for estimation of the relative molecular weight of cyNPDE. (A) Elution profile of cyNPDE activities toward 2',3'-cyAMP (0————0) and 3',5'-cyAMP (O————£) under the sucrose density gradient prepared in 0.1 M Tris—acetate buffer, pH 7.5. Catalase activity was assayed according to the method of Chance et a1. (51). (B) Same condition as described in (A) except the sucrose density gradient was prepared in 0.1 M K-acetate buffer, pH 5.4. (C) Elution profile of cyNPDE activities toward 3',5'-cyAMP (0————O) and 3',5'-cyGMP (0-———0). Tris- acetate buffer, 0.1 M, pH 7.5, was used for the preparation of sucrose density gradient. ACTIVITY (UNITS/FRACTION) 84 (A) Catalase -l.5 {’ “ 1.24 I I o o ziaLcyAMP O l I I 1 . . 3,5-c AMP h1.0 " ‘. Y I I I\ -O.5 /r\. \o/‘F :\a/O\o\ 0.4-J I l .\l I I I ( B I —"5 o o 2Z3'—cyAMP o o 3:5LcyAMP F-I.O —0.5 l l (C ) —O°8 \ I o o o 3,5-cyGMP o o 3:5LcyAMP P04 0 //\’\ L? K Mfi\'\a$2=fié_a ‘ I r I I r l 8 I2 10 2O 24 28 32 36 40 FRACTION NUMBER AA240 I 10 Sec. IFR. 85 of the total activity toward 2',3'-cyAMP and 3',5'—cyAMP, respectively. About 60-70% of enzyme activity was re- covered after centrifugation. Compared to bovine liver catalase (sedimentation constant = 11.3S, molecular weight = 247,500), the major cyNPDE had a sedimentation constant of 14.275 and molecular weight of about 350,000. If acidic sucrose density gradient (prepared in 0.1 M K—acetate buffer, pH 5.4) was used, only one peak of enzyme activity was obtained for either 2',3'-cyAMP or 3',5'-cyAMP (Figure 2-B). Once again, the two activities sedimented in an identical pattern with maximal activity in the same frac- tion, 16. Because it also represented a 60-70% of recovery in enzyme activity, it is suggested that the loss of the two minor peaks of enzyme activity (which appeared in the alkaline sucrose density gradient) may be due to their in- activation by the acidic pH rather than enzyme association. This result also suggested that the dissociation may not occur in alkaline pH as the result shown in Figure 2-A. Three molecular forms of cyNPDE activity against 3',5'- cyAMP are also observed in animal systems (28-32). The elution profiles of enzyme activity toward 3',5'-cyAMP and 3',5'-cyGMP were essentially the same (Figure 2-C). An experiment using 2',3'-cyUMP as substrate gave the same distribution pattern of enzyme activity as for 2',3'-cyAMP in Figures 2-A and 2-B. 86 Polyacrylamide Disc-Gel Blectrgphoresis.-—With 7% polyacrylamide gel as described under "Methods," 0.3 ml quantity of lyphogel concentrated cyNPDE preparation from the Sephadex G-200 chromatogram, containing about 10 ug of protein and 3.0 units toward 2',3'-cyAMP and 1.2 unit toward 3',5'-cyAMP, was subjected to electrophoresis at pH 8.5. The staining pattern of protein and the distribution pattern of enzyme activities are shown in Figure 3. It is evident that the enzyme preparation still contained a number of protein species. The enzyme activity of cyNPDE was separated from the contaminated enzymes such as 3'- nucleotidases, RNase and most of the acidic phosphatase. However, enzyme activities toward both 2',3'-cyAMP and 3',5'-cyAMP were still found to be associated. About 23% and 15% enzyme activities were recovered for hydrolysis of 2',3'-cyAMP and 3',5'-cyAMP, respectively. Electrofocusing Column Chromatography of cyNPDE.-- Using the same procedures described in the methods of the preceding paper, 10 ml of pH 5.0 treated and dialyzed enzyme preparation, containing 85 units toward 2',3'-cyAMP and 26 units toward 3',5'-cyAMP per ml, was applied for an electro- focusing experiment. Ampholyte with pH values from 3.0 to 6.0 was used as protein carrier. After operation in 350 volts for 36 hr, 80-drop fractions were collected and dialyzed. Following dialysis, enzyme activities were assayed according to the standard methods. The elution 87 Figure 3.--Staining pattern of protein and dis- tribution of enzyme activities within a polyacrylamide gel after electrophoresis. An 0.3 ml of cyNPDE preparation containing 10 ug of protein was applied to a 7% polyacrylamide gel as de- scribed under "Methods." Electrophoresis was conducted at pH 8.5 at 4° for 45 min with an applied current of 2 mA per tube. The gel was cut in half; one half was stained with Coomassie blue dye, and the other half was cut in 3 mm segments. Enzyme assays for hydrolysis of 2',3'- cyAMP and 3',5'-cyAMP were performed in the alternative segments as described in "Experimental Procedures." x I o2 UNITS/SEGMENT 88 2:32-cyAMP suasnwe '//////A 3,’5’—cyAMP 20:— 15— IO— RNase SLNUCLEOTIDASE HI 5 SLNUCLEOTIDASE 1 l OJ // m—LL, [tillwlvlllllllilllluy 23 24 20 H H?) ORIGIN 89 profile of pH gradient and enzyme activities is shown in Figure 4. Activity toward 2',3'-cyAMP was found at three points, pH 4.8, 4.6, and 4.3. With 3',5'-cyAMP as sub— strate, enzyme showed isoelectric point at pH 4.8. Re- coveries were 10% of the activity toward 2',3'-cyAMP and 8% of the activity toward 3',5'-cyAMP. Characterization of the Reac- tion Products Action on 2',3'-cyclic Nucleoside Monophosphates.-- Qualitative evidence that 3'-AMP and 3'-UMP were the imme- diate products formed by the hydrolysis of 2',3'-cyAMP and 2,3'-cyUMP respectively was provided by the coupled assays with 3'-nucleotidase (Table 3). As described in the pre- ceding part, pea 3'—nucleotidase I and II were rather spe- cific for 3'-nucleoside monophosphates with activities toward both 3'-AMP and 3'—CMP. However, 3'-nucleotidase from rye grass (Sigma) has been shown to be without activi- ties toward pyrimidine 3'—nucleoside monophosphates. The enzyme activity found in the control reaction was due to the presence of small amount of 3'-nucleotidase in cyNPDE preparation from Sephadex G-200 chromatogram. Further evidence that the reaction product was ex- clusively 3'-AMP was obtained from ion exchange chromato- gram. The enzyme preparation from the sucrose density gradient (fraction 16 in Figure 2) was incubated with 0.1 m1 of 0.01 M of 2',3'-cyAMP and 0.05 ml of 0.2 M K-acetate 9O .Qlll .0§>0-.m..m flOllie .mzamo-.m..m $03300 momzmo coflumcHEumuwo may 00m pom: mumuumnsm .maflmoum mm may mucmmmummu maonflo use Ignaz m>u50 Uflaom =.mconuwzg map was uxmu on» Ca cm>flm mum monopmooum cmaflmumo .mchSUOMOMuomHm o» pmuoanSm mm3 mzdmol.m..m pumzou muflcs com com mz¢mol.m..m GAMBOu mafia: omm mcflcflmucoo coflumnmmmum mfiwucm Umummuu o.m mm one .mcmmumoumfionco cESHoo mcflm500mouuomam cm Eouw momzwo mom wo maflmonm coflusamll.v musmflm 91 3'-cyAMP si-cyAMP I I 2 3 (NOILDVIH lSlINfl) A1IAI13V 30 FRACTION NUMBER 20 92 TABLE 3.--Enzymatic analysis of the hydrolysis product formed from 2',3'-cyNMP.a Substrate Assayed Addition 2',3'-cyAMP 2',3'-cyUMP x 10 umoles Pi released None 1.105 1.500 3'-nuc1eotidase I (pea) 3.815 4.291 3'-nucleotidase II (pea) 3.647 4.145 3'-nuc1eotidase (rye grass) 3.600 1.638 aActivity was determined in a 0.5 m1 reaction mix- ture containing 2 mM substrate, 0.1 m1 enzyme solution (concentrated fraction 18 from Sephadex G-200 column chroma- tography, 0.01 mg protein/ml) and 0.1 M K-acetate buffer, pH 5.4. Incubation was performed at 37° for 1 hr. After incubation, the reaction mixture was then heated in boiling water for 3 min to terminate the reaction. Then 3'- nucleotidase in excess amount was added and incubated under its optimal pH for 15 min. Inorganic phosphate released was determined according to the "Methods." 93 buffer, pH 5.4, in a final volume of 0.3 ml. The incu- bation temperature was at 37° (Figure 5). After termination by heating the reaction solution in boiling water bath for 3 min, the whole reaction mixture was applied to an ion exchange column as described in the "Methods." The amount of 3'-AMP formed from 2',3'-cyAMP increased with time while there was no apparent formation of 2'-AMP. The values shown in the figure have been corrected by the values ob- tained in the zero time incubation. Another experiment using 14C-2',3'-cyAMP (prepared from l4C-polyadenylic acid by the action of pea RNase as described in the preceding part) gave the same result shown above without any detect- able formation of 14C—Z-AMP. Action of 3',5'—cyclic Nucleoside MonOphosphates.-— Enzymes specific for the hydrolysis of 3',5'-cyc1ic nucleo— side monophosphates so far isolated and characterized from animal tissues (33-36, 48), slime molds (37), and micro- organisms (13, 38, 39) have been demonstrated to catalyze the formation of 5'-AMP exclusively from 3',5'-cyAMP. It was desirable to know whether the pea cyNPDE was similarly specific. In order to have a more sensitive assay, purified 3H-3',5'-cyAMP was used for this study. The cyNPDE prepa- ration must be free of 3'-nuc1eotidase. This was accom- plished by sedimenting 0.3 m1 of concentrated enzyme prepa- ration from Sephadex G-200 chromatogram, containing 11 94 .000000 000 000003 00000 IHUGH m0 coflumnsocfi How Aucv 0600 one .A IIIIII v mafia Umcmmp on» an pmucmmmummu m0 mz¢>o|.m..m mo uosooum m0m>aouc>c may mo cumuumm coflusam one =.moocum20 0:0 paw uxmu map 20 ownwnommc mum mmHSUmooum pmawmuwo .Amz¢|.m m0 m8 m.o pom mz¢n.m mo 08 v.ov mocsomeoo 000:03050 £003 Umumunfiamo umuflm mmz 30053 050 o.m x m.ov CEsHoo A HOV NxIHI04 pmmoflm m on pmflammm wauomuflo mm3 musuxflfi coauommu pmummc mcu .COHumnoocH 000mm .cowumummmum mahucm Houucoo may mm poms mm3 wudmflm 050m may 500% oa cofluomum .0: ma no 0: m.m 00m ohm um v.m mm .Hmmwsn mumumomum z N.o mo HE mo.o paw mz¢>ol.m..m SE OH NO HE H.o £003 Umumnsocfi mm3 Am musmflh ca 00 coauomumv coflummDMHHucmo 0cmwomum hyamcmp mmouosm Eonm coaumummmnm weancm .m§%UI.m~ .N mo pooponm mammaoupmc on» mo mnmmumoumfiouno mmcmcoxm coHII.m musmfim 95 «mass Z 20. #013: m2an..~ O on oo- BAILV'HU All/“13V 115 enzyme activities were quite stable under acidic pH at 4° or -20°. The optimal pH of pea cyNPDE was different from that of similar enzymes in microorganisms (38, 39) and animal systems (33-35) for which the enzyme has been shown to have a maximal activity in alkaline pH region, pH 7.5 to 8.0. Influence of Various Metal Ions.--"Assay method-l" and 3'-nucleotidase II were used in the assay of the hydrol- ysis of 2',3'-cyAMP in the presence of various metal ions. For the assay of the influence of metal ions on enzyme activity toward 3',S'-cyAMP, purified 3H-3',S'-cyAMP and "Assay Method-2" were used. The effect of various metal ions on enzyme activity is shown in Table 4. It is evident that both activities behaved quite similarly in response to the presence of metal ions concerned. A slight increase in both enzyme activities was observed in the presence of Mn++, Co++, and Zn++ at concentrations of 0.1-1.0 mM. NaF at 1 mM showed a 45% of inhibition on enzyme activity toward 3',5'-cyAMP with no apparent effect on the enzymatic hydrolysis of 2',3'-cyAMP. The EDTA at the concentration of 0.1 mM or 1.0 mM appeared to have no effect on either activity as also observed in Serratia marcescens (38). Effect of Various Concentration of Sulfhydryl Com- pounds and NaF.--Because sulfhydryl compounds such as cysteine and dithiothreitol do not inhibit the activity of 116 TABLE 4.--Effect of inorganic ions on the activity of cyclic nucleotide phosphodiesterase. Cyclic Nucleoside Monophosphate Compound Assayed 2',3'-cyAMPa 3',5'-cyAMPb Activity Remaining (%) None 100 100 Mgc12 , 1 x 10‘3M 101 100 1 x 10'4M 101 101 Mnc12 , 1 x 10’3M 112 138 1 x 10’4M 110 137 Coc12 , 1 x 10'3M 110 132 1 x 10'4M 105 117 ch12 , 1 x 10‘:M 118 130 1 x 10 M 108 114 (NH4)2504 , 1 x 10'3M 107 110 1 x 10'4M 100 101 KCN , 1 x 10'3M 105 107 1 x 10'4M 100 101 KCl , 1 x 10'3M 108 110 1 x 10‘4M 110 119 NaCl , 1 x 10'3M 100 101 1 x 10'4M 101 100 NaF , 1 x 10'3M 117 55 1 x 10'4M 110 78 EDTA , 1 x 10’3M 100 100 1 x 10'4M 100 100 aWith 2 mM 2',3'-cyAMP as substrate, the standard assay was performed under the condition that 3'-nucleoditase was not rate limiting. bWith 2 mM 3H-3',5'-cyAMP as substrate, the details are described in the text. 117 pea 3'-nucleotidase I as described in the previous section, pea 3'-nucleotidase II was used in a coupled enzyme assay system for the study of the effect of reducing reagents on the activity of pea cyNPDE. As shown in Table 5, cysteine and dithiothreitol at concentrations from 0.04 mM to 4.0 mM had similar effects on the two activities with a maximum enhancement of 35-45%. The NaF inhibited the hydrolysis of 3',S'-cyAMP, but not 2',3'-cyAMP. Effect of Urea on Engyme Activity.--Both activities decreased in parallel in the presence of various concentra- tion of urea (Figure 13). However, points of 50% activity were at 6 M urea for activity toward 3',S'-cyAMP, and 8 M urea for the hydrolysis of 2',3'-cyAMP. It was also ob- served that both activities decreased simiarly with respect to various times of preincubation in 6.5 M urea (Figure 14). Optimum Temperature and Heat Stability of Enzyme Activity.--As seen in Figure 15, both activities had an optimum temperature at 40° under the standard assay condi- tion. It is evident that heat inactivation began appre- ciably at 50°. Activity toward 2',3'-cyAMP was apparently more sensitive to temperature than that toward 3',5'-cyAMP. An Arrhenius plot of data taken from Figure 15 displayed a change in sloPe at 40° as shown in Figure 16. With the integrated form of the Arrhenius equation, E _ 2.303 R TlTZ (log k2 - log K1) — I T2"1'1 118 TABLE 5.--Effect of various concentration of reducing re- agents and NaF on the activity of pea cyclic nucleotide phosphadiesterase. Substrate Assayed Additiona 2',3'-cyAMP 3',5'-cyAMP Activity Remaining (%) None Cysteine, 4 x 10'3M 100 100 2 x 10’3M 134 144 4 x 10'4M 119 109 2 x 10’4M 129 121 4 x 10'5M 95 105 Dithiothreitol, 4 x 10’3M 133 113 2 x 10'3M 129 110 4 x 10'“M 126 134 2 x 10'4M 108 105 4 x 10‘5M 120 98 NaF , 4 x 10‘3M 111 37 2 x 10‘3M 116 38 4 x 10'4M 121 43 2 x 10‘4M 114 79 1 x 10’4M 110 79 standard reaction mixture. aAll reagents were added at zero time with the The 3'-nuc1eotidase in excess amount was used for the coupled assay system as described "Methods." under 119 I.m..m “GIIIIQ .mZdon.m..m “pom: mumnumbcm pmcHEHmumo was pmmmmamu mumcmmonm oacmmuoca may mmmoxm .coflumnsocfl uwuwd .H: A new ohm um moms omumnsosfl oumz cfimuoua m1 om mcflcflmucoo mmusuxHE mo mufl>fluom map so mow: mo coflumuucwocoo on» .ollo £2de =.mponumz= Rocco confluomop mm was poops mm3 mmmofluomHODCI.m m0 COHfiManGGOGOU mDOHHm> £HH3 COHuUme CHMUGM#m wSB .mamzmo mom mo pommmmuu.ma musmflm :1 <55 to 20.2.5szon V a 120 _ _ _ m201.m..m nOIIIIG .m2¢>01.m..m “pom: onmuumnsm .moonumE oumocmum on» on Homo“ .mawmumc nonuHSM Mom .musumummfimu mzoflum> um coflumnsocfl H: H nmumm poops mm3 mmmofiuomaoscl.m mmmoxm menu you poms mmz samuonm on on mcwcflmucoo mHDuXHE cowuommn pumoamum .momzwo mom How maamoum muH>HuUMImusumummEmBII.ma musmflm .wosum 124 A Uo Emaémazfi on n# 0V mm on ma ON m — _ _ _ _ _ _ — _ o o\ o\ \ E I. \\ \ x I C \ \ m<< may um cmumnso Icwmum mum3 sawuoum mo m: om mcacwmucoo mousuxfie cofipomwn pumocmum one .momzao mom mo huflaflnwum Dmmmll.na madman 129 72.2 n V manh 2',3'-cyAMP > 3',5'-cyUMP > 2',3'-cyGMP > 3',5'-cyIMP > 3',5'-cyAMP > 2',3'-cyCMP, 3',5'-cyGMP, 3',5'-cyTMP > 3',5'-cyCMP. It is interesting that 2,6- dibutyryl 3',5'-cyAMP, an analog of 3',5'-cyAMP which is insensitive to the animal enzyme (2), was hydrolyzed with at a rate similar to that of 3',5'-cyAMP in pea system. In general, the enzymes so far isolated and partially purified from animal tissues have been shown to be specific for hy- drolysis of 3',5'-cyAMP and 3',5'-cyGMP (2, 49). Although the diesterase from rabbit brain seems to have activity to- ward 2',3'-cyAMP, it may be due to the contamination of a separate enzyme (48). 132 TABLE 7.--Relative cyNPDE activities toward cyclic nucleo- side monophosphates.a Substrate Assayed Relative Activity (%) 2',3'-cyUMP 100 2',3'-cyAMP 83 2',3'-cyGMP 51 2',3'-cyCMP 26 3',5'-cyUMP 68 3',5'-cyIMP 45 3',5'-cyAMP 41 3',5'-cyGMP 26 3',5'-cyTMP 26 3',5'-cyCMP 23 aAssays were carried out as described under "Methods." Two mM substrates were used for assays. 133 Determination of Michaelis Constant (Km).--The Km values for 2',3'-cyNMP and 3',5'-cyNMP were calculated from the experimental data obtained by incubating several dilu- tions of the substrate with 10 ug of protein on cyNPDE preparation at 37° for 1 hr. The reaction was stopped by heating to 100° for 3 min. The reaction mixture was cooled, excess of 3'-nuc1eotidase was added and Pi was measured as described under "Methods." The double recipro- cal plots of 1/S vs. l/V for various substrates by the' method of Lineweaver and Burk (50) are shown in Figure 18 and Figure 19. It is apparent that the affinity constant (1/Km) of the respective substrate decreased in the order as follows: 3',5'-cyUMP > 2',3'-cyUMP > 2',3'-cyAMP > 3',5'-cyAMP > 3',5'-cyGMP > 2',3'-cyGMP > 3',5'-cyCMP > 2',3'-cyCMP. A summary of the properties of the well- characterized 3',5'-cyclic nucleotide phosphodiesterase and the pea cyNPDE is shown in Table 8. Activity Toward Other Organic Phosphates.—-A1though the pea cyNPDE from the Sephadex G-200 contained activities toward RNA, DNA, and other organic phosphates as indicated in Figure 20, such activities were probably due to the con- tamination of specific or nonspecific phosphatases rather than cyNPDE itself having such activities. For instance, evidence for the contamination of RNase and 3'—nucleotidase in cyNPDE preparation was demonstrated in the study of gel electrophoresis as the result shown in Figure 3. Furthermore, 134 .n3onm ma mumuumnsm nomm How EM one .m\a .mo >\H mo uoHa nusmIHm>mm3 Imnflq on» .unmwm .Amv coflumuunmocoo mumnumnsm m5mum> A>V >ua>fiuom meannm mo uon mnu .ummq =.m©onum2= moons confluomwo mm meuomumm mumz mammmm memunm .mumuumnSm 03“. mm A‘v QEUNUI.M~.N .HO sAGV QZD%UI.M~.N sAOV QZUNUI.M~.N ~A.v QEMUI.M~.N M0 mnoHumupnmonoo msowum> paw nfimuonm mo on oa pmcwmunoo musuxwfi cofluommn one .mmmumummflponmmonm mofluomaosn owaomo mom mo mufl>fluom any so noflpmuunmonoo Amzz>on.m..mv mumuumnsm mo uommmmII.mH musmfim 135 A2... 2.3 :\\ nisnmdv< azzxulhu _ n6 0.— ‘9 V. N N. m 06 ”V (sunn) A 136 .nsonm we ououumnsm nomo Hem EM one .m\H .m> >\H no uon nusmuno>oo3onflq onu .unmflm .mnofluouucoonoo oumuumnSm ozono> A>v >ua>wuoo oawuno mo poem onu .umoq =.moonuoz= uoonn nonwuomoo mo ooEHOMHom ouo3 wommo ouonmmonm oesomuonfl onu ono mononoooum poawouoo .ououumnzm onu mo A can neououm no on oa poneounoo ousuxwa nofluooou one .omououmowoonmmonm ooflpooaosn oeaomo mom mo eua>flvoo onu no nofluounnoonoo AmEZMOI.m..mV oumuumnSm mo uoommmII.mH ouamwm 137 :2... oa3< mZZxUJ “~40 — IIflII :25 3.3 :\ Axe—oiwx A2... 3.: u so. (Slan) A 138 Figure 20.--The elution profiles of enzyme activi- ties from Sephadex G-200 column chromatography. All experimental conditions are the same as de- scribed in Figure 1. Enzyme activities for various sub- strates were assayed according to "Methods." Upper, each solid curve represents the enzyme activity for one specific substrate as indicated by the arrow. Dashed line shows the activity toward p-nitrophenol phosphate (PNP) as measured by the absorbance at 410 mu. Each fraction corresponds to the number shown in the lower part of the figure. Lower, solid curve represents protein concentration as measured by the absorbance at 280 mu. Enzyme activities (in units/ ml) on 2',3'-cyAMP (O————4), 3',5'-cyAMP (0 0), 3'-AMP (pH 8.0) (X----X), RNA (A--—-A), and DNA (A-—-—A) were determined as described under "Methods." .sz : 3 ...5.z_zn:o_v< . m 2.: ~22 139 m r o . o 2 A a2xflh_za.mm(0_h°mdgz d mmauom HmEmeE new + .nfiououm mo oE\cflE\noN>Houo>n madeol.m..m moaofin +mz wo oocomoum on» pouwsqou oE>Nno monocon + n A.uo< .mmv >ua>flu04 waHoommm 2140 xooum macs 000.000 "mmmwm uooooo oz 0~.0 monaoooo moo 1Ha0no .NMImm amuuoz ozaq uom Asomav masoco mad- oooofloflooH 00.0 csmum uom noomHv ufimz ozdu cofluflnfincH m0.o unmom moo 30m: halmIIum UCOEESHQ deI cofiuflnfinnH vo.o nwmum ufinnmm Amomae .MMIMM uooouom ozau conufionocH mI0Hx0HI0 00.0 uumom moon oocouomom AHOE\Hmoxvmm .uz .Ho: uommmum ommmwmwm n++ m.uo< .dm oousom .momououmofloonmmonm oofluooausn oaauaoI.m..m oonwuouooumnouaaos no enmeeamnu.m mqmde 141 Figure 21.--The elution profiles of cyNPDE, RNase, and 3'-nuc1eotidases from sucrose density gradient cen- trifugation. For detailed experimental conditions, refer to the description in Figure 2-A. (A) The elution profile of cyNPDE activities to- ward 2',3'-cyAMP (t————O) and RNase (O----). (B) The elution profile of 3'-nuc1eotidases activi- ties. Substrates and buffers used: 3'-AMP, K-acetate 0.1 M, pH 5.4 (0----0); 3'-AMP, Tris-acetate 0.1 M, pH 8.0 (0——O). (C) The elution profile of cyNPDE activity toward 2',3'-cyAMP (0e———0) and 3'-nucleotidase activity toward 3'-AMP (0----0). 0.1 M K-acetate buffer, pH 5.4 was used for assays. ACTIVITY (UNITS! FRACTION) 142 2:31-cyAMP F RAC T ION NUMBER RNase ACTIVITY ( UNITS/FRACTION) NUCLEOTIDASE 143 the sucrose density gradient centrifugation provided evi- dence that pea cyNPDE had no significant activity toward either RNA or 3'-nuc1eotides as the result shown in Figure 21 (the experimental procedures were the same as described in Figure 2). Discussion An enzyme from Alaska pea seedlings hydrolyzes both 2',3'-cyNMP and 3',5'-cyNMP. This cyclic nucleotide phos- phodiesterase was purified approximately 218-fold with a recovery of about 8% of the total activity toward 2',3'-cyAMP. Although the enzyme preparation still had detectable activities toward 3'-nuc1eotides, RNA, DNA, and some organic phosphates, such activities are probably due to the contamination of nucleotidases and other phosphatases rather than to cyNPDE itself. Evidence for this is as follows: 1. Pea cyNPDE activity can be separated completely from the enzyme activities toward nucleotides, RNA, DNA, and various organic phosphates by means of sucrose density gradient centrifuga- tion, and polyacrylamide gel electrophoresis. 2. Compared to the prOperties of pea RNase (3'— nucleotidase II) as described in the first section, pea cyNPDE is quite different with respect to pH optimum, effect of reducing re- agents, acid stability, rate of sedimentation 144 in sucrose density gradient, electrophoretic mobility and molecular weight. Since most, if not all, of the RNases so far char- acterized from higher plants are cyclizing enzymes that yield 2',3'-cyNMP with little further activity toward 2',3'-cyNMP, the pea cyNPDE described here may play a crucial role in the degradation of RNA. I suggest that RNA degradation in higher plants may not follow the scheme that is generally accepted in which RNase (cyclizing enzyme) hydrolyzes both RNA and 2',3'-cyNMP (8, 9). I propose that the degradation of RNA in higher plants is as follows: RNA 1 (RNase, cyclizing enzyme) 2',3‘-cyNMP (Cyclic nucleotide phospho- diesterase) 3'-NMP 1 (3'-Nuc1eotidase) Nucleoside + Pi Because pea cyNPDE catalyzes the formation of 3'-AMP exclusively from the hydrolysis of 2',3'-cyAMP, the 3'-NMP formed from the study of RNase in higher plants as described in the first section strongly suggest that the contamination of cyNPDE in the RNase preparation. In barley seedlings also the 2',3'-cyNPDE activity is differ- ent from RNase. 145 With respect to the hydrolysis of 3',5'—cyAMP, the pea cyNPDE was purified approximately 470—fold with a recovery of about 19% of the total activity present in the crude extract. 4 Unlike the enzyme from animal tissues, the pea enzyme exhibited an acidic pH optimum, and insensitivity to caffeine, theophylline, and imidazole. Furthermore, the pea enzyme activity was not dependent upon the presence of Mg+2. Other metal ions had no effect on enzyme activity. The reason for the inhibition of NaF is not clear. The pea enzyme catalyzes the formation of 3'-AMP mainly from 3',5'-cyAMP rather than 5'-AMP which is the ex- clusive product in the animal system. The pea enzyme catalyzes the formation not only of 3'—AMP but also of 5'-AMP with a ratio of 3'-AMP:5'-AMP of about 7:1. Both products were formed directly from 3',5'-cyAMP; there was no interconversion of these two nucleotides under the assay conditions. The data suggest that the formation of two products from one substrate is probably due to a single enzyme. Evidence for this is based on the following: 1. The formation of the two nucleotides was parallel with a constant ratio throughout the whole time course of the hydrolysis of 3',5'- cyAMP. 146 2. The ratio of the two nucleotides was constant throughout the fractions of the sucrose density gradient. 3. Both 3'-AMP and 5'-AMP showed a similar degree of inhibition of the enzyme activity toward 3',5'-cyAMP. The activities of enzyme toward 2',3'-cyAMP and 3',5'-cyAMP were maintained in a rather constant ratio throughout the purification procedures and were quite simi- lar with respect to pH Optima, metal ions effect, effect of sulfhydryl reagents, heat stability, temperature Optima, and the sensitivity to treatment with urea. Furthermore, both activities had the same electrophoretic mobility, the same rate of sedimentation in sucrose density gradient, the same isoelectric point and the same behavior on gel filtration. Therefore, it is suggested that the hydrolysis of these two cyclic nucleotides was due to the same enzyme molecule. Since attempts to demonstrate the presence of adenyl cyclase and the incorporation of radioactive adenine and adenosine into 3',5'-cyNMP in either the pea or the barley system have been unsuccessful, the possible biological sig- nificance of the presence of an enzyme with activity toward 3',5'-cyNMP in higher plants is not known. 147 Summary An enzyme able to hydrolyze both 2',3'-cyNMP and 3',5'-cyNMP has been purified about ZOO-fold from germinat- ing pea seedlings. The enzyme shows maximal activity at pH 5.4-6.0 with a Km of 0.62 mM for 2',3'-cyUMP, 0.83 mM for 2',3'- cyAMP, 4.34 mM for 2',3'-cyGMP, 5.0 mM for 2',3'-cyCMP, 0.58 mM for 3',5'-cyUMP, 0.90 mM for 3',5'-cyAMP, 1.61 mM for 3',5'-cyGMP, and 1.81 mM for 3',5'-cyCMP. There is no apparent requirement of metal ions for full activity. The enzyme catalyzes the formation of 3'—AMP ex- clusively from 2',3'-cyAMP and the formation of 3'-AMP and 5'—AMP with a ratio of 3'-AMP:5'-AMP of about 7:1 from 3',5'-cyAMP. There is no interconversion of 3'-AMP and 5'-AMP. The formation of the two products from one sub- strate is probably not due to the presence of two different enzymes. The activities of the enzyme toward 2',3'-cyAMP and 3',5'-cyAMP were quite similar with respect to pH optima, effect of metal ions, effect of sulfhydryl reagents, heat stability, temperature optima, and sensitivity of treatment with urea. Furthermore, the two activities had identical physical properties. It is, therefore, suggested that the two activities reside on a single protein molecule. With gel filtration and sucrose density gradient, three enzyme activities toward 2',3'-cyAMP were found. Only 148 one of these (molecular weight about 350,000) had a preferential activity toward 3',5'-cyAMP. Methylxanthines showed no effect on enzyme activity. Activation energy for hydrolysis of 2',3'-cyAMP was 8.6 Kcal/mole and for 3',5'- cyAMP was 7.2 Kcal/mole. Isoelectric points of the three activities were at pH 4.3, 4.6, and 4.8. Because the cyNPDE does not hydrolyze RNA, a new mode of RNA degradation in higher plants has been proposed. 10. 11. 12. 13. 14. BIBLIOGRAPHY Sutherland, E.W., and Rall, T.W., J. Biol. Chem., 232, 1077 (1958). Robison, G.A., Butcher, R.W., and Sutherland, E.W., Ann. Rev. Biochem., 31, 149 (1968). Pastan, I., and Perlman, R., Sciences, 69, 339 (1970). Ide, M., Yoshomoto, A., and Okabayashi, T., J. Bac- teriol., 24, 317 (1967). 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