PURIFICATION AND PROPERTIES OF AN. EXONUCLEASE (PHOSPHODIESTERASE I) FROM CUCUMIS MELO Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY RICHARD E. JAGGER. JR. 1971 3“" LIBRARY Michigan State University This is to certify that the thesis entitled PURIFICATION AND PROPERTIES OF AN EXONUCLEASE (PHOSPHODIESTERASE 1) FROM CUCUMIS MELO presented by Richard E. Jagger, Jr. has been accepted towards fulfillment of the requirements for Ph.D. degree inhiochemmry v V" ‘ Major professor \j Date October 26, 1971 0-7839 800K BINDERY mcI ‘ LIBRARY BINDERSI gammy}. Ilcmsgj - A A ./ f P'. 7.1 ' f ' ABSTRACT PURIFICATION AND PROPERTIES OF AN EXONUCLEASE (PHOSPHODIESTERASE I) FROM CUCUMIS MELO By Richard E. Jagger, Jr. An exonuclease was purified from gucumis melo seeds to an extent of 3,h00-fold with a recOvery of about 1h percent of the total exonuclease activity present in the crude extract. The enzyme displays many of the properties typical of phosphodiesterase I activity. No contamination by nonspecific phosphodiesterase, endonuclease, phosphatase, nucleotidase, adenylic acid deaminase or adenosine deaminase was detected. The enzyme was prepared by an expeditious procedure involving a heat step with a accompanying change in pH, followed sequentially by acetone fractionation, ammonium sulfate fractionation, Sephadex G-100 gel filtration and phosphocellulose chromatography. The enzyme has a pH optimum of pH 9.3, with the activity decreasing quite sharply on either side of this pH. The stability of Q, melo exonuclease was dependent both on temperature and on pH, Optimal conditions being ROG and pH 7.5. The enzyme activity was stimulated by Mg++, Ca++ and Ba++ and was destroyed or reduced in the presence of sulfhydryl compounds, fluoride ion, and EDTA. Hydrolysis of denatured DNA and ribosomal RNA was shown to be exonucleotylic in nature. Using gel filtration techniques, the molecular weight of the enzyme was determined to be about 78,000 daltons. Richard E. Jagger, Jr. An activation energy of 2,800 cal per mole was obtained for the hydrolysis of p-nitrOphenyl-pT. The enzyme hydrolyzed the p-nitrophenyl esters of S'-nucleotides, with pfnitrophenyl-pdG being hydrolyzed faster than Ernitrophenyl-pT or Ernitrophenyl-pdc, and with the hydrolysis of ‘p-nitrophenyl-pdA being the slowest. The S'-deoxyribotide esters were hydrolyzed more rapidly than the ribotide analogues. Denatured DNA and ribosomal RNA were hydrolyzed much more slowly than were the p-nitrophenyl nucleotides. Hydrolysis of native DNA by the enzyme preparation could not be detected. The ribotide homOpolymers of polynucleotides were hydrolyzed at a slow rate by the exonuclease. A strong inhibition by S'-AMP of the hydrolysis of penitrophenyl-pT and penitrophenyl-pdc was observed and determined to be competitive in nature. PURIFICATION AND PROPERTIES OF AN EXONUCLEASE (PHOSPHODIESTERASE I) FROM CUCUMIS NELO BY _.J Richard E? Jagger, Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1971 ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Professor James L. Fairley for his valuable guidance, encouragement, and thoughtful criticism throughout the course of this work. It was he who instilled the constant reminder that a scientist is also a person who shows an interest in our world. The author also wishes to thank Fritz M. Rottman, Allan J. Morris, Clifford J. Pollard, and Hans A. Lillevik for serving on his guidance committee. He is also grateful to John E. Wilson for his assistance with the activation energy determinations and in the subsequent computer analysis, and to Miss Elizabeth Lessard for assisting in the preparation of the figures. The continual encouragement and faith of my parents is appreciated. The author is especially grateful to his wife, Pat, for typing this manuscript and to both his wife and daughter, Trudy, for their patience and love throughout the course of this work. ii TABLE OF CONTENTS Page INTRODUCTION ..................................................... 1 MATERIALS AND METHODS Enzyme Source ................................................ Enzyme Substrates ......... ..... .......... ...... ......... ..... Enzymes ........................................ ....... ....... Resins .................................... ..... .............. Reagents ..................................................... Protein Determinations .................. ...... ............... Phosphate Determinations ..................................... \oooooooal—qmm Exonuclease Assays ........ ....... ............. ...... ......... H O Nucleotidase Assay .... ..... .................. ........... ..... Deaminase Assay ................ ...... ........................ 10 Gel Filtration ............................................... 11 Isoelectric Focusing ......................................... 11 Polyacrylamide Gel ElectrOphoresis ........................... 12 Paper Chromatography ......................................... 12 EXPERIMENTAL RESULTS Purification of the Exonuclease .............................. 13 Preparation of Cucumig melo Seeds ......................... 15 Preparation of Soluble Enzyme ............................. 13 pH Adjustment and Heat Coagulation Precipitation .......... 13 Acetone FraCti-onation COO...O..0.COOOOOOOOOOOOOOCCCCOOOOOOO 11+ iii Page Ammonium Sulfate Fractionation ............................ 15 6-100 Sephadex Gel Filtration ............................. 15 Chromatography on Phosphocellulose ........................ 18 Comments on the Isolation Procedure ....................... 22 Properties of Muskmelon Exonuclease ........................... 25 The Enzyme Assay .......................................... 25 Effect of pH on Enzyme Activity ........................... 25 pH Effect on the Stability of the Exonuclease ............. 2S Isoelectric Point of Muskmelon Exonuclease ................ 30 Effect of Temperature on Muskmelon Exonuclease ............ 51 Effect of Storage and Dialysis ............................ 31 Effect of Activators and Inhibitors on Exonuclease Activity 5h Effect of EDTA upon Activity .............................. 56 Reversal of EDTA Inhibition by Metals ..................... 36 Effect of Sulfhydryl Compounds ............................ kl Specificity of Muskmelon Exonuclease on Ip-nitrophenyl Substrates .................... ...... ........ hl Kinetic Characteristics ................................... AS The Effect of Nucleotides on the Rate of Hydrolysis ....... h8 Type of Inhibition Displayed by S'-AMP .................... 50 Hydrolysis of Polynucleotides ............................. SO Contaminating Activities .................................. SS Mode of Degradation of Denatured DNA and Ribosomal RNA .... 55 Hydrolysis of Oligouridylic Acid .......................... 59 Polyacrylamide Gel Electrophoresis ........................ 60 Determination of the Molecular weight ..................... 61 iv Page Determination of the Activation Energy of makmelon ExonuCIease 000......00.000.000.00.0.00.0000...O. 61 DISCIJSSION 000......OOOOOOOOOOOOOOOOOO...0.0.0.0000000000000000000 67 SUMY 0.0.00.0...OOOOOOOOOOOOOOO0.00000000000COOOOOOOOOOO'00.... 76 BIBLIOGMPHY 00.0.0000...OOOOOOOOOOOOOOOOOOO0.00000000000000000... 78 Table II. IIIa. IIIb. IV. VI. VII. LIST OF TABLES Page Summary of the purification of exonuclease from 600 g (dry weight) of muskmelon seeds ........................... 21 Effect of activators and inhibitors on exonuclease activity ...................................... 3S Protocol for experiment displaying metal ion reversal of EDTA inhibition ............................... 39 Effect of divalent metal ions in restoration of EDTA inhibited exonuclease activity .................... 39 Effect of sulfhydryl compounds on activity ................ #2 Specificity of muskmelon exonuclease on E-nitropheny1'8Ubstrates OOOOCOOOOOOOOOO00.0.0.0...0.0.0... uh The effect of nucleotides on the rate of hydrolysis Of E-nitropheIIYI-pT 0.000000000000000...00000000000000.0000 ’49 Hydrolysis of polynucleotides ............................. Sh vi LIST OF FIGURES Figure Page 1. Elution pattern of Sephadex G-100 gel filtration ........ 16 2. Elution pattern of the phosphocellulose ion eXChange colum OOOOOOOCOOOOOOO0.0.00.00.00.00.0...... 19 3a. pritrophenyl-pT hydrolysis by C, melo exonuclease asafunction Of tilne OOOOOOOOOCOOOOOOOOOOOOOOOOOOOOOOOOOO 26 3b. ‘EfNitrOphenyl-pT hydrolysis by C, melo exonuclease as a function of enzyme concentration .................... 26 A. Effect of pH on exonuclease activity ..................... 28 5. Effect of temperature on exonuclease activity ............ 32 6. Inhibition of exonuclease activity by EDTA ............... 37 7. The relationship of substrate concentration to reaction velocity as shown by a Lineweaver-Burk plot for muskmelon exonuclease ........................... R6 8. Competitive inhibition by S'-AMP of the hydrolysis of pfnitrophenyl-pT by the exonuclease from C. melo ...... 51 9a. Chromatography on Bio-Rad P-6 of denatured DNA at stages in its digestion by venom phosphodiesterase ....... 57 9b. Chromatography on Bio-Rad P-6 of denatured DNA at stages in its digestion by micrococcal nuclease Of -S-. aureus 0.0.0....0.0...OOOOOOOOCOOCOOOOOOOCOOOOOOO0.0 57 9c. Chromatography on Bio-Rad P-6 of denatured DNA at stages in its digestion by C, melo exonuclease ........... 57 9d. Chromatography on Bio-Rad P-6 of ribosomal RNA at stages in its digestion by C. melo exonuclease ........... 57 10. Determination of molecular weight by gel filtration ...... 62 ll. Arrhenius plot for muskmelon exonuclease ................. 6S vii INTRODUCTION In 1903, Araki observed that a substance extracted from animal tissues caused changes in isolated nucleic acid (1). In the subsequent sixty-eight years, a wealth of information has been obtained about the detection of, the isolation of, and the nature of the substances which catalyze the chemical hydrolysis of nucleic acids. Enough material has been published to warrant at least one review article per year for the last few years dealing in part or entirely with the enzymes which degrade DNA and/or RNA. These enzymes have been called nucleases, polynucleotidases, RNases and DNases, to name a few. With the mountain of reports describing an enlarging number of enzymes of this nature, a meaningful classification of such enzymes was needed. Laskowski (2) has proposed nine criteria for use in classifying DNases, and Bernard (3) has modified it for classifying RNases. With these two reviews some order to the classification of degradative enzymes of nucleic acid has been achieved. Among the enzymes considered in the reviews is a class of enzymes commonly known as phosphodiesterases. In a general sense phosphodi- esterases are enzymes which hydrolyze the bond between a phosphoryl function and one of two ligands attached to it, the latter possessing an alcoholic function through which the phosphate ester bond is formed. It is usually, but not always, assumed that one of the two ligands to the phosphoryl group is a nucleoside. Hydrolysis of both the inter-ribonucleotide and/or the 1 inter-deoxyribonucleotide linkages with the stepwise formation of 5'- or 3'-nuc1eotides is characteristic of a class of phosphodiesterases called exonucleases. Phosphodiesterase however is still generally used interchangeably with exonuclease, presenting a small state of confusion. In this thesis the term phosphodiesterase is assumed to be completely analagous to the term exonuclease. Phosphodiesterases have been found to be ubiquitous in nature, having been detected and isolated from E. Eflll (h, S), bacteriOphage A (6), Erlich Ascites Tumor cells (7), snake venom (8), salmon testis (9), mammalian organs (10-13), and higher plants (IA-16) for examples. Exonuclease I, II, and III from E. ggli and an DNA exonuclease from A degrade only DNA while other exonculeases from E. 221$ and from Erlich Ascites Tumor cells degrade only RNA. The phosphodiesterases (exo- nucleases) from other sources tend to be nonspecific toward the sugar moiety, and have been defined by Razzell (17) as phosphodiesterase I and phosphodiesterase II by a criteron based on the specificity of hydrolysis. Phosphodiesterase I, of which venom phosphodiesterase (8) is an example, exhibits an absolute specificity for a nucleoside with a S'- phosphoryl residue and a 3‘-hydroxyl function. This enzyme liberates nucleoside-S'-phosphates from a number of substrates including DNA, RNA, coenzymes and a number of synthetic substrates, i.e. pfnitrophenyl nucleoside-5'-phosphates. The hydrolysis of these synthetic substrates demonstrates unequivocally the presence of phosphodiesterase I activity in the presence of other polynucleotidases (17). The complementary exonuclease to phosphodiesterase I is phosphodiesterase II. For enzyme activity the substrate must have a nucleoside 3'-phosphory1 residue and a free S'-hydroxy1 function. The typical synthetic substrate for this type of activity is either Tp-p-nitrOpheny1* (18) or 2,h-dinitrophenyl-thymidine-3'-phosphate (9). The former has been used to demonstrate the unequivocal presence of this type of activity (17). Phosphodiesterase II hydrolyzes the Tp-pf nitrOphenyl substrates to nitrophenol and 3'-TMP. Phosphodiesterase II activity was originally found in extracts of calf spleen (19); the enzyme from this source has been purified and some of its properties have been reported (18, 20, 23, 2h). The enzyme has also been found in animal tissues (10), salmon testis (9), and extracts from Lactobacillus acidophilus (21, 22). The pH Optimum ranges from pH 5.5 to 7.8, and the enzyme activity is not inhibited by the presence of EDTA. The moiety attached through the 3'-phosphoryl linkage has considerable influence on the rates of hydrolysis. The rate of hydrolysis by enzymes from.L, acidOphilus and salmon testis is markedly lower when a nucleotide or oligonucleotide replaces the penitrophenyl group, as in Tpr, DNA or RNA. The effect is reversed for the spleen enzyme; that is, the rate of hydrolysis is greater for dinucleotides or RNA than that observed for Tp-prnitrophenyl. The activity for both spleen and salmon is higher using Tp-2,h-dinitropheny1 than the p-nitro- phenyl ester. The hydrolysis of oligonucleotides by Phosphodiesterase *The abbreviations used in this manuscript are those adopted by the Journal of Biological Chemistry. Other abbreviations are Tp-pfnitro- phenyl, pfnitrophenyl thymidine-3'-phosphate; pfnitrophenyl-pT, pf nitrophenyl thymidine-5'-phosphate,lp-nitrophenyl-pdc; p-nitrophenyl deoxycytidine-S'-phosphate; pfnitrophenyl-pdc,.p-nitrophenyl deoxy- guanosine-S'-phosphate;‘pfnitrOphenyl-pdA; penitrOphenyl deoxyadenosine— 5'-phosphate; p-nitrophenyl-pA, penitrOPhenyl adenosine-S'-phosphate; penitrophenyl-pU, penitrophenyl uridine-5'-phosphate; m units, milliunits. II begins at the 5'-terminus, sequentially liberating nucleoside-3'- phosphates (17). Little if any base preference is indicated. There exists, however, an unusual property of phosphodiesterase II worth mentioning at this point. With the three enzymes which have been purified, all possess nucleotide transesterification activity. The enzymes are able to produce significant amounts of longer oligonucleo- tides from TpT. The homogeneity of any of the three phosphodiesterase II preparations has not been established. Many of the properties of the enzyme activity are being investigated as new enzyme sources are being detected and the activities subsequently purified. Several reviews on phosphodiesterases describe some additional general properties (17, 20, 23-25). Phosphodiesterase I, the most extensively investigated class of phosphodiesterases (exonucleases), was first reported by Uzawa in 1932, (26) as an activity contained in snake venom. The work produced in the following 28 years was principally concerned with the purification and properties of the activity from snake venom (8, 27-32) and from intestinal mucosa (19, 25). Since 1960, phosphodiesterase I was examined in numerous plant and animal sources including hog kidney and liver (33, 3h), animal and human tissues (10), rat liver (12), rat intestinal mucosa (13, 35), peas, corn and potato (3h, 36), malt (1h), carrot (15, 37), and 53222 leaf tissue (16). The venom of various snakes has been the principal source of the enzyme for biochemical investigations. Numerous approaches to purification have been attempted (38-h6). The reaction catalyzed by the exonuclease proceeds with the libera- tion of nucleoside-5'-monophosphates from substrates of the form Xp-nucleoside. When X is the penitrophenyl group as in the case of pr nitrophenyl-pT, the hydrolysis yieldst-nitrophenol and 5'-TMP. The enzyme will also hydrolyze bisjp-nitrophenyl-phosphate; so a nucleoside is not essential as part of the substrate. Although venom exonuclease possesses a pH optimum of pH.8.9 to 9.5, it has been shown to hydrolyze normally resistant Xpo type substrates at low pH, pH 5, the resistance of the 3'-phosphoryl moiety being lowered by the reduction of the negative charge at the 3'-phosphoryl groups (A7, A8). The phospho- diesterase activity is enhanced by the presence of divalent metal ions, especially Mg++, and is inhibited by metal chelators i.e. EDTA and also sulfhydryl compounds. Other general properties of the enzyme are discussed in several review articles (17, 20, 23, #9). Previous results of an investigation by the author on phosphodi- esterases (exonucleases) (50) showed that phosphodiesterase I activity with a pH optimum near pH.8.9 and a nonspecific phosphodiesterase activ- ity with a pH optimum near pH 5.0 were present in extracts of all animal and plant sources tested. A preference in the rate of hydrolysis of p- nitrOphenyl derivatives of pyrimidine nucleoside-5'-phosphates over those of purine nucleoside-5'-phosphates was shown by all tissue extracts. Two members of the family Cucurbitaceae, muskmelon and cucumber, showed an unusual activity based on the pyrimidine/purine ratios. The ratio is set up as the rate of hydrolysis of pfnitrOphenyl-pdT + pfnitrophenyl— pdC/pfnitrophenyl-pdc +qp-nitropheny1-pdA. The pyrimidine/purine ratios were 2-fold higher than that of the average value of the plants tested. Because of this unusual activity and also because Cucumis melo served as a source for another nuclease in this laboratory, an investigation was undertaken to purify this enzyme activity and to examine its prOperties and specificity more closely. MATERIALS AND METHODS Enzyme Source Mbskmelon seeds (Cucumis melo), a Honey Rock variety, were purchased in a 100 pound quantity from Farm Bureau Services, Lansing, Michigan. Enzyme Substrates The p-nitrOphenyl-thymidine-5'-phosphate and the nitrophenyl- thymidine-3'-phosphate used to compare hydrolysis rates were products of Raylo Chemical Company, Edmonton, Alberta, Canada. The pfnitrophenyl- thymidine-5'-phosphate used in the enzyme purification assays as well as sodiumepfnitrophenyl-phosphate, bisjp-nitrophenyl-phosphate, DNA from calf thymus (Type I), mononucleotides, and Oligouridylic acid were products of Sigma Chemical Company, St. Louis, Missouri, The poly- nucleotides, Poly A, Poly U, Poly G and Poly C were obtained from Schwarz-Mann Bioresearch, Inc., Orangeburg, New York. pritrophenyl-cytidine-S'-phosphate, penitrOphenyl deoxyadenosine- 5'-phosphate, pfnitrOphenyl-deoxyguanosine-5'-phosphate, pfnitrOphenyl- adenosine-5'-phosphate and pfnitrOphenyl-uridine-S'-phosphate were synthesized in this laboratory using the method of Borden and Smith (51). "Crude" rRNA, a gift from Dr. R. C. Slabaugh, was prepared from rabbit recticulocyte ribosomes by the following procedures. Ribosome suspensions were treated with 6 M LiCl and 1+ _M urea in 0.05 M sodium acetate pH 5.6 and 0.05 M 2—mercaptoethanol. This mixture precipitated rRNA after a 16 hour hoc incubation (52). The "crude" 10,000 x g rRNA 6 precipitate from the above incubation was dissolved in 0.01 M.Tris C1, pH'7.5. One-tenth volume of 20% potassium acetate pH 5.0 was then added followed by 2 volumes of absolute ethanol. After letting the mixture stand overnight at -30°C, the precipitate was collected by centrifuga- tion. The precipitate was dissolved in water and lyophilized to a powder. The lyophilized powder was designated rabbit reticulocyte rRNA. Enz 9 Alkaline phosphatase EC 3.1.3.1 (E, coli, BAPC), snake venom phosphodiesterase EC 3.1.h.1 (Crotalus adamanteus, VPH) and micrococcal nuclease EC 3.1.h.7 (Staphoccus aureus, NFCP) were obtained from Worthington Biochemical Corporation, Freehold, New Jersey. 38.8.1113 Sephadex G-100 (MO-120 H) was purchased from Pharmacia Fine Chem- icals, Inc., Piscataway, New Jersey, and was prepared according to the instructions of the manufacturer. Cellulose phosphate cation exchange resin, 0.8 meq per gram, was obtained from Sigma Chemical Company and prepared in the following manner. The resin was suspended in water and the fines were removed. The process was repeated three times. The resin was then washed alternatively with 0.3 M KOH and water until both washes were colorless. The resin was then washed extensively with water and finally stored in water with .0017% Zepharin HCl as a bacteriostatic agent. The resin was resuspended in 0.01 M Tris acetate and washed with the same solution before packing in the column. Reagents All chemicals used in the course of this work were reagent grade materials purchased from Mallinckrodt Chemical WOrks, Fisher Scientific Co., or J. T. Baker Chemical Co. unless otherwise specified. Tris- (hydroxymethyl)aminomethane (Trisma Base, reagent grade) cacodylic acid and , monothioglycerol (crystalline, free acid) were products of Sigma Chemical Co. B-Mercaptoethanol was a product of Eastman Organic Chemicals, Rochester, New York. Thioglycolate was from Calbiochem, Los Angeles, California. Lanthanum nitrate (C. P.) was obtained from Sargent Welch Co., Detroit, Michigan. Acrylamide, N,N,N'N'-tetramethy1- enediamine (Temed) and N,N'-methy1enebisacry1amide (Bis) were obtained from Canalco, Rockville, Maryland. ‘Protein Determinations Throughout the purification of the enzyme, the concentration of protein was determined by the method of Lowry £5 _a_1_. (53). Bovine serum albumin (Fraction IV) was used as a reference standard. The absorbance determinations were made on a Bausch and Lamb Spectronic-2O calorimeter at 750 nm using an infrared filter. On purified muskmelon exonuclease, because of the extremely low concentrations of protein, the tannic acid method of MejbaumeKatzenellenbogen and Dobryszyca (5h) was used for estimating the protein concentration. The absorbance was determined at 650 nm in the Spectronic-20. Phosphate Determinations Phosphate determinations were carried out by a modification of the method of Dreisbach (55). Samle aliquots 0.5 ml were mixed with 0.5 m1 of water-saturated phenol. The aqueous and organic phases were separat- ed by centrifugation (1,100 x g) for 10 minutes to separate the layers. The A310 of the organic (upper) phase was then determined in a Beckman DB Spectrophotometer in cuvettes fitted with glass caps. From the A310 values, a standard curve was constructed for each assay. Exonuclease Assays Assay I, used primarily for determining exonuclease activities at different conditions of pH, was a procedure similar to that of Razzell and Khorana (8). The incubation mixture in a final volume of 0.3 ml contained 0.25 “moles of penitrOphenyl-pT, 100 “moles of an appropriate buffer, 12 ”moles of magnesium acetate and 100 pl of enzyme previously diluted with 0.01;M Tris-acetate pH.7.5 to an appropriate activity. This mixture minus enzyme was preincubated for 5 minutes at 37°C, then the enzyme was added. Aliquots (0.05 ml) were removed at timed inter- vals and added to 1.0 ml of 1.0 N NaOH. The absorbance was determined at h00 nm in a Beckman DB Spectrophotometer. The molar extinction of .p-nitrophenol under these conditions was determined to be 17,100. Assay II, used for following enzyme activity during purification procedures and used for comparing the rates of hydrolysis of various pf nitrophenyl-pdx substrates, was essentially the procedure of Razzell and Khorana (18). The reaction was carried out in a 37°C constant tempera- ture cell compartment on a recording spectrophotometer system consisting of a Hitachi Mbdel 139 monochromator and a Gilford Mbdel 220 absorbance indicator. The reaction mixture consisted of 0.25 ”moles of substrate (penitrophenyl-pT), 50 “moles of Tris acetate pH 8.9 and 0.5 “moles of magnesium acetate and enzyme in a 0.5 ml volume. The mixture minus 10 enzyme was preincubated 5 minutes at 37°C, the reaction being initiated by the addition of the enzyme solution. An increase in absorbance of 5.3 indicates the hydrolysis of 1.0 “mole/ml based on a molar extinction of 15,800 in the recording spectrophotometer at h00 nm. A unit is defined as the hydrolysis of one umole of substrate/hour at 37°C. Specific activity is defined as units per mg of protein. Nucleotidase Assgy The assay used to determine the release of inorganic phosphate frOm several isomers of adenosine monophosphate was set up as described below. The buffered nucleotide solution in a volume of 2.0 ml, formed from 1.9 ml of 0.11 M Tris acetate (either pH.7.2 or 8.9, adjusted with acetic acid) and 0.1 m1 of 0.0’4- y nucleotide (5'-AMP, 3'-AMP or 2'-AMP), was mixed with 0.06 ml of enzyme. The final enzyme concentration was 1h units per ml. The assay mixture was incubated 60 minutes. Duplicate samples of 0.5 ml were removed at 60 minutes and assayed for inorganic phosphate by the modified method of Dreisbach. The zero time assays were handled similarly except that samples were withdrawn for phosphate determinations immediately after adding the enzyme. Deaminase Assay Adenosine deaminase and adenylic acid deaminase activities were measured by the increase in A285 nm by a procedure similar to that of Smiley, Berry and Suelter (56). Contained in 0.5 m1 were20 “moles of sodium cacodylate buffer pH.6.8, 0.5 umoles of substrate (5'-AMP or adenosine) and 0.5 umoles of B-mercaptoethanol. The mixture was pre- incubated in a constant temperature cell of the recording 11 spectrophotometer at 37°C. After 5 minutes the reaction was initiated by the addition of 2.9 units of enzyme. The increase in absorbance at 285 nm was followed with time. Gel Filtration Experiments designed to establish whether the enzyme action on denatured DNA and ribosomal RNA was endo- or exonucleotic were performed by the method of Birnboim (57). The G-100 column, however, was replaced by a column (1.2 x 2h cm) of Bio-Rad P-6 polyacrylamide gel, Bio-Rad Laboratories, Richmond, California. The fractionation range is 1,000 to 6,000 daltons. The flow rate was maintained at 1 ml per minute with a peristalic pump and the elution pattern was monitored automatically with an ISCO Model UA-2 Ultraviolet Analyzer. Isoelectric Focusing The apparatus was an LKB Electrofocusing unit, type 8100, with a type 8101 column (110 m1 volume). A density gradient of sucrose, in which Ampholine carrier ampholytes in the pH range from.3-10 were dissolved, was prepared according to the manufacturer's instructions. The sample was introduced when one-half of the gradient has been layered in the apparatus. A potential of 300 volts was applied to the column for 36 hours at ADC using a Krohm-Hite Model UHR-2h0 constant voltage power supply. The ampholyte solution was drained from the bottom of the column (the cathode) by pumping water into the top with a peristalic pump at a rate of about 2 ml per minute. One minute fractions were collected. The pH of each fraction was determined with a combination electrode on a Leeds and 12 Northrup pH Meter. Polyacrylamide Gel Electrophoresis Disc gel electrophoresis in acrylamide gels was carried out in 0.5 mm ID tubes and was essentially the procedure of Davis (58) except that the sample gel was eliminated. Samples of 0.5 ml were mixed with 0.05 ml of glycerol and placed on top of the spacer gel. A current of 5 ma per tube was applied to the gels for 3 hours at ADC. The gels were stained via two methods. The activity stain was essentially the procedure of Sierakowska and Shugar (59) modified by Lerch (60). Gels were placed in a 10 ml solution of 0.1 M Tris buffer pH 8.9 containing 10 mg of Fast Red TR salt (Sigma) and 0.05 mg of abnaphthyl-thymidine-S'-phosphate. The enzyme activity is localized as a maroon precipitate formed by the coupling of the a-napthol released with the diazotate. The gels were also stained overnight for protein using Coomassie blue 0.3% in 10% TCA containing 30% methanol after the protein was fixed using 12.5% TCA. The gels were destained using 10% TCA containing 30% methanol and were stored in 10% TCA. Paper Chromatography The separation of oligonuCleotides from bacterial alkaline phospha- tase was carried out using descending chromatography on Whatmmn 3 MM in 95% ethanol, 1;! ammonium acetate, pH'7.5 (7:3, v/v). This system, solvent system I, was also used for the chromatography of the Poly U digest by muskmelon exonuclease. EXPERIMENTAL RESULTS Purification of the Exonuclease _Preparation of gggggis mglo Seeds Muskmelon seeds (700-800 g) were placed in a three liter beaker and washed continuously for one-half hour with distilled water. The seeds were drained through a double layer of cheesecloth and dried in an oven for 3 hours at 35-h00C. Preparation of Soluble Enzyme Washed seeds (600 g) were allowed to imbibe for 12 hours in dis- tilled water. They were subsequently drained through a double layer of cheesecloth and resuspended in 2 liters of cold (hoe) distilled water. The resuspended seeds were homogenized at too in a commercial Waring Blendor (Mbdel CB-5) for 15 seconds at 16,500 rpm followed by #5 seconds at 20,500 rpm. The crude homogenate obtained was centrifuged at 1h,OOO x g for 20 minutes in a Lourdes Centrifuge (Model A-2) equipped with a 1350 rotor. The supernatant liquid was then filtered through glass wool to remove unwanted suspended and floating material. The filtered supernatant liquid was designated the Step I enzyme fraction. pH Adjustment and Heat Coagulation Precipitation The Step I enzyme was made 0.1 M in ammonium acetate and the pH was carefully adjusted to pH 5.0 using glacial acetic acid. The solution 13 1h was heated to 60°C with occasional stirring in an 80°C hot water bath. The enzyme solution was then chilled in ice until the temperature had dropped below 20°C. Unwanted material was removed by centrifuging 20 minutes at 16,300 x g in a Sorvall RC-2B using a GSA rotor. All sub- sequent centrifugations were accomplished using a Sorvall refrigerated centrifuge. The supernatant liquid was again filtered through glass wool to remove floating material. This gave a clear, light yellow solution designated as the Step II enzyme fraction. Acetone Fractionation After chilling in a slated ice bath, Reagent grade acetone at -200C equal to 30% of the enzyme volume was added drOpwise (10 ml per minute) from a reservoir cooled with ice and dry ice via six polyethylene deliv- ery tubes (0.085 inch I.D.) to the Step II enzyme. Upon completion of the addition of acetone, the mixture was stirred for an additional 15 minutes and then centrifuged lh,000 x g for 15 minutes. The precipitate was discarded. To the supernatant fluid acetone was again added so that the final acetone volume was equal to 60% of the starting enzyme volume. Upon completion of acetone addition, the suspension was stirred for 15 minutes. The enzyme was collected by repeated centrifugation at h,000 x g in four 250 m1 centrifuge bottles. The precipitate was taken up in 0.01 M Tris-acetate buffer, pH 8.0, containing 10"4 M magnesium acetate and resuspended with the use of a glass Potter-Elvejehm homogenizer. The resulting suspension was stirred 3 hours to ensure that the enzyme was dissolved completely. Centrifugation for 20 minutes at h0,000 x g (SS-3h rotor) removed the insoluble material. The clear, yellow supernatant fluid was designated as the Step III enzyme fraction. 15 Ammonium Sulfate Fractionation The Step III enzyme solution was adjusted to pH 5.0 with glacial acetic acid. Ammonium sulfate (.25 g/ml of Step III enzyme) was added slowly while the solution was being mechanically stirred. Additional ammonium sulfate was added until a 55% saturated solution of ammonium sulfate was obtained as determined by refractive index on a Bausch and Lamb Abbe-3L Refractometer. This procedure was necessary because of the varying amounts of salt carried over during the acetone precipitation step. After 15 minutes of stirring, the solution was centrifuged h0,000 x g for 20 minutes and the precipitate was discarded. Ammonium sulfate (.15 g per ml) was added to the supernatant as in the first precipita- tion. Additional ammonium sulfate was added until a 75% saturated solution of ammonium sulfate was again determined via refractive index. The suspension was centrifuged h0,000 x g for 20 minutes and the supernatant was discarded after assaying for activity. The clear, brownish tinged liquid was termed the Step IV enzyme fraction. G-100 Sephadex Gel Filtratign The Step IV enzyme fraction was immediately layered on the tOp of a G-lOO Sephadex column (5.0 x 93 cm) previously equilibrated with 0.01 M Tris acetate buffer, pH'7.5, containing 10.4 M magnesium acetate. Use of sucrose to increase the density of the sample was not required due to the amount of ammonium sulfate present in the enzyme solution. The column was eluted at room temperature overnight with the same buffer in a descending manner. Flow rate was adjusted 1 ml per minute, and 10 minute fractions were collected. Figure 1 from a typical preparation, shows the phosphodiesterase activity, nonspecific phosphodiesterase Figure l. 16 Elution pattern of Sephadex G-100 Gel Filtration. To a column of G-100 Sephadex (5 x 93 cm) previously equilibrated with 0.01 M Tris acetate buffer, pH 7.5 containing 10-4 M magnesium acetate, a 10 m1 sample containing the Step IV enzyme was applied. The column was eluted with the same buffer. Ten minute fractions of about 10 ml were collected. The fractions containing the enzyme were pooled to yield the Step V enzyme fraction. Phosphodiesterase activity, o—o; nonspecific phosphodiesterase activity A—‘; and absorbance 280 nm, O—c—o. 17 (lw/suun) Amway (g V N 0 d d r l 7‘ C '0' C ./ .I I.’ d . .’.’ .‘p—r.’ 0", .0’ ' s~ .\ 0.. ‘9 C 'o \ .\. \ 0‘. \.‘ .\ .0 I / C —O of ‘0‘ ,4/ o~ ..,O 4“ .’_o ‘ ‘< \d .\ 0‘. ,5 .‘. C l l l 1 st. 00 N. "- o o 0 <5 033v ISO 100 I25 75 Fraction Number 50 25 18 activity and 280 nm absorbance profiles. The tubes which contained phosphodiesterase activity were pooled. This pooled fraction was designated as the Step V enzyme fraction. Chromatography on Phosphocellulose The pooled fraction from the G-lOO Sephadex column was diluted 1:1 with 0.01 M Tris acetate, pH 7.6, and washed onto a 2 x 25 cm phospho- cellulose column previously equilibrated at room temperature with 0'01.§ Tris acetate buffer, pH 7.6. The elution rate was about 1 ml per minute and 15 minute fractions were collected. Following enzyme absorption onto the resin, the column was washed with 230 m1 of the same buffer. Next a 500 m1 linear gradient of 0 to 0.5 M NaCl in Tris acetate buffer was started. The elution rate was kept at about 1 ml per minute and the fraction collector was changed to collect 5 minute fractions. Typical phosphodiesterase activity and 280 nm absorbance profiles are shown in Figure 2. The fractionscontaining the second peak of phosphodiesterase activity were pooled and subsequently concentrated to a small volume using an Amicon Model 602 Diaflo cell with a PMplO membrane (Amicon Corp., Lexington, Massachusetts). The concentrated fraction is the Step VI enzyme fraction. The results of a typical 600 g preparation is shown in Table I. The final specific activity was 13,080 with over a 3,000-fold enrichment. A lh.0% recovery of activity was obtained. Figure 2. 19 Elution pattern of the phosphocellulase ion exchange column. The phOSphocellulose column (2 x 25 cm) was equilibrated with Tris acetate buffer pH'7.5. The Fraction V enzyme (120 ml) was applied and the column was washed with 230 m1 of the Tris buffer. A linear gradient of 0.0 to 0.5 M NaCl was applied at a flow rate of about 1 ml per minute. Five minute fractions were collected. The total volume of the gradient was 500 ml..—. , absorbance at 280 nm;O-—-O, exonuclease activity. 20 IOO (Ina/sum) MM“ av 75 Fro ction Number 50 25 21 oee.m ooo.m~ ea ome.n ma eueeeeeee eeoaefiaeo .H> mmn mnm.a em oom.e~ owe xeeeeeem oo~-o .> m.m m.en me oom.om m «sewage aeeeeee< .>H e.m m.mm em oom.mm m.mm coaueeoeuueue eeoueo< .HHH m we.» mm oom.em cmeea eweeee me was use: .HH H me.n ooH oom.em 0mm.H «vane .H ea, in mvmom cofioexmnz mo Auswwoa %uov w 000 scum omoosoacoxm mo cowuwuamwunm osu we %umaa:m H mqm2>Zo< r m I —. — I—$—A —A / 0 10 6.0 7.0 8.0 5.0 pH 30 0.10 M. Two and four-tenths enzyme units were diluted to 2.0 ml in ammonium acetate pH h.0 and pH 5.0, ammonium cacodylate pH 5.0, pH 6.0, and pH 7.0, Tris acetate pH 7.0, 8.0 and 9.0, and ammonium acetate pH 9.0 and pH 10.0, and were incubated for 2, h, 8, 16, 32 and 6h hour periods at 37°C. Enzyme activity was determined at each of the periods by assaying at pH 8.9. The results show that storage for even short periods of time at pH h.0 (2 hours) completely destroys enzyme activity. Mest buffers at the various pH values tested caused a small initial drop in activity over a 12 hour period. Yet after 12 hours, there was little if any change in activity with time for any of the buffer systems, except for ammonium acetate buffer at pH 10.0 which displayed a continuous decline of activity after an initial activation. Enzyme incubated in Tris-acetate buffer or ammonium acetate buffer at pH 9 and Tris-acetate buffer at pH 7 or 8 retained the highest levels of activity. Isoelectric Point of Muskmelon Exonuclease The isoelectric point of the muskmelon exonuclease was determined early in the purification procedure using the LKB 8100 electrofucusing apparatus. This information was needed to ensure more complete precipitation in the subsequent acetone and ammonium sulfate fraction- ation procedures. With Step II enzyme, a single zone of activity at pI = 5.3 was obtained using a pH 3 to 10 gradient. In later experiments using Step IV enzyme, no activity could be recovered from the electro- focusing column even after 260 units were initially applied. 31 Effect of Temperature on Muskmelon Exonuclease To determine the effect of temperature on exonuclease activity at different pH's, the following experiment was devised. One-hundred units of enzyme were diluted to 5.0 ml in 0.1 M Tris buffers at various pH's and heated to 80°C for 15 minutes. The pH's used were 7.5, 8.0, 8.5, and 9.0. When the samples were chilled in ice and subsequently assayed a pH dependent loss Of activity was observed. At pH 7.5, no loss of activity was seen in the 15 minute incubation but at higher pH's a h% to 17% loss was seen after the 15 minute incubation. When a 0.1 M carbonate- bicarbonate buffer was used, 12% and 3h% decreases in activity were seen at pH 8.5 and 9.0. The incubations at pH 9.0 and 10.0 in carbonate- bicarbonate buffer led to 87% and 100% losses of activity respectively. Thus the enzyme appears to be much less stable at a given pH in a carbonate-bicarbonate buffer than in a Tris acetate buffer. To investigate the effect of temperature on exonuclease activity at pH 8.9, aliquots containing 33 units of the enzyme were diluted to 10.0 ml with Tris acetate 0.1 MipH 8.9. One ml volumes were incubated for 30 minutes at various temperatures (2h°, 37°, 50°, 60°, 70°, 80°, 85°, and 90°C). After cooling in ice, the various samples were assayed using assay II described in Methods and Materials. The results are shown in Figure 5. Above 70°C there was marked loss of activity after the 30 minute incubation period. At 90°C there was no detectable activity remaining. Effect of Storage and Dialysis Aliquots (0.1 ml) of a 20-fold diluted Step VI enzyme were placed in 0.6 m1 test tubes under three conditions: refrigeration at hOC, 32 Figure 5. Effect of temperature on exonuclease activity. Muskmelon exonuclease (33 units) was diluted to 10 ml with 0.10 M Tris acetate buffer pH 8.9. Aliquots of 1.0 ml each were incubated for 30 minutes at 7 different temperatures. The samples were then cooled in ice for 5 minutes after which they were assayed as in Assay II. The results are graphed as percent activity remaining based on the room temper- ature sample as 100%. 55 Gov eczoeang 9.022.350:— illl - — _ — O /./ O/ O /O /O l. ON 0v 0 'O O Q wnv % 8M 3# frozen at -25°C and standing room temperature. The 0 time sample possessed an activity of 6#0 units per ml. After 10 days at these conditions, the samples were brought to room temperature and assayed using Assay II. The refrigerated sample had an activity of 5#0 units per ml. Both the frozen sample and the room temperature sample possessed activities of 500 units per ml. There is very little difference in the conditions of storage since the values of 5#O and 500 units per m1 amounted to a 16% and 22% respective loss Of activity in 10 days. Dialysis against pH 7.5 Tris buffer (0.01 M) for #8 hours produced no detectable loss of activity over the nondialyzed sample. In this experiment a lower concentration of enzyme was used. Triplicate samples (1.0 ml) containing 19.5 units of enzyme were placed in small dialysis tubing and dialyzed #8 hours. There was a decrease of 8.8% of the starting activity, but the nondialyzed sample also had a decrease in activity of 8.8%, yielding 17.8 units of enzyme. There was no decrease in activity of the dialyzed sample over the control sample. Effect of Activators and Inhibitors on Exonuclease Activity The effects of metals, activators, and inhibitors upon exonuclease activity is shown in Table II. All additions gave a final concentration of 10.3 M with the exception Of ammonium sulfate whose final concentrathan was 0.10 M (this was also made pH.8.9 with NH4OH before addition to prevent a pH change). Sixty-five m units (20 ul) of exonuclease were added to the assay to initiate the reaction. Among the cations tested, only Mg++, Ca++, and Ba++ evoked a stimulatory effect on activity. Co++, Zn++, and Hg++ exhibited a marked inhibitory effect displaying greater than 25% inhibition. s£++, Mn++, Cd++, Ni++, and Cuf+ yielded slight 35 TABLE II Effect of Activators and Inhibitors on Exonuclease Activity Reactions were carried out as described in Materials and Methods under Assay II, except that the enzyme reaction contained millimolar concentrations of the metal listed. Addition of Step VI enzyme initiated the reaction. A__* Addition (10.3 M final concentration) % of Control none 100 M3c12 106 CaC12 107 BaC12 109 Coc12 68 Sr012 99 Mn012 93 ZnC12 68 CdCI2 81 N1012 79 C11012 75 H8C12 72 NaF 16 NaCl 100 (NH4)2504* 79 *0.1 M final concentration. 36 inhibitory effects on exonuclease activity ranging from 1% to 25%. A monovalent anion, fluoride, provided the strongest inhibition of activity, 8#%, whereas chloride had no effect. Ammonium sulfate (0.1 M) displayed an 21% inhibition of activity unlike the ammonium sulfate activation seen with the malt enzyme (l#). Effect of EDTA.upon Activity Characteristically EDTA and other metal chelators are inhibit- ors of phosphodiesterase I activities (8, 13, 15). In order to determine whether EDTA exhibited an inhibiting influence upon muskmelon exonuclease activity, measurements of activity in the presence of EDTA were performed. In a volume of #00 pl the following were contained: 50 umoles of Tris acetate buffer pH 8.9 EDTA of varying concentrations, and 31.9 milliunits of enzyme. This was incubated for 5 minutes at 37°C in a constant temperature compartment of the spectrophotometer. The enzyme reaction was initiated upon the addition of 0.25 umoles of pfnitrophenyl-pT (100 pl). Activity was followed as the increase in A400 with time. Figure 6 shows the inhibitory effect of EDTA as a function of concentra- tion. EDTA in extremely low concentrations (2 x 10.5 M) inhibited enzyme activity completely. Fifty percent inhibition was displayed by 7 x 10-6 M EDTA. This data suggests that EDTA binds to a metal which is essential for enzyme activity. _Reversal of EDTA Inhibition by Metals The protocol for the experiment to examine the possible reversal of EDTA inhibition by metals is shown in Table IIIa. Based on the data 37 Figure 6. Inhibition of exonuclease activity by EDTA. The assays were performed as described in the text with constant amounts of substrate (p-nitrOphenyl-pT) and enzyme (31.9 milliunits). 38 .9. OO— A mmv 1° 95 39 TABLE IIIa Protocol for Experiment Displaying Metal Ion Reversal of EDTA Inhibition - 5 minute incubation 13). 10'2 .1! EDTA -[120x Tris acetate 0.1 M pH 8.9 201 Enzyme 12 hours d incubation -O.IO ml MeH 10'2 1_4 cuvette 0.50 mu total volume r100). 2.5 x 10":3 M p-nitrOphenyl-pT 501 1.0 M Tris acetate pH.8.9 1100). HOH TABLE IIIb Effect of Divalent Metal Ions in Restoration of EDTA Inhibited Exonuclease Activityl Addition m units of activizy % of Control none2 0 0 MgC12 35.6 119 N1012 0 0 CdC12 0 0 SrC12 36.6 122 COC12 21.2 71 MnC12 1.9 6 CUC12 O O BaC12 2.8 9 CaC12 33.7 112 an12 O 0 Control3 30.0 100 1Assays were performed as described in the text and outlined in the above protocol. 2N0 metal ion addition. 3Neither EDTA nor metal was added. #0 from the above assays, .01 umoles of EDTA is needed to inhibit 31.9 milliunits of enzyme in a 0.5 ml assay volume. With a 6.5-fold scale-up in enzyme, a value of 0.065 “moles was calculated to insure inhibition by EDTA. Subsequent addition of 1.00 “moles of Me'H' would yield an 61-fold excess of metal ions. For each metal ion examined, 160 milliunits of enzyme was mixed with 0.065 moles of EDTA and 50 umoles of Tris acetate, pH 8.9, in a volume of 600 pl. After incubating this solution for 5 minutes at 37°C, 0.#0 ml of the metal ion, 10-2 55 was added. This 1.0 m1 mixture was incubated for 12 hours at 37°C. The concentrations of EDTA and Me++ during this incubation were .065 mM and #.0 mM, respectively. Two- hundred-fifty pl of the metal-EDTA enzyme mixture was mixed in a cuvette containing 0.25 umoles of p-nitrophenyl-pT, 50 “moles of Tris acetate buffer, pH 8.9, in a total volume of 0.250 ml. The concentrations of EDTA and Me++ in the final reaction mixture were 0.016 pM and 100 uM, respectively. The increase of absorbance at #00 nm was followed with time. The results of the metal ion reactivation of enzyme activity is shown in Table IIIb. The enzyme control having a value of 30 milliunits was carried through the identical manipulations except neither EDTA nor metal ions were added. The case where no metal was added displayed complete inhibition. Mg++, Sr++, and Ca++ were able to restore enzyme activity to 35.6, 36.5, and 33.7 milliunits, respectively. Cobalt +1- ++ restored 21.2 milliunits of activity while divalent Ni++, Cd , Mn , H Cu++, Ba++, and Zn restored little or no activity. #1 Effect of Sulfhydryl Compounds The effect of the sulfhydryl compounds tested upon exonuclease activity is shown in Table IV. Cysteine, thioglycolate, thioglycerol, B-mercaptoethanol, and dithiothreitol were the reagents used. The assay was assay II with the exception that various sulfhydryl reagents were added to the preincubation mixture, the addition of enzyme (.095 units) initiating the reaction. All sulfhydryl solutions which were tested inhibited enzyme activity. The inhibition displayed by these sulfhydryl compounds is reflected in the concentration of sulfhydryl needed to evoke a 50% inhibition. Fifty percent inhibition was Observed at 6.8 x 10.5 M for cysteine, #.0 x 10-4 M for dithiothreitol, 5.# x 10-3 M for B-mercaptoethanol, 5.7 x 10-3 M for thioglycerol and 2.0 x 10”2 M for thioglycolate. Cysteine inhibited the enzyme strongly while the other compounds inhibited enzyme activity to varying degrees. Specificity_of Muskmelon Exonuclease ongp:nitrophenyl Substrates The specificity assays using p-nitrophenyl derivatives were carried out at pH 8.9 and pH 5.5. Assay II was used except thatthe reaction mixture was carried out in a final volume of 1 m1, doubling the amount of all components so that the final concentrations were the same as in Assay II. In the pH 8.9 assays for most of the substrates, the components were preincubated at 37°C for 5 minutes and the reaction was then initiat- edby the addition of 72 milliunits of enzyme (0.05 ml). The assays with p-nitrophenyl-phosphate, bis-p-nitrophenyl-phosphate, and p-nitrophenyl- thymidine-3'-phosphate at this enzyme concentration showed very little hydrolysis. Accordingly experiments were performed with these substrates using 2.9 units of enzyme, a #0-fold increase. A multiplier of 0.025 was #2 TABLE IV Effect of Sulfhydryl Compounds on Activity Condition of the assays are as described in the text. Values reported are the rates of hydrolysis expressed in “moles of p-nitrophenyl-pT hydrolyzed per hour. Where values are omitted, assays were not performed. Activity Final Molar Concentration of Addition Sulfhydryl Compound in Assay lo'2 14 10'3 y 10'4 y 10'5 y Cysteine -- .OOO (oo)* .021 ( 22) .095 (100) Thioglycolate .089 (9#) .093 (98) .095 (100) -- Thioglycerol .019 (20) .089 (9#) .091 ( 96) -- B-Mercaptoethanol .018 (19) .091 (96) .095 (100) .095 (100) Dithiothreitol -- .026 (27) .08# ( 88) .095 (loo) *The second value given is the percent of the control value. to used to adjust the Observed rates of hydrolysis for only a rough compari- son with the 72 milliunit enzyme assays. The assays at this enzyme concentration may have been outside the range where the rate of hydrolysis of the substrate is proportional to enzyme concentration. The relative rates of hydrolysis of the p-nitrOphenyl derivatives are tabulated in Table V. The rate of hydrolysis of p-nitrophenyl-pdG was the highest, 87 umoles per hour, at pH.8.9. The rates of hydrolysis for the other deoxynucleotide derivatives, dC, dT, and dA were 76, 72, and 11 m “moles per hour, respectively at this pH. The ribonucleotide derivatives were hydrolyzed more slowly, p—nitrophenyl-pU being hydrolyzed at a rate comparable to one-half that for p-nitrophenyl-pT and p-nitro- phenyl-pA being hydrolyzed at a rate of only 7% that of p-nitrOphenyl-pT. Bis-p-nitrophenyl-phosphate, Tp-p-nitrophenyl, and p-nitrophenyl-phosphate gave extremely low rates of hydrolysis. A #0-fold increase in enzyme concentration in the enzyme assay gave values of 1#0, .023 and 0 munoles of substrate hydrolyzed per hour. A #0-fold increase in the hydrolysis of p—nitrophenyl-pT would yield a value of 2880 mumbles of substrate hydrolyzed per hour. Table V shows, in parentheses, the rates of hydroly- sis of the compounds normalized to 72 milliunits of enzyme. The pH 5.5 assays were performed to detect possible contaminating phosphodiesterase and phosphatase activities. The substrate, in an amount Of 0.25 “moles was mixed with 50 umoles of sodium acetate buffer at pH 5.5 in a total volume of 0.5 m1. No magnesium or metal ions were added. Again 2.9 units of enzyme were added to this mixture following a 5 minute preincubation at 37°C. The reaction was terminated after 20 minutes by the addition of 0.5 ml of 1.0 M NaOH. The absorbance at #00 nm was read in a Beckman DB spectrOphotometer. The pH 5.5 assays ## TABLE V Specificity of Muskmelon Exonuclease on p-Nitrophenyl-Substrates The conditions of the assays are discussed in the text. The activity values reported are rates of hydrolysis of the p-nitrophenyl esters obtained using 72 milliunits of exonuclease. The figures in parentheses are rates of hydrolysis obtained with 2.9 units of exonuclease which have been normalized to a 72 milliunit value. Activity at pH.8.9 Substrate mumoles % of per hour p-nitrophenyl-pT p-nitrophenyl-pdT 72 100 Ip-nitrOphenyl-pdc 76 105 ‘p-nitrophenyl-pdG 87 121 lp-nitrophenyl-pdA ll 15 p-nitrophenyl-pA 5 7 p-nitrophenyl-pU 36 50 Bis-p-nitrophenyl-phosphate 3 (3.5) # (3) Tp-p-nitrophenyl 0 (0.6) O (0.1) p—nitrophenyl-phosphate 0 (0) 0 (O) #5 were fixed point assays and cannot be compared directly to the pH 8.9 assays. The 80-fold increase in enzyme concentration over the 72 milliunit/ml concentration used in the hydrolysis of p-nitrOphenyl-pT at pH 8.9 was able to cause some hydrolysis of the p-nitrophenyl-phosphate, p-nitrophenyl-pT, Tp-p-nitrOphenyl and bis-p-nitrophenyl-phosphate at pH 5.5. The values for the rates of hydrolysis of these esters are 0.36, 72, 1.8, and 7.8 mimoles per hour respectively under the conditions described. It is interesting, however, that there was some hydrolysis even though the rates were low. This has been reported by other investigators for similar activities (16, #8). Kinetic Characteristics The assay substrate, p-nitrophenyl-pT, used in the bulk of the experiments was shown to be degraded by muskmelon exonuclease to 5'-dTMP and p-nitrOphenol by use of paper chromatography system I. Experiments were performed with Step VI exonuclease to determine the kinetic para- meters of the reaction. The Km value Obtained from the Lineweaver-Burk plot (61) shown in Figure 7 was found to be 3.6 x 10-5 M. The Vmax was l#,900 umoles per hour per mg of protein. Kinetic parameters for hydrolysis of the other deoxynucleotide-nitrophenyl substrates were also determined: ‘p-nitrophenyl-pdG, Vmax is 18,900 umoles per hour per mg protein and Km is 1.# x 10-4 M; p-nitrophenyl-pdA, vmax is 8,660 “moles per hour per mg protein and the Km is 2.# x 10"5 M; and pfnitrOphenyl- pdC, Vmax is 16,600 ”moles per hour per mg protein and the Km is 3.8 x 10-5;M. These also reflect the rates of reaction seen in Table V. The values of the Km's differ by less than a factor of 1.6 with the exception of that for p-nitrophenyl-pG which is about #-fold higher than the Figure 7. #6 The relationship of substrate concentration to reaction velocity as shown by a Lineweaver-Burk plot for muskmelon exonuclease. The assay was basically enzyme assay II described in Methods except that all components were doubled (volume - 1.0 ml). The substrate concentration was varied from 0.025 mM to 0.75 mM. The mixture was preincubated at 37°C, the reaction being initiated by addition of 60 munits of Step VI enzyme. The velocity is expressed in umoles of substrate hydrolyzed per hour per mg Of enzyme. #7 Roe x E\* 0v 0m ON Or 0 q . - 10v .\ow / o\ 1 M o\ m. \o\ 9 o TONP O \ three Others. The Effect of Nucleotides on the Rate of Hydrolysis The effect of various nucleotides on the rate of hydrolysis of lp-nitrophenyl-thymidine-S'-phosphate is summarized in Table VI. The largest degree of inhibition was displayed by adenosine-5'-phosphate. At a concentration of mM 5'-AMP, no hydrolysis of p-nitrophenyl-pT could be detected. All derivatives or analogues of 5'-AMT tested yielded a slight to a marked inhibition of activity. At millimolar concentrations, deoxyadenosine-S'-phosphate, the closest analogue of 5'-AMP, produced 98% inhibition. Other moieties attached to the basic 5'-AMP nucleotide decreased the degree of inhibition, i.e., AMP>ADP>ATP and AMP>DPN. The presence of a 2'- or 3'-phosphate attached to adenosine lowered the amount of inhibition as evidenced by the appreciable rate of hydrolysis of p-nitrophenyl-pT in the presence of 2'- or 3'-AMP. Similarly, TPN+ caused only half the inhibition displayed by DPN+ at millimolar concen- trations. Oxidized flavin adenine dinucleotide (FAD) is an exception in that it caused very strong inhibition at 10.4 M, being equal to that exerted by 5'-AMP at the same concentration. The product of the hydrolysis of p-nitrophenyl-pT, 5'-dTMP, lowered the hydrolysis rate to 3#% of the control. The addition of 5'-dCMP or 5'-dGMP gave values of 39% and #7% of the control value, respectively. When p-nitrophenyl-pdG was used as substrate, no hydrolysis of ‘p-nitrOphenyl-pdG could be detected at mM concentration of 5'-AMP. Addition of deoxynucleotides 5'-TMT, 5'-dGMP, 5'-dCMP, and 5'-dAMP at mM final concentrations gave rates of hydrolysis of 67%, 35%, 26% and 0% of the control rate, respectively. Addition of the two isomers #9 TABLE VI The Effect of Nucleotides on the Rate of Hydrolysis of p-NitrOphenyl-pT The assay was essentially that described in assay II in Materials and Methods, 100 milliunits of enzyme were added to initiate reaction. Additions of nucleotides and nucleotide derivatives were made such that a final concentration of 10'.3 M or 10”4 M was obtained. Activity is expressed in umoles of substrate hydrolyzed per hour. Enzyme activity at two inhibitor concentrations Addition 10"3 y 10‘4 54 Activity % of Control Activity % of Control none .090 100 5'-dGMP .O#2 #7 5'-dCMP .035 39 5'-dTMP .031 3# 5'-dAMP .002 2 .017 19 5'-AMP .000 0 .007 7 ADP .008 8 .058 #3 ATP .01# 16 .0#7 53 3'-AMP .073 81 .071 79 2"AMP 0%]. 68 .066 73 DPN .003 3 .016 18 TPN .052 58 .071 79 FAD - - .007 7 50 of 5'-AMP, 2'-AMP and 3'-AMP gave rates of hydrolysis equal to 71% and 82% of the control. Type of Inhibition Displayed by_5'-AMP Because of the marked inhibition exerted by 5'-AMP, determination of the type of inhibition exerted became desirable. The assay used had a final volume of 1.0 ml containing 100 “moles Of Tris acetate pH 8.9, 1.0 ”moles of magnesium acetate and substrate, 'p-nitrOphenyl-pT, of three different concentrations: 0.5 mM, 0.25 mM, or 0.125 mM. Each mixture was incubated 5 minutes at 37°C, and the reaction was initiated by the addition of 100 milliunits of enzyme. The inhibitor (5'-AMP), when present, was at various concentrations from 0.1 mM to 7.5 NM. The data obtained was plotted as shown in Figure 8, the method described by Dixon (62). The Dixon plot indicated a clear case of competitive inhibition. The K1 for 5'-AMP using ‘p-nitrOphenyl-pT as substrate was 6 x 10-7 M. Hydrolysis of Polynucleotides To determine if there is a marked difference in the rates of hydrolysis of different polynucleotides, natural and synthetic poly- nucleotides were subjected to hydrolysis by muskmelon nuclease. To a 7.8 ml solution of each of the synthetic polynucleotide substrates (1 mg/ml), 0.78 ml of Tris acetate 1.0 M pH 8.9, 0.08 ml of 1.0 M M3012, and 750 units (0.5 ml) of enzymes were added. At 20 minute periods 0.6 ml aliquots were removed and mixed with 0.6 m1 of cold lanthanum nitrate-HCl reagent (0.02 M La( N03)3, 0.2 M HCl). This was chilled on ice for 10 minutes and then centrifuged 20 minutes. The Figure 8. 51 Competitive inhibition by 5'-AMP of the hydrolysis of p-nitrophenyl-pT by the exonuclease from 9.31312. The conditions of the assay are described in the text. Substrate concentrations of p-nitrophenyl-pT were .---0, 0.5 mM; O---O, 0.25 mM; and I---l, 0.125 mM. The enzyme used was the Step VI enzyme. Velocity is expressed as umoles of p-nitrophenyl released per hour. 52 - _ 10 S3 absorbance at #00 nm of the supernatant solution was determined in a Beckman DB SpectrOphotometer. The assays for the hydrolysis of DNA, denatured DNA and ribosomal RNA (rRNA) were similar except that 90 units of enzyme was used and time periods were 15 minutes. Initial velocities were calculated based on changes in A260 nm with time. The calculation Of the millimicromoles of acid soluble nucleotide released was based on the extinction coefficients of the various bases. The extinction coefficient for calf thymus DNA was based on the following nucleotide composition of DNA: dAMP and TMP, 28.5%; dGMP and dCMP, 21.9% (63). The percentages of each nucleotide multiplied by its respective extinction coefficient gave an average extinction value of 11,100 per mole of DNA nucleotide. An extinction coefficient for ribosomal RNA based on the base ratios of rRNA of M..ggli (6#) when calculated gave an average extinction which deviated less than 1% from the value Obtained for DNA. Thus, 11,100 was also used in calculations for rRNA. The results in Table VII, show denatured DNA was the preferred substrate, releasing 2,500 millimicromoles of acid soluble nucleotide per hour under conditions of the assay. The rate of hydrolysis of rRNA was 53% that of dDNA while native DNA showed no increase in A250 nm over the blank value after 2 hours. The hydrolysis of the synthetic poly- nucleotides ranged from 0.9% to 1#.8% that of denatured DNA. Poly G formed a fibrous DNA like precipitate in the buffer at pH 8.9 and there was no appreciable hydrolysis over blank by muskmelon exonuclease on this polymer. 5# TABLE VII Hydrolysis of Polynucleotides The assays are described in the text. The values for the rates of hydrolysis of native DNA, denatured DNA and rRNA have been normalized for an additional of 750 units of enzyme. A value for the extinction coefficient of calf thymus DNA and rabbit reticulocyte rRNA was calculated to be 11,100. Substrate nuEIzgiizeofieI:Agegoézglhour % denatured DNA (calf thymus) 2,500 100 native DNA (calf thymus) 0 0 rRNA (rabbit reticulocytes) 1,320 52.8 Polyadenylic acid 23.5 0.9 Polycytidylic acid 118 #.7 Polyuridylic acid 369 l#.8 55 ContaminatipgfiActivities The Step VI enzyme was assayed for contaminating adenosine mono- phosphate nucleotidase and deaminase activities. The nucleotidase assay used was that described in Methods and Materials. The 60-minute assay was performed with enzyme concentrations of 1# units per ml and substrate concentrations of 2 mM. The possible hydrolysis of 5'-AMP, 3'-AMP and 2'-AMP by phosphatases or nucleotidases was examined at pH 8.9 and pH 7.2. The results show that there was essentially no release of inorganic phosphate, less than 0.02 umoles per hour, at the above enzyme concen- tration. This value was normalized to less than one percent of the rate of hydrolysis of p—nitrOphenyl-pT found in Table V. The deaminase assays were run in cacodylate buffer at pH 8.9 using mM 5'-adeny1ic acid and mM adenosine concentrations. Addition of 2.9 units of enzyme to the 37°C preincubated assay mixture produced no detectable increase in absorbance at 285 nm for 30 minutes. Under these conditions, the deamination of 0.1 umole of AMP or adenosine would have caused an increase of absorbance of 0.03 at 285 nm. Mode of Dggradation of Denatured DNA and Ribosomal RNA To establish that the mode of attack by Q, Mglg_exonuclease was in fact an exonucleolytic rather than an endonucleolytic type of degradation, the method of Birnboim (57) was used except that Bio-Rad P-6 polyacryl- amide resin replaced Sephadex G-100. Reaction mixtures contained 8 m1 of denatured DNA or ribosomal RNA (20 A280 units per ml), 0.8 m1 Tris acetate 1.0 M pH 8.9 and 0.08 ml magnesium acetate 0.36 M. The reactions were initiated by the addition of enzyme and incubated at 37°C. At various intervals after initiation of hydrolysis, 0.5 ml of the reaction 56 mixture was removed and added to 0.5 ml of the lanthanum nitrate-HCl reagent as described in the Experimental Results section under Hydrolysis of Polynucleotides. At the same time, 0.5 ml was also removed and added to 0.05 ml of glacial acetic acid to stop the reaction. This mixture was then diluted with 0.5 m1 of 0.1 M sodium acetate. A half milliliter of this solution was withdrawn for chromatography on the P-6 polyacrylamide column. The elution patterns Obtained from the U.V. monitor at various stages of hydrolysis are shown in Figure 9. The stages were determined by using the lanthanum nitrate-HCl assay and the results are expressed as a percentage of the total acid soluble products. After typical patterns of exonucleolytic and endonucleolytic digestion of denatured DNA were established using venom phosphodiesterase (Figure 9a) and micrococcal nuclease from S. aureus (Figure 9b), a digestion of denatured DNA with exonuclease from muskmelon was performed. The resulting pattern, shown in Figure 9c, was similar to that produced by venom phosphodiesterase rather than the micrococcal endonuclease, in that intermediate sized products were absent. Thus on denatured DNA the mode of action of the muskmelon enzyme appeared to be an exonucleolytic type of digestion. The conditions for the digestion of ribosomal RNA were identical to those used for denatured DNA except that the enzyme concentration was increased 50%. Again,after appreciable degradation of RNA by the muskmelon exonuclease, the characteristic double peak appearance could be seen in the U.V. monitor tracings indicating an exonucleolytic type of degradation of ribosomal RNA. Figure 9A. Figure 9B. Figure 9C. Figure 9D. 57 Chromatography on Bio-Rad P-6 of denatured DNA at stages in its digestion by venom phosphodiesterase. The reaction mixture contained 100 ug of venom phosphodiesterase per ml. The A254 elution profiles correspond to the percent hydrol- ysis as determined by acid soluble products in the lanthanum nitrate-HCl assay: a) 0%,.; b) 28%,0; c) #6%,A; and d) 85% A. Chromatography on Bio-Rad P-6 of denatured DNA at stages in its digestion by micrococcal nuclease of S. aureus. The reaction mixture contained 50 pg per ml of micrococcal nuclease. The A254 elution profiles correspond to the percent acid soluble products: a) #%,O; b) 12%,0; c) #0%, A ; and 70%, A . Chromatography on Bio-Rad P-6 of denatured DNA at stages in its digestion by g. gelp exonuclease. The reaction mixture contained #00 units of exonuclease per ml. The A254 elution profiles correspond to the percent acid soluble products: a) 0%,0; b) 162,0; c) 29% A; and d) 6396,11. Chromatography on Bio-Rad P-6 of ribosomal RNA at stages in its digestion by‘§.lgglg exonuclease. The reaction mixture contained #00 units of micrococcal nuclease. The A254 elution profiles correspond to the percent acid soluble products: a) 0%,.; b) 37%,0; and c) 65%, A. A 254 58 o 0 40 Time (minutes) 50 60 59 Hydrolysis of Oligouridylic Acid Oligouridylic acid (25 pmoles) was treated with 10 units of alkaline phosphatase in 0.05 ml of 0.02 M Tris acetate pH 8.9 for 12 hours at 37°C. The hydrolysis mixture was then spotted on Whatman 3 MM paper and chrom- atographed for 56 hours as described in the Methods section. The spot containing the majority of the A250 units was cut out and eluted using ammonium bicarbonate, 0.01 M, pH 8.5. The eluant was concentrated to dryness using a Buchler rotary evaporator. No trace of alkaline phosphatase could be detected in the sample using p-nitrophenyl-phosphate as substrate. Six micromoles of diphosphorylated Oligo-U was dissolved in 0.2 m1 of 0.05 M Tris acetate, pH 8.9, and 100 units of (#0 pl) exonuclease were added. The reaction mixture was incubated at 37°C. At intervals of 30 minutes, 10 p1 aliquots of the mixture was removed and spotted on Whatman 3 MM paper. Chromatography with appropriate standards was carried out in a descending manner for 16 hours using 95% ethanol: l‘M ammonium acetate (pH.7.5) (7:3, v/v). The chromatogram showed that in early stages of hydrolysis, only spots corresponding to UMP could be detected. NO dinucleotide or trinucleotide spots could be visualized. Samples incubated for 3 and # hours produced ultraviolet absorbing material corresponding to uridine, UMP, a light spot localized between uridine and UMP (probably UpU) and long streak of products near the origin (most probably intermediate sized oligonucleotides). Spots corresponding to UMP in the 2# hour digest were converted to a faster migrating material when treated with 5'-nuc1eotidase. The chromatograms obtained above reinforce an exonucleolytic type degradation which produces mononucleotides. No di- or trinucleotides were present in early stages of hydrolysis. The direction of sequential 6O cleavage by §.'gglg exonuclease was suggested by the data to be from 3‘ to 5'. If initial cleavage of the dephosphorylated Oligouridylic acid had been from the 5' end releasing 5'-products, uridine would have been the first product Of the reaction. Hydrolysis from the 3' to 5' end would yield only mononucleotides as early products which agrees with the experimental data. Polyacrylamide Gel Electrophoresis Polyacrylamide gel electrophoresis was used for two reasons. It was desirable to have an estimation of the contamination by additional proteins of the protein containing exonuclease activity and also to determine if there were more than one band of activity which could be resolved by gel electrophoresis. The gels were prepared and run as described in Methods and Materials. Samples of 100 pg of protein (0.1 ml) were prepared by concentrating the Step V enzyme in dialysis tubing using Sephadex G-100 to absorb the moisture. Aliquots of 0.1 ml were applied to gels which were to be stained for protein and 0.020 ml aliquots were applied to gels which were to be stained for activity. After electrOphoresing, the gels stained with Coomassie blue showed one broad dark band of protein and six light bands which migrated faster in the gel than the former. The activity stain showed a maroon double band Of activity in the region of the slowly migrating dark band. This may suggest more than one band of diesterase activity, either by contamination by another exonuclease, or by resolution of two isozymes of the exo- nuclease activity. Polyacrylamide gels showed no protein bands when electrOphoresed using concentrated Step VI enzyme. Only 20 pg of protein was applied in the sample and this result was anticipated. The activity 61 stain showed only one intensely colored band which migrated slowly in the gel. Determination of the MOlecular Weight Estimations of the molecular weight of the exonuclease from Cucumis .9212 were Obtained from the gel filtration method of Andrews (65). A column (2.5 x 38 cm) of G-lOO Sephadex (#0-120 p bead size) was prepared and equilibrated with 0.02M; Tris acetate buffer pH 7.1 containing 10-4.! magnesium acetate. A flow rate of about 0.5 ml per minute was established by the use of a peristaltic pump. Fractions of 2.55 ml were collected at 5 minute intervals. The sample in a volume of 0.50 to 0.75 ml was layered under the tap buffer above the resin bed and eluted with the above Tris buffer, the sample containing several standard proteins of known molecular weight. There was less than 2% variation in the elution volume of the same protein using consecutive passes through the column. Based on the known molecular weight of the standards used, extrapolation from the curve shown in Figure 10 gives a value of molecular weight of muskmelon exonuclease of 87,000. The molecular weight estimation via the gel filtration method has a maximum uncertainty value of i 10% for globular proteins in the range of 10,000 to 150,000 daltons. Determination of the Activation Energy of Muskmelon Exonuclease The method used to determine the activation energy was the procedure of Wilson (66). The reaction mixture of 1.0 m1 contained 100 pmoles of sodium bicarbonate buffer pH 9.5, .10 pmoles of magnesium acetate, and 0.50 pmoles of p-nitrophenyl-pT. The assay mixture was pre-equilibrated at room temperature in a quartz microcuvette. The reaction was initiated Figure 10. 62 Determination of molecular weight by gel filtration. The semi-log plot of molecular weight of standard proteins versus the elution volume of that protein is shown. ‘ Procedure is described in the text. Standard of known molecular weight were A, cytochrome c, 12,#00 (69); A, DNase 1, 31,000 (70); D, ovalbumin, #5,000 (71); I, bovine serum albumin, 67,000 (72); O, alkaline phosphatase, 80,000 (73); and C, lactate dehydrogenase, 135,000 (71+). The muskmelon exonuclease (a Step VI preparation) had an elution volume of 92 m1 giving it an estimated molecular weight of 87,000. 63 ON no: 2203 uo_:uo.o<< 9m 0 v, N 0 Jeanne. . . J6m— 4 it (lul) OwflIOA uoglnlg .\ no: 4 O 0 L O N 6# by the addition of aliquots of enzyme (130 milliunits per ml) as indicated in Figure 11. The cell was immediately stoppered with a Teflon plug equipped with the MTl micrOprobe of a BAT-# micrOprobe thermometer (Bailey Instrument Co., Saddle Brook, New Jersey). The cell was then placed in the sample compartment of a Turner Model 330 Spectrophotometer, thermostatted at #5°C. The output of the micrOprobe thermometer was followed with time on a Sargent SR recorder while the absorbance at #00 was followed on a Sargent SRL recorder. The reaction was followed through an increase of approximately 20°C in temperature, about 5 minutes. Rates of reaction were determined by drawing tangents to the absorbance curve at points corresponding to known temperatures. The data, rates of hydrolysis, and temperatures were analyzed by the CDC 6500 computer programmed to fit 1n r vs. 1/T to the "best" straight line, determined by the least squares criterion. The Ea was calculated from the slope of this line. The energy of activation determined from this procedure gave values of 2.5 Kcal per mole and 2.8 Kcal per mole- for 601 and 501 aliquots of the enzyme respectively as seen in Figure 11. Bicarbonate buffer system was used for this determination because of the larger change in pH with changes in temperature using Tris buffer. 65 Figure IL Arrhenius plot for muskmelon exonuclease. Data for the curve was Obtained from the analysis of the curves as explained in the text. Determinations shown are of two differing enzyme concentrations 50)., O---O; and 60)., C---.. The line is the least squares "best" line through the data points. The correlation coefficient for the two enzyme concentrations are 0.890 and 0.937 for 60x and 501, respectively. ”9:: . mmd 00m mmd ON.m mam d d u u d Nd mm 0.0 u QN QN DISCUSSION In early experiments dealing with an examination of phosphodiester- ase activities in higher plants, it was found that two members of the family Cucurbitacea possessed an activity which differed significantly from that of the other sources tested. For these preparations, pyrimi- dine derivatives were found to be hydrolyzed more rapidly than the purine analogs. One of these unusual enzymes, that of Cucumis.melp, was selected for further study. An exonuclease or phosphodiesterase I from Cucumis melo, the fourth enzyme of this type from higher plants which has been isolated and characterized, has now been purified about 3,#00-fold. The preparation has a specific activity of 13,000 units per mg protein, thus being one of the more active preparations of exonuclease. Two steps in the isolation procedure were major steps leading to the purification of the enzyme to its present specific activity. Removal of the lipid-like material by lowering the pH of the crude enzyme solution and heating to 60°C permitted the subsequent use of ammonium sulfate fractionation and column chromatography in the purification scheme. The phosphocellulose column apparently separated two exonuclease activities. The exonuclease activty which was not eluted in the wash fractions was eluted with NaCl, yielding a preparation which contained an extremely small amount of protein. The enzyme differs from all other purified phosphodiesterase I in that the enzyme does not bind to DEAE cellulose in the pH range from.7.5 to 8.5. A major attribute of the 67 68 .S'.EEl2 enzyme is that the step VI enzyme can be prepared expeditiously from the imbibed seeds in 2 days using only two column procedures. Values for protein concentration obtained by the Lowry method of protein determination were shown to be somewhat unreliable at low protein concentration in the presence of Tris buffer. This was due to the extremely shallow lepe of the protein standard curve in Tris buffer. The average value for protein concentration using the method of Lowry on Step VI enzyme was 50 pg per ml. The tannic acid method of protein determination displayed no interference from Tris buffer even at low concentration. Values for the Step VI protein concentration was 20 pg per ml, the method being linear below the 10 pg per m1 level. The enzyme demonstrates properties typically characteristic of phospho- diesterase type I (17). The pH activity curve for muskmelon exonuclease is similar to that obtained for other phosphodiesterases from plant and animal sources in that the pH activity curve appears to be quite symmetrical around the Optimum pH 9.3 and falls off quite sharply on either side of this pH. At pH 8.9, the pH of the assay conditions, the activity drOps to 69% of that at the pH optimum. In Tris acetate buffer at pH 8.9, this activity is only slightly lower. Since the initial use of p-nitrophenyl-pT with venom phosphodiesterase in 1959, hydrolysis of this compound at pH 8.9 has become a standard assay for comparison of enzymes with various sources. For this reason the assays for muskmelon exonuclease were also performed at pH 8.9. There was essentially no activity at pH's below pH‘7.5 in the buffers tested. Stability as well as activity was pH dependent, but the 9. £312 enzyme like the other exonucleases from plant sources was more heat 69 stable than its animal counterparts. At 37°C the enzyme activity was destroyed completely at pH #.0 after 2 hours. The pH of optimal stability ranged from pH 7.0 to 9.0 for 37°C incubation. After 15 min- utes incubation at 80°C, the enzyme in Tris acetate buffer at pH 7.5 retained all of its activity. In Tris at higher pH's and in sodium carbonate-bicarbonate buffers (in the pH range of 8.5 to 10.0) consid- erable amounts of activity were lost. NO attempt was made to determine the change in pH from 2#°, the temperature at which the buffers were prepared, to 80°C. The significant negative temperature coefficient of Tris buffer and the loss of 002 from a bicarbonate-carbonate buffer system at 80°C may have played a large role in the loss of activity seen. At the pH of the enzyme assay, pH 8.9, the enzyme lost a high percentage of activity when the temperature was raised above 70°C, until at 90°C no detectable activity remained. Exonucleases (phosphodiesterase I) have been shown to hydrolyze a spectrum of substrates, ranging from polynucleotides to substituted nucleoside components. 9. 9212 exonuclease exhibited a high rate of hydrolysis for p-nitrophenyl nucleoside-5'-phosphates in comparison with oligonucleotide substrates. A preference in the rate of hydrolysis of ‘p-nitrophenyl-pdG over the rate of hydrolysis of p-nitrophenyl-pdc and p-nitrophenyl-pT, and a low rate of hydrolysis of p-nitrophenyl-pdA was observed. In contrast to the two phosphodiesterases from other sources which have been examined (8, 3#), ribotide derivatives of p-nitrOphenol were hydrolyzed by the Q, 2512 enzyme more slowly than their analogous deoxyribonucleotide derivatives. There was however no apparent correla- tion between the rate of hydrolysis and the Km for the substrates tested. Rates of hydrolysis of p-nitrophenyl esters other than p-nitrOphenyl 70 nucleoside-5'-phosphates were extremely low. The nonspecific phospho- diesterase substrate, bis-p-nitrophenyl-phOSphate, gave a rate only #% that of p-nitrophenyl-pT. The hydrolysis of Tp-p-nitrophenyl and p- nitrophenyl-phosphate could not be measured at an enzyme level of 72 milliunits. Even upon a #0-fold increase in enzyme concentration above 72 milliunits, no nonSpecific phosphatase activity could be detected using p-nitrOphenyl-phosphate as substrate. There was an extremely small but measurable hydrolysis of p-nitrOphenyl esters at pH 5.5 which agrees with reports describing exonucleases from other sources. This is quite unusual since this pH is almost 5 pH units from the pH optima of the enzyme. Use of this unique property has been made by Richards and Laskowski with venom exonuclease at pH 5.0 for the hydrolysis of Xpo to X and po, a reaction which at pH 8.9 is highly unfeasable due to the inhibition of the reaction by the ionized 3'-phosphoryl group (#8). The addition of various nucleoside monophosphates to the assay mixture lowered the rates of hydrolysis with p-nitrOphenyl-pT or p- nitrophenyl-pdC, the only substrates tested. Low rates of hydrolysis of both esters were seen in the presence of adenosine-5'-monophosphate and deoxyadenosine-S'-monophosphate, the greater inhibition being offered by 5'-AMP. The ability of 5'-AMP to strongly inhibit the enzymatic activity of phosphodiesterases has only been suggested in an earlier report on kidney phosphodiesterase I. The type of inhibition exerted by 5'-AMP was determined to be competitive in nature with the aid of a Dixon plot. The low value for the K1, 6 x 10.7 M, suggested that a strong complex exists between the enzyme and 5'-AMP. Observations with Q. 2212 enzyme represents the only examination in some detail of this phenomenon. This also may explain the low rate of hydrolysis of 71 p-nitrophenyl-pdA and p-nitrophenyl-pA by Q, Mglg_exonuclease. The products of these reactions, 5'-dAMP and 5'-AMP, are probably bound tenaciously to the enzyme at or near the active site, inhibiting new incoming substrate from entering the region of the catalytic center and resulting in a low rate of hydrolysis. All ligands attached to 5'-AMP lower the inhibitory effect exhibited by adenylic acid itself. The presence of the phosphoryl group in the 2'- or 3'-position of the ribose sugar rather than the 5'-position also lowers the inhibition. The rate of hydrolysis of polynucleotide was lower than that seen with the p-nitrophenyl derivatives. This is an observation which agrees with data of workers using exonuclease from other sources. Oligonucleo- tides, polynucleotides, and even dinucleotides were hydrolyzed at a lower rate than the respective p-nitrophenyl-nucleotides tested (8, 15, 16). A marked preference for heat denatured DNA was shown by the enzyme. Native DNA was not hydrolyzed under the assay conditions. Rabbit reticulocyte rRNA was hydrolyzed at about one-half the rate of denatured DNA. The slower hydrolysis of rRNA may have been due in part to the secondary structure attributed to ribosomal RNA. However, the low rate of hydrolysis of RNA compared with DNA was similar to the results obtained earlier with p-nitrophenyl derivatives of ribo- vs deoxyribo- tides, suggesting that the deoxyribotide moiety is indeed a preferred substrate. The rates of hydrolysis of the synthetic polymers were lower than these found for either natural polymer. Poly A was hydrolyzed at about one-third the rate for Poly U. This again is consistent with the results obtained earlier with the p-nitrophenyl esters of AMP and UMP. Poly C was hydrolyzed comparatively much more slowly than Poly U. This may have been due to the existence of considerable secondary structure 72 in Poly C at the pH of the assay conditions. A possible explanation for the slower hydrolysis of the presumably natural substrates compared with the more rapid hydrolysis of p-nitro- phenyl nucleotides can be proposed based on differences in the rate of the release of products following hydrolysis. The active center of the enzyme probably has at least two binding sites. The first is for the nucleoside-5'-phosphory1 group (8) and a second for the group or moiety attached to the 5‘-nucleotide. For synthetic substrates, this group would be p-nitrophenol and for more "natural" substrates the group would be a nucleoside unit of an oligonucleotide. Binding at the first site would be similar for either substrate. It may be that the nonionized nitrOphenyl group binds at site 2 with a binding constant similar to that of the nucleoside unit of an oligonucleotide. Upon hydrolysis, however, the negatively charged nitrOphenoxide may be bound less strongly and therefore released more rapidly from site 2 than the non-charged nucleoside moiety. The release process may be the rate determining step in the reaction. In this connection it may be significant that the hydrolysis of pT pT is faster than the hydrolysis of TpT for enzymes from two different sources (8, 15). Again this is a situation in which a charged group in site 2 gives a higher rate of hydrolysis. g. 9312 exonuclease benefits from the addition of certain divalent metal ions, i.e. Mg++, Ca++, and Ba++. EDTA, a metal chelator, had an inhibitory effect upon activity of the exonuclease, a concentration of 2 x lO-S‘M.completely inhibiting enzyme activity. Fluoride ion, also a complexing agent, reduced enzyme activity 8#% at mM concentration. If the fluoride was acting as a metal complexing agent, rather than binding to the enzyme itself as seen with adenylic acid deaminase (67), this 73 would be additional evidence that the exonuclease is a metal enzyme and that tightly bound metal ions are necessary for enzymatic activity. Inhibition of enzymatic activity by EDTA was restored by incubation of the enzyme with Mg++, sr++, or Ba++. The inhibition of exonuclease activity by sulfhydryl reagents reported with venom and rat intestinal mucosa exonucleases was also seen with the 9°.9212 enzyme. The inhibition exerted by these compounds may again be by formation of chelators, since these complexes are formed by sulfhydryl compounds (6#). The stimulation of enzymatic activity by ammonium sulfate as reported for the malt enzyme (1#) was not seen with g. EELS exonuclease, an inhibition of activity being observed. There was no detectable contamination of the exonuclease by adenosine deaminase or adenylic acid deaminase. Commercial preparation of spleen exonuclease, however, are known to contain a contaminating deaminase activity (68). No phosphatase or nucleotidase activity could be detected in Step VI exonuclease when assayed at pH 7.2 or pH 8.9 with 5'-, 3'-, or 2'-AMP or with p-nitrophenyl-phosphate at pH 8.9. The hydrolysis of bis-p- nitrOphenyl-phosphate at pH 8.9 was most probably an indication of the nonspecific nature of the enzyme activity rather than a separate contaminating phosphodiesterase. Phosphodiesterase II activity was not detected using Tp-p-nitrOphenyl as substrate. The enzyme activity has an exonucleolytic attack on denatured DNA, ribosomal RNA and Oligouridylic acid. This was expected since this has been shown previously with similar phosphodiesterases from other plant and animal sources. The direction of hydrolysis appears to be from the 3' to the 5' end, 5'-mononucleotides being liberated. No production of di- or trinucleotide could be seen. An estimate for the molecular weight 7# of C. melo exonuclease via gel filtration was 78,000. This is much larger than the molecular weights for most nucleases but is somewhat smaller than the molecular weight of .98 to 1.15 x 105 obtained for carrot phosphodiesterase I (37). The activation energy of the muskmelon enzyme was about 2,800 cal per mole. This is quite small in comparison with the activation energy of most enzymatic reactions which range from about 8,000 cal per mole for citrate synthetase to 12,000 cal per mole for brain hexokinase (66). The exonuclease from 9:.9212 like the enzymes from Avena leaves (16) and carrot (15) is apparently not compartmentalized as has been found with the acid hydrolases nor is it bound as part of a small particle complex. Rather, the enzyme is most probably located in the cytosol of the plant cell. It is difficult at this time to postulate a biological role for the enzyme which has any experimental support. If no inhibitory control mechanism is in Operation, the enzyme may be performing a continuous catabolic function within the cell. Native DNA usually located in the nucleus, in mitochondria, or in chloroplasts resists hydrolysis by the enzyme, but single stranded DNA which may enter the cytosol from a compartmentalized source or from the environment would be readily degraded. Ribosomal RNA is not available for hydrolysis since it is usually an integral part of the ribosome. Messenger RNA and single stranded viral RNA may be excellent substrates for the exonuclease providing that the 3' terminus is free, particularly after partial degradation by an endonuclease. Transfer RNA which may be partly degraded from its 3' hydroxyl terminus by exonuclease has a repair system which may replace the terminal cytidylic and adenylic acid units. The purified preparation of exonuclease from C. melo shows no 75 detectable contamination by nucleotidase, nonspecific phOSphodiesterase, endonuclease, adenosine deaminase or adenylic acid deaminase activities. Accordingly, the enzyme may be a useful tool in nucleic acid biochem- istry, particularly as an acid in sequence determination of oligo- nucleotides. SUMMARY An exonuclease from Q'.E212 seeds has been purified to an extent of 3,#00-fo1d with a recovery of about 1#% of the total exonuclease activity present in the crude extract. The enzyme displays many of the properties typical of phosphodiesterase I activities. No contamination by endo- nuclease, nonspecific phosphodiesterase, phosphatase, nucleotidase or deaminase activities has been detected. The enzyme was prepared by an expeditious procedure involving a heat step with a change in pH, acetone fractionation, ammonium sulfate fractionation, Sephadex G-100 gel filtration and phosphocellulose chromatography. The enzymatic activity shows a pH optimum of pH 9.3 with activity decreasing quite sharply on either side of this pH. The stability Of the activity was found to be dependent both on temperature and on pH, optimal conditions being #00 at pH 7.5. The enzyme activity was ++and Ba++. Enzymatic activity stimulated by the addition of Mg++, Ca was reduced or destroyed by the presence of sulfhydryl compounds, fluoride ions, and chelating agents. The C. melo exonuclease was sensitive to extremely small amounts of EDTA. Reversal of the EDTA ++ 4+ inhibition was Obtained with Mg++, Ca and Sr . The purified enzyme readily hydrolyzed p-nitrOphenyl esters of 5'-nucleotides. Ip-Nitrophenyl-pdG was hydrolyzed much faster than 2- nitrophenyl-pT or p-nitrophenyl-pdc which hydrolysis of p-nitrophenyl- pdA was extremely slow. 5'-Deoxyribonucleotide esters of p-nitrophenol 76 77 were hydrolyzed faster than their ribotide analogues. Esters of ‘p-nitrophenol other than 5'-mononucleotides were hydrolyzed at a rate of less than #% of that seen for the hydrolysis of p-nitrophenyl-pT. Denatured DNA and ribosomal RNA, presumably more natural substrates, were hydrolyzed at a rate much slower than that seen for the p-nitro- phenyl-5'-deoxynucleotides. Native DNA was not hydrolyzed under the assay conditions. 0f the homOpolymers examined, Poly U was hydrolyzed much faster than Poly A or Poly C. Poly G was not hydrolyzed under the conditions of the assay. A possible explanation for the rapid hydroly- sis Of the p-nitrOphenyl-S'-nucleotides in comparison with oligonucleo- tides was prOposed. An exceedingly strong inhibition by 5'-AMP of the hydrolysis of p-nitrophenyl-pdG and p-nitrophenyl-pT was observed and determined to be competitive in nature. An extremely small Ki for 5'-AMP was found. Hydrolysis of denatured DNA and ribosomal RNA by the g, Mglp_enzyme was shown to be exonucleolytic in nature. The molecular weight of the enzyme was determined to be about 78,000 using gel filtration. 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