THE PURiFICM‘EON AND PARTIAL CHARACTEREZATIUN 0F ASSOC|ATED DEGXYRIEDNUCLEASE RIBONUCLEASE AND 3' -NUCLEDTiDASE ACTWITHES 0F WHEAT SEEEUNGS Thesis for the Degree of Ph. D. MICHIGAN STATE UNNERSITY DOUGLAS M. HANSON 19:58 LIBRARY TH 5508 Michigan Sm UnivetSi‘Y This is to certify that the thesis entitled The Purification and Partial Characterization of Associated Deoxyribonuclease, Ribonuclease, and §'-Nuc1eotidase Activities of Wheat Seedlings presented by Douglas M . Hanson has been accepted towards fulfillment of the requirements for Ph.D. degree in Biochemistry MEL/gee Major professor! Date Chill}, 9) 1968 0-169 , 800K BINDERY INC. m LIBRARY amoens amends av V DAB 8: SUNS’ INFO". IIGIIGIJ ABSTRACT THE PURIFICATION AND PARTIAL CHARACTERIZATION OF ASSOCIATED DEOXYRIBONUCLEASE, RIBONUCLEASE, AND 3'-NUCLEOTIDASE ACTIVITIES OF WHEAT SEEDLINGS by Douglas M. Hanson In a preliminary investigation of the deoxyribonuc- lease activities of a variety of plant sources it was observed that wheat seedlings contained a relatively high level of deoxyribonuclease activity towards denatured DNA. This thesis reports work done in an attempt to purify and to determine the Specificity of this enzyme activity. It was hoped that the enzyme might exhibit a relative degree of base specificity and thus be useful in studies on the structure and base sequence of DNA. A nuclease has been purified from germinating wheat seedlings to an extent of over BBQ-fold. The purified enzyme preparation hydrolyzes denatured DNA, rRNA, and the 3'-ph03phoester linkage of 3'-AMP at similar rates. The preparation is free of detectable contaminating enzyme activities. The deoxyribonuclease, ribonuclease, and 3'-nucleo- tidase activities have remained associated throughout a variety of purification procedures including chromatography on several ion-exchange resins, gel filtration, and poly- acrylamide disc electrophoresis at pH 9.5 and 8.3. The ratios of the three activities remain essentially constant throughout the last four steps in the purification procedure Douglas M. Hanson as well as throughout all of the above mentioned procedures. The three enzyme activities exhibit a great degree of simi- larity with reSpect to a variety of properties. It is ten- tatively concluded that the three associated enzyme activ- ities are either properties of the same protein or of a very stable complex of two or more proteins. The deoxyribonuclease activity is highly preferential for denatured DNA. The hydrolysis of denatured DNA was found to be endonucleolytic in manner by a number of criteria. The mononucleotides and presumably the oligonucleotides pro- duced bear a 5'-phOSphoryl group. At all stages of digestion investigated, dAMP was the predominate mononucleotide compo- nent. The dAMP level was always about 2-fold greater than any of the other three deoxymononucleotides. A very low level of dGMP was observed throughout the digestion and this level actually decreased as a percentage of the total mono- nucleotide fraction as the digestion proceeded. Essentially all of the deoxyguanylate residues could be accounted for in the oligonucleotide fraction. Thus it would appear that the wheat deoxyribonuclease exhibits a relative degree of base Specificity with bonds involving deoxyadenylate residues being preferentially cleaved and bonds involving deoxyguany- late residues being relatively resistant. The mode of action of the wheat ribonuclease on rRNA was found to be exonucleolytic and the mononucleotides produced appear to be the 2',3'-cyclic compounds. The association of deoxyribonuclease, ribonuclease, Douglas M. Hanson and 3'—nucleotidase activities seems common in plants and may also be common in other biological sources as well. The biological significance of this association is not clear, but the observation that these activities exist in relatively high levels in germinating, rapidly growing seedlings suggests that these enzymes may play a role in the process of DNA repair or replication. THE PURIFICATION AND PARTIAL CHARACTERIZATION OF ASSOCIATED DEOXYRIBONUCLEASE, RIBONUCLEASE, AND 3'-NUCLEOTIDASE ACTIVITIES OF WHEAT SEEDLINGS By ‘. Douglas M. Hanson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1968 ACKNOWLEDGMENT The author would like to thank Dr. James L. Fairley for his advice and assistance during the work on this prob- lem and for his valuable contribution to my development of a philosophy toward biochemistry and biochemists. He would also like to thank Dr. Fritz M. Rottman, Dr. John E. Wilson, Dr. Willis A. Wood, Dr. Armon F. Yanders, and Dr. Clarence H. Suelter who kindly and patiently served on his guidance committee. His appreciation is also given to Larry and Nancy Muschek and to Mrs. Shirley Randall for their assistance in preparation of this manuscript and to everyone in the Department of Biochemistry of Michigan State University who discussed, criticized or helped with this research in any manner. He also wishes to eXpress his appreciation to The National Institutes of Health for providing the funds to support this study. D. M. H. ***** ii Dedicated to Lorraine and Michael 111 TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . . . . 10 Materials . . . . . . . . . . . . . . . . . . . . 10 Methods . . . . . . . . . . . . . . . . . . . . . 13 DNase Assay . . . . . . . . . . . . . . . . 13 RNase Assay . . . . . . . . . . . . . . . . 18 3'-Nucleotidase Assay . . . . . . . . . . . 18 Gel Filtration . . . . . . . . . . . . . . . 20 Assay for TCA-Soluble Ribose and Deoxyribose O O O O O O O O O O O O O O O O 20 Paper Chromatography . . . . . . . . . . . . 21 DEAE-Sephadex Chromatography . . . . . . . . 22 Protein Determination . . . . . . . . . . . 23 Polyacrylamide Disc Electrophoresis . . . . 24 RESULTS . . c o c c o o o . o o o . . . . . . . . . . 25 Purification of Enzyme . . . . . . . . . . . . . 25 Growth of Wheat Seedlings . . . . . . . . . 25 Homogenization of Seedlings . . . . . . . . 25 Ammonium Sulfate Fractionation . . . . . . . 29 Ethanol Fractionation . . . . . . . . . . . 29 Heat Fractionation . . . . . . . . . . . . . 30 PhOSphocellulose Chromatography . . . . . . 31 iv Concentration of PhoSphocellulose Fraction and P-BO Chromatography . Concentration of the P-30 Fraction Preparation of Enzyme for Storage Further Attempts at Separation of the Three AOtiVltj-es O O O O O O O O O O O O O 0 Batch Treatment With Various Adsorbents DEAE-Cellulose Chromatography at pH 8 ECTEOLA-Cellulose Chromatography . pH Gradient on Phosphocellulose . Chromatography on Bio-Gel P-6O . . Polyacrylamide Disc Electrophoresis Properties of the Enzyme Preparation . Contaminating Enzyme Activities . pH Optimum . . . . . . . . . . . . Effect of Sulfhydryl Compounds . . Effect of Zinc Ion . . . . . . . . Stability of Enzyme to Storage . . Effect of Inhibitors . . . . . . . EDTA Effect . . . . . . . . . . . Effect of Metal Ions . . . . . . . Effect of Amino Acid Modifying Reagents Temperature Effect and Heat Inactivation Ratio of Enzyme Activities During Purifi- cat10n.............. Effect of DNA and RNA on RNase and DNase ACtiVitieS O O O O O O O O O O O 0 Effect of Sulfhydryl Compounds and 8 M Urea on Gel Electrophoresis Pattern V Page 34 37 37 38 38 38 39 39 39 40 45 us 45 48 53 54 54 56 56 60 61 63 63 69 Studies on Enzyme Specificity . . . . . . . . Activity Toward Native and Denatured DNA Mode of Action on Denatured DNA . . . . Determination of PhOSphate Position in Mononucleotides Obtained from Denatured DNA HydrOIij-S O O O O O O O O O O O O O Mononucleotides Produced from Denatured DNA at Various Stages of Digestion . . . Activity Towards rRNA . . . . . . . . . Effect of Zn++ on rRNA Hydrolysis . . . Identification of Mononucleotides Produced from rRNA Digestion . . . . . . DISCUSSION 0 O O O O O O O O O O O O O O O O 0 O 0 SUMMARY BIBLIOGRAPHY O O O I O O O O O O O O O O O O O O 0 vi Page 70 7O 73 83 85 88 91 92 97 121 124 Figure 1. LIST OF FIGURES Non-linearity of lanthanum nitrate-acid soluble product assay for DNase activity Elution pattern from a phOSphocellulose column of protein, DNase, RNase, and 3'-nucleotidase activity . . . . . . . . Elution pattern from a combination phos- phocellulose P-BO column of protein, DNase, RNase, and 3'-nculeotidase activ- ity O O O O O O O O O O O O O O O O O O Elution pattern from a P-6O column of protein, DNase, RNase, and 3'-nucleoti- dase activity . . . . . . . . . . . . . Staining pattern of protein and distri- bution of enzyme activity within a poly- acrylamide gel after electrophoresis . . pH-aCtIVIty curve 0 o o o o o o o o o 0 Effect of EDTA on activity of DNase and RNase O O O O O O O O O O O 0 O O O O 0 Time course of hydrolysis of denatured and native DNA and of rRNA as measured by the lanthanum-acid soluble product assay 0 O O O O O O O O O O O O O O O O a. Chromatography on P-lOO of denatured DNA at various stages in its diges- tion by wheat DNase . . . . . . . . b. Chromatography on P-1OO of a mixture of denatured DNA and mononucleotide c. Chromatography on P-lOO of denatured DNA at various stages in its diges- tion by snake venom phoSphodiester- age 0 D O O O O O O O O O O O O O 0 vii Page 15 32 35 41 43 46 57 71 7h 74 74 Figure Page 10. Time course of hydrolysis of denatured DNA by wheat DNase and venom phOSpho- diesterase as measured by the DEAE assay . . 77 11. Elution pattern from a DEAE-Sephadex A-25 column of mono- and oligonucleo- tides produced during the hydrolysis of denatured DNA by wheat DNase . . . . . . . . 81 12. a. Chromatography on P-6 of a mixture of rRNA and mononucleotide . . . . . . . 89 b. Chromatography on P-6 of rRNA at various stages in its digestion by wheat RNase . . . . . . . . . . . . . . 89 viii Table I. II. III. IV. V. VI. VII. VIII. IX. XI. XII. LIST OF TABLES a. Summary of purification of DNase from 7-kilogram of wheat seedlings . . . . . b. Summary of purification of RNase from 7-kilogram of wheat seedlings . . . . . c. Summary of purification of 3'-nucleo- tidase from 7-kilogram of wheat seedlings o o o o o o o o o o o o o o 0 Effect of removal of Zn++ or cysteine on DNase, RNase, and 3'-nucleotidase activ- itiesatpHLl'QScococoon-coco. Time course of dithiothreitol inactivation of the three enzyme activities at pH 8 . . Effect of various sulfhydryl compounds at pH 8 O O O O O C O O O O O O O O 0 O O O 0 Effect of inhibitors on DNase and RNase aCt1V1tieS O O O O O O O O O O O O O O O 0 Effect of cations on activity . . . . . . . Effect of some common amino acid modifying reagents on the three enzyme activities . . Enzyme activity with reSpect to heat treat- ment at various temperatures . . . . . . . Enzyme activity with reSpect to duration theatingat7oooooooooooocoo Comparison of the ratios of RNase and 3'- nucleotidase activities to that of DNase after various procedures . . . . . . . . . Effect of DNA and RNA on RNase and DNase aCt1v1ties O O O O O O O O O O O O O O O 0 Identification of phoSphate position in mononucleotides from the hydrolysis of denatured DNA . . . . . . . . . . . . . . . ix Page 26 27 28 49 50 51 55 59 62 64 65 66 68 84 Table Page XIII. Composition of the mononucleotide fraction at various stages in the digestion of denaturedDNAcccooccococo-coo87 XIV. Effect at Zn++ on the hydrolysis of rRNA byWheatRNase 01.000.000.000... 93 INTRODUCTION Friedrich Miescher, a postdoctoral student of Hoppe- Seyler, was the first to report the extraction of nucleic acid material from the cell (1). That was in 1871 and iiiescher called the nuclear material that he had isolated In 1889, Altmann (2) proposed from pus cells "nuclein." for this material. The years fol- tlie name "nucleic acid" Ilcrwing the discovery of "nuclein" were filled with attempts determine the chemical composition and structure of this t<> Since biochemists seem to have a knack for dis- material. rupting things, especially molecules, it was not long before the major components of the nucleic acid material were deter- As is usually the case for most biological materials InfiLrumi. ‘Vlitich have been isolated, enzymes were soon discovered which “’EBJre capable of degrading the newly discovered nucleic acid nnéifberial. Thus in 1903, Araki (3) observed that extracts of 1:1Lssues such as liver, thymus, and Spleen had the power to :1~1~<;uefy gels of DNA. Levene and Medigreceanu (3a) introduced tzliles collective term "nuclease" for all enzymes which were fiLl"Involved in the metabolism of nucleic acids or their degrada- t::L-<:n products or precursors. The pancreatic Juice of dogs EVEELES also found to contain enzymes which caused the liquefac- t:'-:IL«::n of nucleic acid gels (4, 5). This liquefaction occurred v'wh‘JL-‘tzhout the liberation of nitrogenous bases or inorganic 2 phOSphate. Feulgen (6) made the important observation that degradation of DNA by pancreas preparations stopped at the formation of oligonucleotides and did not yield many mono- nucleotides. Laskowski (7) and McCarty (8) achieved a con- siderable purification of the enzyme from pancreas and in 1950 Kunitz (9) crystallized the enzyme and introduced the term "deoxyribonuclease" for those enzymes which are capable The pancreatic deoxyribonuclease (DNase) of degrading DNA . The term DNase I is presently was referred to as DNase I. used to denote a whole class of enzymes from various sources wTrich exhibit optimal activity at alkaline pH. A DNase which has an optimum pH in the range of 4.5 to 5. 5 was reported in Spleen by Catcheside and Holmes (10). Maver and Greco (11, 12) reported that a large portion of the llblsase activity of thymus gland was due to an enzyme which was different from DNase I-type enzymes. The Spleen enzyme has lDEBean purified to homogeneity by Bernardi and Griffe (13) and 1181s3 been called DNase II. DNase II-type enzymes, with acid IJEI optima, have been isolated and purified from a variety of Sc3"..1rces. Microorganisms have also been a source of a wide ‘reictriety of DNase activities. ‘These enzymes have recently be36311reviewed by Laskowski (14) and Lehman (15). In 1942 Greenstein reported the presence of DNase at<=“t:ivity in extracts of the embryos of corn, wheat, pumpkin, s: 11l’lflower, and lima bean. These activities were capable of d. ‘3’]E>olymerizing thymus DNA. The fact that these plant enzymes 0. <:"Clld degrade animal DNA was strong evidence that the previous 3 distinction which had been made between "plant" and "animal" DNA (16, see p. 3) was no longer tenable. It was also of interest that these DNase activities were relatively high in germinating, rapidly growing seedlings. Brawerman and Chargaff (17) partially purified a DNase from germinating barley, using commercial malt diastase as a starting material. {The enzyme contained RNase and 3'-nucleotidase activities as ‘weJJ.as DNase activity. This enzyme has been more exten- sively studied by Holbrook gt 11;. (29). Shuster prepared a This plant source was also a DNase from rye grass (18). DNase activity has good source of 3'-nucleotidase activity. £13.80 been reported in soybean Sprouts (19). A DNase from mung bean Sprouts was reported by £31sockx and Van Parijs (20). It was also observed that an Iiblase and a 3'-nucleotidase activity were associated with likle mung bean DNase (21). These activities have been fur- 13k1er studied by Sung and Laskowski (22). The properties of these enzymes have been extensively studied by Walters and I¥<>cring (23, 24) who concluded that the DNase activity was E3111ythe DNase activity decreased on storage of the enzyme (I33E‘eparation. Other interpretations of these results, how- e"V‘er, are also possible. Mukai (25) has reported the presence of a DNase with IDESTease I-type properties in rice bran. At least two distinct :EDJNTaase activities exist in germinating garlic (26). Bjork jEMF-ins separated and partially purified two endonucleases from .a ‘1‘ 7n Fan. Li potato tubers (27). Adams and Fairley have reported the purification of a DNase from muskmelon seed (28).” This DNase, an endonuclease, appears to be closely associated with an RNase and a 3'-nucleotidase activities (see foot- note 5). A complete listing and discussion of all the DNases ‘whdch have been isolated since their discovery in 1903 VHDUId fill several volumes due to the vast number and vari- erty'of activities which have been demonstrated. DNase axztivities have been reported in almost every possible type <31? biological source and it is a safe generalization to say 'tlmat no source has been found which is completely devoid o f DNase activity. The classification of DNase activities was origin- ally quite simple and four major properties of DNases were 3elected for the purpose of classification (30). These 131?operties were: 1. Specificity toward the sugar moiety. 2. Exo- versus endonucleolytic mode of action. 3. Cleavage of the internucleotide bond on the 3'-P versus the 5'-P side, thus forming products bearing either 5'- or 3'-monophOSphate. 4. Nature of the base adjacent to the susceptible linkage; i.e. base Specificity. While these criteria were sufficient for some years JL'RZ' soon became evident that several other properties of Itilxlzases should also be considered for the purpose of classi- 5 fication (14). These additional properties are: 5. Specificity toward the secondary structure of the DNA; i.e., native (double-stranded) versus dena- tured (single-stranded) DNA. 6. Inability to attack the DNA from the same Species. 7. Inability to hydrolyze dinucleotides. 8. Inability to attack either native or denatured DNA but capacity of hydrolyzing oligonucleotides. 9. Ability to hydrolyze both strands of native DNA simultaneously at the same locus. It is becoming evident that no classification of DNase activities can be absolute. The wide variety of enzymes Pfkllch have been isolated indicate that many of these proper- 1:1.es mentioned above overlap and that while a particular terlzyme may be like other DNases in some of its properties it nasty be greatly different with re8pect to some other properties. 15:1.80 the observation by a number of workers (14) that the SI>ecificity and mode of action of many DNases changes dur- ing the course of DNA hydrolysis further complicates the IDIJEacement of a particular enzyme in a definite classification. 0f the criteria listed above only number 3, the cleav- ‘E‘éE:e of internucleotide bonds to produce produCts which bear £3"fiL‘ther a 5'- or 3'-monophoSphate terminus, remains absolute. TO data no enzyme has been found which can split the inter- 111leleotide bond on either side of the phoSphorus atom. For 53’ ilong time it was felt that exo- versus endonucleolytic (3 :lueavage was a reliable criteria for classification. It now ". 'U 6 appears, however, that under Specific conditions an endo- nuclease may favor an exonucleolytic activity. This has been shown for micrococcal nuclease (31) and may eXplain why endonucleases such as the one from NeuroSpora crassa (32) and the wheat endonuclease reported in this thesis produce a significant amount of mononucleotides at all stages in the digestion of DNA. Many DNases have been shown to possess a distinct preference for DNA in either the native (double-stranded) state or for DNA in the denatured (single-stranded) state. Some of these enzymes which exhibit Specificity toward the secondary structure of the DNA have recently been reviewed (14, see p. 181). To underline the complexity of nuclease action let us consider the case of DNases which are relatively pref- erential for native DNA. It might be eXpected that the Overall mode of action of all DNases of this category would be the same. That this is not the case has been decisively Shown by Young and Sinsheimer (33), who compared the early ac tion of DNase I and DNase II on native DNA. By studying uI'Zlder alkaline conditions the density gradient centrifuga- tion patterns of DNA treated with these two enzymes, they were able to Show a distinct difference in their mode of ac tion. Splenic DNase II degraded the DNA by simultaneous cleavage of both polynucleotide chains at or near the same pt>Sition. With DNase I, however, single-stranded cleavages Q Q-Qurred: and, on an average, only one in four cleavages b5 Suited in scission of both chains at the same locus. 7 Subsequent studies have shown that similar mechanisms exist for other nucleases as well. One of the major goals of nuclease research has been the isolation of specific nucleases which exhibit an abso- lute base Specificity and can be used in the determination of base sequences in nucleic acids. A milestone event occurred in 1965 when Holley and co-workers reported the complete base sequence of alanine-SRNA (34. 35). This was determined with the extremely Specific Tl-RNase. The same type of sequence determination is theoretically possible for DNA, however, DNases are notoriously non-Specific. To date no enzyme has been found which exhibits the degree of base Specificity observed with some of the RNaseS. Several enzymes have been reported which are relatively Specific, at least in the early stages of DNA hydrolysis, so it is Still possible that specific DNases will someday be isolated. The most Specific DNases observed to date are all endonuc- leases. An endonuclease from the hepatopancreas of OctoEus .Vulgaris (36) is preferential for the bond, pX‘pC, where X 1 :3 any deoxynucleoside residue. The mung bean DNase men- tioned previously (22) has preferential activity for bonds involving deoxyadenylate residues, while the endonuclease I from potato tubers is preferential for both deoxyadenylate a~1'1 120 280 —_. 200 a_ 160 ~—- 120 ___ 80 40 240 (rm/serum) fiqtntqov 140 40 60 80 100 20 Fraction Number 34 Concentration of PhoSphocellulose Fraction and P:30 Chromatography The pooled material from the phOSphocellulose column (330 ml) was diluted with Buffer B until it was 0.1 M in NaCl as determined from the refractive index of the solution. This usually required at least an equal volume of buffer. A column (2.2 x 76 cm) of Bio-Gel P-30 was packed under gravity flow and a pad of wet glass wool 1 mm thick was placed on top of the gel bed. A bed of phOSphocellulose (2.2 x 3 cm, 1 g dry weight of resin) was layered on top of the glass wool pad. This combination column was then washed with 300 m1 of Buffer B. The enzyme fraction was applied to the column at a flow rate of 1 ml per minute and then the column was washed with Buffer B until the effluent was free of NaCl (about 320 ml). The column was washed with 45 ml of Tris-maleate buf- fer, 0.05 M, pH 6.5, 0.1 mM in zinc acetate, 2 mM cysteine, 0.7 M NaCl, at a flow rate of 0.5 ml per minute. The high concentration of salt and the pH change were found to be sufficient to elute the enzyme from the phoSphocellulose as a very Sharp yellow band which then entered the P-30 gel in a very small volume. The column was then washed with Buffer B at 1 ml per minute. AS shown in Figure 3, the enzyme activ- ity was eluted in fractions 15 through 23. Ultraviolet absorbing material, presumably the buffer components, began to appear in fraction 41 and NaCl in fraction 48. Figure 3: 35 Elution pattern from a combination phos- phocellulose P-30 column of protein, DNase, RNase, and 3'-nucleotidase activ- ity. A column, 2.2 x 76 cm, was packed with P-30 and a 1 mm pad of wet glass wool was placed on top of the bed of gel. A bed of phOSphocellulose (2.2 x 3 cm. 1 g dry weight of resin) was layered on top of the pad. The column was washed with 300 ml of Buffer B. The diluted phOSphocellulose fraction (660 ml) was applied to the column at a flow rate of 1 ml per minute. The column was washed with Buffer B until free of NaCl. The protein was eluted with 45 ml of Tris- maleate buffer, 0.05 M, pH 6.5. 0.1 mM in zinc acetate, 2 mM in cysteine, 0.7 M in NaCl, at a flow rate of 0.5 ml per minute. The column was then washed with Buffer B. The three enzyme activities were eluted from the column in fractions 15 through 23. Buffer components began to elute in fraction 41 and NaCl in frac- tion 48. The enzyme assays were performed as described in "Methods and Materials." Protein, I___l; DNase, EJ---C] ; RNase, 0"‘0: 3'-nucleotidase, A-.-A . Activity (Units/ml) 240 200 160 120 80 40 36 25 Fraction Number .'\D l FfiaeLaael I 35 45 .60 .50 .40 .30 .10 A280 37 Concentration of the P-30 Fraction Fractions 15 through 23 (55 ml) from the P-30 column were pooled and concentrated with a rotary flash evaporator, adjusting the vacuum to minimize foaming. This step was carried out at 280 and usually took about 1 hour. The volumn of the P-30 fraction was routinely reduced by a fac- tor of 10. However, the solution was never allowed to go completely to dryness as this resulted in a greater than 90% loss of enzyme activity. The concentrated fraction was then dialyzed for a total of 15 hours against two 1 liter portions of Buffer B, changing the buffer after the first six hours. The average degree of purification of the concentrated P-30 fraction was about 830-fold and the average recovery was about 12% of the total DNase activity present in the crude homogenate. This fraction was used in most cf the SXperiments to be discussed below and will be designated as the P-30 fraction. Preparation of Enzype for Storage The dialyzed concentrated enzyme solution was routinely filtered through a sterile Millipore Filter (GSWP 025, 0.22 n) to minimize bacterial contamination. One or two ml portions were placed in sterile, screw-cap culture tubes. These tubes of "sterile" enzyme solutions were then stored either at -200 or 4° until use. 38 Further Attempts at Separation of the Three Activities Batch Treatment with Various Adsorbents In an attempt to find an ion-exchange resin or adsor- bent which would effect a separation of the three enzyme activities the following eXperiment was performed. A total of 8 ion-exchange resins (phoSphocellulose, carboxymethyl- cellulose, DEAE—cellulose, DEAE-Sephadex, ECTEOLA-cellulose, Dowex-i, Dowex-50, and Amberlite-50) and 2 adsorbents (wood- cellulose, and Bentonite) were used in a batch-wise manner to treat enzyme solutions at pH 4.5, 6.5, and 8.0. After 15 minutes of mixing the resins were centrifuged and the super- natant solutions assayed for the three enzyme activities. Within the accuracy of the assay procedures, no separation of the three activities was found. The activities either did not bind to the resin or were bound to the same degree. DEAE-Cellulose Chromatograppy at_pH 8 A column of DEAE-cellulose, 1.5 x 10 cm, was packed and washed with 0.025 M Tris-chloride, pH 8.0, 0.1 mM in zinc acetate. Enzyme from the P-30 fraction was applied to the column in the same buffer at a rate of 1 ml per minute. A linear salt gradient from 0 to 0.5 M NaCl (total volume of 200 ml) was then applied to the column. The three enzyme activities appeared in a single peak at 0.12 M NaCl concentration. This peak also showed a tail of activity on the higher salt side of the peak. This tail was essen- tially the same for all three activities. 39 ECTEOLA-Cellulose Chromatography The P-30 fraction was also chromatographed on an ECTEOLA-cellulose column (1 x 10 cm) with Tris-maleate buf- fer, 0.05 M pH 6.5, 2 mM in cysteine, 0.1 mM in zinc acetate. A linear salt gradient from 0 to 0.5 M NaCl was applied and again the three activities appeared as a single peak at approximately 0.1 M salt concentration. pH Gradient on PhOSphocellulose Treatment of the enzyme preparation with phOSphocel- lulose at pH 6.5 indicated that the three activities were not retained by the resin at this pH. Since this resin gave a good purification at pH 4.5 when a salt gradient was applied to the column it was decided to study the behavior of the activities in reSponse to a pH gradient. The P-30 enzyme at pH 4.5 was added to a 1 x 10 cm column of phoSpho- cellulose previously equilibrated with Buffer B. A pH gradient consisting of 50 ml each of Buffer B and 0.05 M Tris-maleate buffer, pH 6.9. 0.1 mM in Zn++, 2 mM in cysteine was applied to the column at a flow rate of 1 ml per minute. The three activities were eluted from the column at approxi- mately pH 5.5 as a Single peak, with the pattern of the three activities throughout the peak again being essentially the same 0 Chromatography on Bio-Gel P-60 A 2 x 80 cm column of Bio-Gel P-60 (50-150 mesh) was packed under gravity flow in Buffer B. A sample of enzyme 40 from the phoSphocellulose fraction was applied and chromato- graphed at a rate of 1 ml per minute in the same buffer. The enzyme was retained by this gel as shown in Figure 4. The three activities were obtained in a single peak at about the same elution position as pancreatic RNase dimer. This suggests a possible molecular weight of about 30,000. A small peak of material adsorbing at 280 mu was also found which was retained to a greater extent by the P-60 column. This material,was, however, completely devoid of any enzyme activity. Polyacrylamide Disc Egectrophoresis An aliquot of the P-30 fraction containing up to 400 ug of protein was examined by electrophoresis using a 10% polyacrylamide gel at pH 9.5, as described in "Methods and Materials." Half the gel was stained with Coomassie Blue Stain by the method of Chrambach gpugl. (44). The other half of the gel was cut into 4 mm segments. Each segment was placed in 1 ml of Tris-chloride, 0.025 M, pH 8 and allowed to stand overnight at 4°. The solutions were then assayed for the three enzyme activities. Figure 5 shows the staining pattern of the proteins and the distribution of the three activities within the gel. Judging from the number of bands obtained, the preparation still contains a number of protein Species; however, the three enzyme activ- ities were found to be associated with a single band and were distributed through this band in essentially identical 41 .dllfl .ommpapomHoSStR ao...o .omczm .DIIIU .ommzm ”QIIIO .adopoam =.mHmHaopcz was escapes: SH confluence mm ooaaomaoa cams mashed mahusm .opaaaa Hod as a no open 30am a as m Homwsm ad posamawopSEOHSO end poaaaam was Soapomah omoHaHHooosamoaa on» mo AHS oav madame 4 .m Mommam suds dogmas and deacon was oolm Hooloam mo .ao om N N .SasHoo < .modpabdpos ommoapoodoaal.m use .ommzm .ommzm .aaopona mo :aSHoo omlm m Soak Savanna Soapdam a: oaswdm 42 082v 0a. ON. on. 0:. on. Honadz Soapomam II ONH .Il 00H II com (Im/SfiTufl) £41A14°V Figure,5: 43 Staining pattern of protein and distribu- tion of enzyme activity within a poly- acrylamide gel after electrophoresis. A sample (0.2 ml) of the P-30 fraction con- taining 400 ug of protein was applied to a 10% polyacrylamide gel as described in "Methods and Materials." Electrophoresis at pH 9.5 was performed at 4° for 4 hours with an applied current of 5 MA. The gel was cut in half, one half was stained with Coomassie Blue stain, and the other half was cut into 4 mm segments. Each segment was placed in 1 ml of Tris- chloride buffer, 0.025 M, pH 8 and allowed to stand overnight at 4°. The solutions were assayed for enzyme activity as described in "Methods and Materials." 44 3'-Nucleotidase RNase DNase _ O 2 F _ 0 0 h». 3 Ha\npaaa _ 0 1.. 0 _ _ 0 0 3 2 40.. Ha\mpaao . _ 0 1.. 0 40.. _ _ 0 0 3 2 aS\mpaaa _ o 1.. Origin 45 fashion. A similar result was obtained upon electrophoresis at pH 8.3. It is apparent that a significant purification can be achieved by electrophoresis. EXperiments applying prepara- tive electrophoresis to the P-30 fraction are now in progress. Properties of the Enzyme Preparation Contaminating Enzyme Activities Aliquots of a P-30 fraction, each aliquot containing 8.8 DNase units with a specific activity of 418, gave no detectable hydrolysis at pH 5 of 5'-AMP, bis-p-nitrophenyl phOSphate, 5'-p-nitrophenyl thymidylate, or 5'-p-nitropheny1 adenylate after 24 hour incubation at 37°. These assay con- ditions represent a ten-fold higher enzyme concentration and a 144-fold longer incubation period than that used under the normal assay conditions for DNase activity. Under the same conditions a Slight amount of hydrolysis of p-nitrophenyl phoSphate and of 3'-p-nitropheny1 thymidylate was observed. On examination of DNA digests, however, neither 3'-nucleo- tides, nucleosides, nor P1 could be detected. From these results it was concluded that the purified enzyme prepara- tions contained little if any 5'-nucleotidase, phoSphodi- esterase, and non-Specific phosphomonoesterase activities. pH Optimum Figure 6 Shows the similar pH-activity curves. in the absence of added Zn++, for the DNase, RNase, and 3'-nucleo- Figure 6: 46 pH-activity curve. The assays were per- formed as described in "Methods and Materials." All points are for 0.05 M sodium acetate buffer adjusted to the appropriate pH with either 1 M acetic acid or KOH. DNase, O___O; RNase, O——-O. 3'-nucleotidase, 0———O. 47 100 _ 80 apabapod 5833: ac pSoohom 20 L— 4.8 5.6 5.2 4.4 4.0 pH 48 tidase activities. Maximal activity for DNA hydrolysis by the P-30 fraction was between pH 4.8 and 5.3, for RNA hydrol- ysis between 4.9 and 5.5, and for 3'-AMP hydrolysis between 4.8 and 5.8. The 3'-nuc1eotidase curve also had a shoulder at about pH 4.7. The meaning of this is not clear at present. If similar SXperiments are conducted with Zn++ present at 1 mM concentration the three curves were unchanged below pH 5.1. Above this pH, all three activities were markedly decreased. A precipitate is apparent under these conditions. Effect of Sulfhydryl Compounds The three enzyme activities were found to require the presence of sulfhydryl compounds for maintenance of maximal activity at pH 4.5. Table II shows the decrease in the activities after dialysis overnight at 4° against buffer lacking sulfhydryl compounds. At pH 8, however, the presence of such compounds caused complete loss of all three activities in approximately 24 hours at 4°. A time course of inactiva- tion by 1 mM dithiothreitol at pH 8 is Shown in Table III for the three activities. The curves were found to be very Similar with 50% inactivation being reached at 2.3, 2.1, and 2.9 hours after dithiothreitol addition for the DNase, RNase, and 3'-nuc1eotidase activities reSpectively. This effect at pH 8 appears to be a general sulfhydryl effect as can be seen in Table IV. Essentially the same results were obtained regardless of the sulfhydryl source, i.e. dithiothreitol, mercaptoethanol, or cysteine, provided that the total sulf- hydryl concentration was the same in each case. All three 49 Table II Effect of the removal of Zn++ or cysteine on DNase, RNase and 3'-nucleotidase activities at pH 4.5 Samples (1.0 ml) of enzyme were placed in dialysis tubing and dialyzed against two 2-liter changes of buffer at 4° for 15 hours. The control buffer was Buffer B. The other buffers were the same as B but lacked the component indicated. The assay procedures were those described in "Methods and Materials." Percent of Original Activity Remaining Compound deleted DNase RNase 3'-Nucleotidase Control 100 100 100 Zn++, cysteine 7 29 9 Cysteine 80 90 63 Zn++ 71 65 66 50 Table III Time course of dithiothreitol inactivation of the three enzyme activities at pH 8 To a sample (4.0 ml) of enzyme in 0.025 M Tris-chloride buffer at pH 8 was added 80 31 of 50 mM dithiothreitol to give a final concentration of 10' M dithiothreitol. The enzyme solution was allowed to stand at 4° and samples (0.4 ml) were removed at the times indicated below. These samples were assayed as described in "Materials and Methods." Percent of Original Activity Remaining Hours after addition DNase RNase 3'-Nucleotidase 0 100 100 100 1 78 66 81 3 41 41 49 6 24 24 20 12 9 9 3 24 1 1 0 51 Table IV Effect of various sulfhydryl compounds at pH 8 Samples (0.5.m1) of enzyme in 0.025 M Tris-chloride buffer at pH 8 were allowed to stand at 4° for 15 hours in the presenc of the sulfhydryl compound at a final concentra- tion of 10' M, except that the molarity of the dithiothreitol was half this value. The samples were then assayed as des- cribed in "Methods and Materials." Percent of Original Activity Remaining Sulfhydryl _addition DNase RNase 3'-Nucleotidase None 100 100 100 Dithiothreitol 3.4 1.5 1.4 Cysteine 8.9 20.6 0.9 Mercaptoethanol 8.9 10.3 2.8 52 activities were stable to storage at pH 8 in the absence of sulfhydryl compound to the same extent that they were stable at pH 4.5 in the presence of sulfhydryl compound. Titration curves fbr activity versus the sulfhydryl concentration over the range of 2 x 10'2 to 10"5 M were essentially the same at pH 4.5 for the three activities. Similar curves were obtained regardless of whether dithio- threitol or cysteine was used as the sulfhydryl compound provided that the total sulfhydryl concentration was the same. Removal of cysteine from the inactivated enzyme prepa- ration by dialysis against 0.05 M Tris-chloride, pH 8.0 caused no detectable reactivation of the DNase activity. Dialysis of this same material against Buffer B resulted in approximately doubling the activity in 24 hours at 4°. This increase, however, represented only about 5% of the initial DNase activity. Whether additional increase in activity occurs with longer periods of time at pH 4.5 was not determined. The point in the purification procedure at which cysteine or dithiothreitol is added was found to be very critical. Routinely the sulfhydryl compound was first added to the system after the ethanol fractionation and before the heat step. In the heat step and subsequent procedures the enzyme was not stable in the absence of sulfhydryl compounds. It was found, however, that addition of sulfhydryl to any step prior to the ethanol fractionation resulted in a com- .l I.".? ‘1 .- 9” (J 53 plete loss of enzyme activity in only a few hours at 4°. The explanation of this observation is obscure, but one possibility is that sulfhydryl compounds activate one or more proteases which are removed by the ethanol step. Effect of Zing_Ipp The addition of Zinc acetate to a final concentra- tion of 1 mM in the assay mixture caused about a 20% increase in DNase activity with the less purified fractions (up to and including the heat step). Addition of Zn++ at this con- centration to the assay mixture of subsequent. more highly purified fractions led to as much as a 25% decrease in DNase activity. Slight inhibition of the purest fractions was found with Zn++ at concentrations as low as 10'5 M. For this reason Zn++ was not routinely added to the assay mix- tures. The stability of the three enzyme activities to storage and to subsequent treatments was, however, strongly dependent on the presence of Zn++ in the enzyme medium. Removal of the Zn++ from Buffer B resulted in about the same degree of loss of all three activities in 15 hours at 4°. This is shown in Table II. The figure also shows that removal of both Zn++ and cysteine gave a much greater decrease Of the activities than did removal of the Zn++ or cysteine alone. Because of this stabilizing effect of Zn++, the puri- fication steps subsequent to ethanol fractionation and stor- age of the enzyme were carried out in buffer that was at 1938t 10'“ M in Zn++. Attempts to reactivate enzyme prepara- tions from which both Zn” and cysteine were removed by 54 dialysis at pH 4.5, by restoring both of these substances were only partly successful. About 35% of both DNase and RNase activities could be regained. The nucleotidase activities were not examined in this experiment. At pH 8 added Zn++ did not appear to be required for stability of the three activities. Stabiligy of Enzyme to Storage The heat fraction was stable in Buffer A at 4° for several weeks with no appreciable loss of DNase activity. The phoSphocellulose fraction has been stored in Buffer B for up to 5 months at either 4° or -20° with less than 5% loss of the three enzyme activities. Repeated freezing and thawing of the phosphocellulose fraction was observed to result in a small but significant loss of activity. To minimize this loss, the enzyme solution was divided into portions of 1-2 ml and stored in sterile culture tubes until use. Effect of‘InhibitorS Table V shows a typical set of data for the inhibi- tion of the DNase and RNase activities by KH2P°4° The effect of phoSphate was at least qualitatively the same for both the DNase and RNase activities. Approximately 50% inhibition of the DNase activity was obtained at a phos- phate ion concentration of 1 x 10'3 M whereas 5 x 10"3 M phoSphate ion was needed to inhibit the RNase to the same extent. 55 Table V Effect of inhibitors on DNase and RNase activities Reactions were carried out at pH 4.5 as described in "Methods and Materials" with inhibitors added to the reaction mixture at 4° five minutes before adding enzyme. Percent of Original Activity Remaining Molar Inhibitor concentration DNase RNase None --- 100 100 KH2P04 1 x 10'5 117 96 1 x 10'” 78 86 1 x 10"3 53 78 1 x 10"2 0 3 1 x 10"1 0 0 None --— 100 100 NaF 1 r 10'5 78 86 1 x 10'” 108 62 1 x 10'3 70 69 5 x 10"3 21 38 1 x 10‘2 o 15 56 A similar effect was obtained when sodium fluoride was used as the inhibitor. As indicated in Table V fluoride ion affected both DNase and RNase activities in much the same way, with 50% inhibition occurring at approximately 2 x 10"3 M and 3 x 10-3 M respectively. The effect of both of these inhibi- tors on the 3'-nucleotidase activity could not be assessed because of interference with the assay procedure. EDTA Effect The effect of EDTA on the DNase and RNase activities was examined over the range of concentration of EDTA from 10"8 to 2 x 10"2 M. The titration curves are shown in Figure 7 and demonstrate a region of concentration between about 10'5 M and 10"2 M EDTA in which the degree of inhibition was found to be constant for each activity. Note that the RNase activity was inhibited more strongly at intermediate concen- trations of EDTA than was the DNase activity. The curves do, however, Show the same overall pattern for both the activ- ities. No eXplanation for the unusual shape of this curve is readily available. Effect of Metal Ions The effect of various metal ions on the three enzyme activities was studied with the results Shown in Table VI. A P-30 fraction with a Specific activity of 418 was used for this study. The metals (all as chlorides) were added to the incubation mixture to a final concentration of 10"3 M. The solutions were allowed to stand at 4° for 5 minutes before 57 .OIIIo .ommzm .OIIIO .ommzm .Hoapaoo me women was damn peeps mo unwound can :a hpabupom one :.mHmaaopmz use meoSpoz= ad confluence we poaaouaoa macs madame can one poops was oaauno no poSUAHc a4 .mopaaaa u Mom 0: as serum on dozoaam one: moaapwaa on» our oopmoaoaa maodp imapcooaoo Honda can on mohdeaa SoaPSDSoSa on» on poops was «Ham .owmzm use ommzm mo hpabdpom :0 «Sam mo vacuum an onswam 58 auoa IOH N mica a «sea eo apaaeaoz Shea _ mica _ mica _ uloa _ at“... a It 0N 0: .00 om .looa oma Agrariov torquoo go queoxea 59 Table VI Effect of cations on activity Reactions were carried out at pH 5, as described in "Methods and Materials," with cations added as indicated five minutes before adding enzyme. Percent of activity in absence of cation Addition to assay3 DNase RNase 3'-Nucleotidase None 100 100 100 M8C12 92 133 89 Mn012 68 71 76 ZnCl2 76 8 5 88 C0012 54 39 66 CaClz 93 109 91 FeClz 61 56 -- NiClz 58 54 79 CdClzb 9 3 52 NaCl 113 107 80 KCl 115 128 92 NHucl 103 113 91 8The final concentrations of the additi as were 10"3 M except for NaCl. KCl and NHuCl which were 10' M. bThe experiments with Cd++ may not be valid in that obvious precipitates formed in all cases. 60 adding enzyme, incubating the mixture and assaying for enzy- matic activity. The DNase was inhibited to some extent by all the di- valent ions tested, although only Slightly by Mg++ and Ca++. Relatively strong inhibition was obtained with Mn++, Zn++, Co++, Fe++, Ni++, and Cd++. The RNase activity was stimu- lated by Mg++ and Ca++ Mn++ and relatively strongly inhibited by , Co++, Fe++, Ni++, and Cd++. The 3'-nuc1eotidase activ- ity was also relatively strongly inhibited by Mn++, Co++, Ni++, and od+*. The experiments with Cd++ may not be valid in that obvious precipitates formed in all cases. - The addition of 10"2 M KCl, NaCl, or NH#Cl to the incu- bation mixture resulted in a slight stimulation of the DNase and RNase activities. These same salts caused a slight inhibition of the 3'-nuc1eotidase activity. In general the various ions resulted in qualitatively the same changes in rates of hydrolysis for all three activ- ities. The stimulatory effect of Ca++ and Mg++ on the RNase activity may be due to an effect of these metals on the sub- strate and not necessarily an effect on the enzyme itself. Effect of Amino Acid Modifying Reagents The effect of a Series of compounds which are known to react with Specific amino acid residues of a protein was determined with the P-30 enzyme fraction. An aliquot of enzyme in Tris-chloride buffer, 0.025 M, pH 8, 0.1 mM in zinc acetate was treated with a final concentration of 10"3 M modifying reagent and allowed to stand at 4° for 12 hours. 61 The treated enzyme was then assayed for each of the three enzyme activities in the standard manner. Appropriate con- trols were also performed to correct for any effects of the modifying reagents on the various substrates or assay pro- cedures. Table VII presents the data resulting from these experiments. At pH 8 all three enzyme activities were essentially completely destroyed by N-bromosuccinimide. None of the enzyme activities were decreased by treatment with iodoacetic acid, iodoacetamide, N-ethylmaleimide, or sodium arsenite. The enhancement of the RNase activity by all of these compounds is at present not readily SXplain- able 0 Temperature Effect and Heat Inactivation The three activities were found to possess very Similar heat stabilities. The phoSphocellulose fraction was used for this study. This represented enzyme which had been previously heated to 75° during purification. Aliquots of enzyme (0.5 ml) were placed in conical centri- fuge tubes and heated at selected temperatures for 8 minutes. The tubes were then placed in ice-water to achieve rapid cooling. Above 85° all three activities were essentially completely destroyed in 8 minutes. The temper- ature values for 50% reduction of activity for the DNase, RNase, and 3'-nuc1eotidase activities were 76°, 75°, and 75° respectively. Stimulation of all three activities 62 Table VII Effect of some common amino acid modifying reagents on the enzyme activities Samples (0.5 ml) of enzyme in 0.025 Tris-chloride buffer at pH 8, 0.1 mM zinc acetate and 10' M in modifying reagent were left at 4° for 12 hours. The samples were then assayed as described in "Methods and Materials." Percent of original activity remaining Modifying ,reagent DNase RNase 3'-Nucleotidase None 100 100 100 N-bromosuccinimide 1 1 0.5 Iodoacetic acid 101 126 100 N-ethylmaleimide 101 139 101 Iodoacetamide 101 119 100 Sodium arsenite 101 125 100 63 between 65° and 70° was also observed. This may be similar to the temperature effect observed by Eley and Both (45) with the chicken pancreas nuclease. Our results are shown in Table VIII. The results of experiments examining the effect on enzyme activity of the duration of heating at 70° were also found to be very similar for the three enzyme activities (Table IX). The Q10 values for the increase of the three activ- ities in the range of 27° to 47° were determined. The RNase activity exhibited the highest Q10 value of 4.2 to 4.5. The DNase and 3'-nucleotidase activities exhibited values that were somewhat lower, near 3.4 and 2.1 reSpectively. Ratio of Enzype Activities During Purification The ratios of the total RNase and 3'-nucleotidase activities to the activity of the DNase at various stages of the purification procedure are presented in Table X. It is purely fortuitous that these ratios are both near one, since each enzyme activity is calculated in arbitrary units dependent, for example, on incubation time. Clearly, both ratios range only from 1.0 to 1.3 throughout these various procedures. Effect of DNA and RNA on RNase and DNase Activities The following experiment was performed to determine whether the addition of rRNA to the denatured DNA assay would inhibit the DNase activity. Since the rRNA would 64 Table VIII Enzyme activity with reSpect to heat treatment at various temperatures Samples (0.5 ml) of enzyme were heated at the temper- atures indicated for 8 minutes as described in the text. The samples were assayed as described in "Methods and Materi;ls." The control values for unheated enzyme are taken at 100 c Percent of control activity remaining Temperature DNase RNase 3'-Nucleotidase 65° 75 78 64 70° 96 87 92 75° 61 53 51 80° 14 1 7 15 85° 1 0 0 65 Table IX Enzyme activity with reSpect to duration of heating at 70° A sample (2.5 ml) of enzyme in Buffer B was heated in a water bath at 70°. Samples were removed at the times indi- cated and were rapidly cooled in ice-water. These samples were then assayed as described in "Methods and Materials." Percent of zero time activity remaining Minutes at 70° DNase RNase 3'-Nucleotidase 10 70 73 84 20 61 62 71 30 42 44 52 4o 36 35 39 60 18 15 10 90 9 6 0 66 Table X Comparison of the ratios of RNase and 3'-nucleotidase activities to that of DNase after various procedures The ratios are of the Specific activity of DNase to RNase or to 3'-nucleotidase activity, reSpectively. Ratioa Procedure DNase: RNase DNase: 3'-Nucleotidase Ethanol fractionation 1.0 1.1 Heat fractionation 1.2 1.1 PhoSphocellulose (salt gradient) 1.3 1.1 P-30 column 1.1 1.1 P-60 column 1.3 1.0 PhoSphocellulose (pH gradient) 1.0 1.1 DEAE-cellulose, pH 8 1.2 1.1 ECTEOLA-cellulose, pH 6.5 1.0 1.3 Gel electrophoresis 1.1 1.2 aThe average value for these ratios was 1.1 for both the DNase: RNase and DNase: 3'-Nucleotidase. 67 presumably also be hydrolyzed during the DNase assay it was not possible to use the lanthanum nitrate-acid soluble pro- duct assay for this experiment. For this reason the DNase activity was followed by measuring the release of TCA- soluble deoxyribose as determined by the diphenylamine reaction as described in ”Methods and Materials." The release of TCA-soluble deoxyribose material was linear up to 20 minutes as shown in Table XI. During this same period the addition of an amount of rRNA equilivalent to the amount of denatured DNA in the reaction mixture was found to cause approximately 42% inhibition of the DNase activity. After 60 minutes incubation the hydrolysis of denatured DNA in the presence of RNA had reached 99% of the maximum value which was obtained in 30 minutes without added RNA. A Similar eXperiment was performed to determine the effect of added denatured DNA on RNA hydrolysis. In this experiment the release of TCA-soluble ribose measured by the orcinol reaction was used as a measure of RNase activ- ity. The orcinol assay was performed as described in ”Methods and Materials” and the appropriate correction for the orcinol reaction with deoxyribose was made for all values. These results are also Shown in Table XI. It was found that the addition of an equivalent amount of denatured DNA caused a 60 to 70% inhibition of the RNase activity dur- ing the initial stages of rRNA hydrolysis. After 60 minutes incubation the inhibition had dropped to approximately 42%. 68 Table XI Effect of DNA and RNA on RNase and DNase activities DNase and RNase assays were performed in the standard manner with the following exceptions. To the DNase incuba- tion mixture was added an equivalent amount (1 mg) of rRNA and to the RNase mixture was added an equivalent amount (1 mg) of denatured DNA. At the times indicated 1 ml of each reaction mixture was placed in 1.2 m1 of cold 20% TCA. The mixture was allowed to stand at 4° for 15 minutes and then was centrifuged for 20 minutes at 1,100 x g at 4°. Aliquots, 0.2 ml for ribose determination and 1.0 ml for deoxyribose determination, were removed and assayed for TCA-soluble ribose or deoxyribose as described in "Methods and Materials." Control values for the ug of each sugar made TCA-soluble at each period of time in the absence of added nucleic acid are taken as 100%. All values for TCA-soluble ribose in the RNA digests containing DNA were corrected for the contribution of.TCAesoluble deoxyribose to the orcinol color reaction. Effect of RNA on DNase Effect of DNA on RNase Minutes of Percent of Percent of incubation control activity control activity 10 57 31 20 58 36 30 77 41 60 99 58 69 Longer periods of digestion of RNA in the presence of dena- tured DNA were not investigated. Effect of Sulfhydryl Compound and 8 M U323 on Gel Electrophoresis Pattern The inactivation of the three activities by sulfhydryl compounds at pH 8 and the stabilizing effect of Zn'“+ suggested that the enzyme might be composed of two or more subunits. The staining pattern of the proteins after gel electrophoresis was examined for samples of the P-30 fraction which had been treated both with dithiothreitol, and with dithiothreitol and 8 M urea at pH 8. The samples of the P-30 fraction (0.2 ml) were brought to 1 mM in dithiothreitol and solid urea was added to one of the samples to a final concentration of 8 M. These samples were allowed to stand at 4° for 18 hours and then were subjected to electrophoresis on 10% polyacryl- amide gel as described in "Methods and Materials." The gels were stained with Coomassie Blue as described previously. The staining pattern of the sample treated with dithiothreitol at pH 8 showed a marked reduction in the intensity of the band corresponding to the active band in untreated samples. In the sample which was treated with both dithiothreitol and 8 M urea this band was completely absent. The effect of 8 M urea alone was not investigated. iii as fire 1 ievelc the l ‘4 pr: 70 Studies on Enzyme Specificity Activity Toward Native and Denatured DNA One of the initial findings with crude wheat extracts was the relatively high level of activity toward denatured DNA as well as towards native DNA. The purification proce- dure for the wheat nuclease under investigation here was developed to maximize the activity toward denatured DNA. A time course of the hydrolysis of native and denatured DNA by purified wheat preparation is shown in Figure 8. After an initial lag period, the hydrolysis of dena- tured DNA was linear with time. A similar lag period was also evident in the hydrolysis of native DNA. In early stages, the apparent rate of hydrolysis of native DNA was an appreciable fraction of that for denatured DNA-10% in the 15 to 25 minute period. However, the rate of liberation of lanthanum nitrate-acid soluble materials from native DNA decreased with time, so that in the 80 to 100 minute period, the rate was only 1.3% of that observed for the denatured DNA. No decrease in the rate of hydrolysis of the denatured DNA was apparent by the end of the experiment. Controls incu- bated in the absence of enzyme for the same periods of time Showed essentially no increase in lanthanum nitrate-acid soluble material with either native or denatured DNA, thus demonstrating the absence of exogenous nuclease activity at pH 4.5 in the salmon sperm DNA preparation. From these results it appears that the DNase activity is highly prefer- Figure 8: 71 Time course of hydrolysis of denatured and native DNA and of rRNA as measured by the lanthanum nitrate-acid soluble product assay. Each substrate was incu- bated under standard conditions, as described in "Methods and Materials," with 0.3 units of enzyme. Reactions were terminated at the times indicated by placing the reaction mixture in ice- water and then the standard assay pro- cedure was performed. Activity on: denatured DNA, O———O: native DNA, O———O: rRNA. D'—D O 72 4.0 - 300 " A260 1.03 I - j ‘ L + 20 (+0 60 80 100 120 Time (Minutes) 73 ential for denatured DNA, although this preference may not be absolute. Mode of Action on Denatured DNA In order to determine whether the hydrolysis of dena- tured DNA proceeded by an endo- or exonucleolytic mechanism, the following eXperiment was performed according to the method of Birnboim (66). A column, 1.5 x 10 cm, was packed with Bio- Gel P-100 in 0.15 M NaCl, 0.015 M sodium citrate at pH 7 and then washed with about 200 ml of the same solution. The column was attached to an ISCO ultraviolet monitor and a peristaltic pump was employed to regulate the flow rate at 1 ml per minute. A sample containing 500 ug of denatured DNA and 1 mg of 5'-dAMP in 0.5 ml of sodium acetate buffer, 0.05 M, pH 5, was chromatographed on the column. The elution pattern obtained gave the reference positions for denatured DNA and mononucleotides. Denatured DNA was incubated under standard assay conditions with 0.2 units of wheat DNase for various periods of time. At each time period 0.5 ml of reac- tion mixture was transferred to a conical centrifuge tube and heated at 100° for 3 minutes to inactivate the enzyme. The sample was cooled on ice and chromatographed on the P-100 column. The elution patterns at various incubation times are shown in Figure 9a and the standards are shown in Figure 9b. A Similar experiment was performed with snake venom phoSpho- diesterase (Cortalus adamanteus venom) to determine the elu- tion pattern from a known exonuclease. The incubations were the same as above except 50 ug of phosphodiesterase was added Figure 9: a. C. 74 Chromatography on P-100 of denatured DNA at various stages in its diges- tion by wheat DNase. Denatured DNA was incubated under standard assay conditions with 0.2 units of wheat DNase at 37°. Aliquots (0.5 ml) were removed at the times indicated and placed in a boiling water bath for 3 minutes. The sample was then cooled and chromatographed on a P-100 column, 1.5 x 10 cm, as described in "Methods and Materials." The elution patterns shown are for incubation periods of a) 0 minutes; b) 3 minutes; c) 6 minutes; d) 20 minutes. The arrow marks the position of the known mononucleotide, dAMP. Chromatography on P-100 of a mixture of denatured DNA (dDNA) and mononuc- leotide (dAMP). Chromatography on P-100 of denatured DNA at various stages in its diges- tion by snake venom phOSphodiesterase, a known exonuclease. Incubation con- ditions were the same as above except that the buffer was 0.05 M Tris- chloride, pH 8.8, 0.03 M in MgCl and 50 ug of snake venom phOSphodies er- ase was added to the reaction mixture. At the times indicated samples (0.5 ml) were removed and placed in a boil- ing water bath for 3 minutes, then cooled and chromatographed on the P-100 column. The elution patterns Shown are for incubation periods of a) 0 minutes; b) 1 minute: 0) 10 minutes; d) 60 minutes. The arrow marks the position of the known mono- nucleotide, dAMP. 75 a 0.8 — A 0 l ' 10 15 20 Volume (ml) ' ' b 0.8 a 1’ A260 001+ *— 0 I 20 Volume (ml) 0.8 .. a ‘1’ ° A . 2600.,+ _ A 0 ‘ n \ l 5 10 15 20 Volume (ml) 76 instead of the wheat enzyme and longer incubation periods were required. The elution patterns obtained for venom phosphodiesterase hydrolysis of denatured DNA are shown in Figure 9c. The decrease in the height of the denatured DNA peak and the appearance of a mononucleotide peak is exactly what would be expected from an exonuclease producing mainly mononucleotides. In contrast the patterns from the wheat enzyme digest shows a decrease in the height of the dena- tured DNA peak and broadening of the peak in intermediate stages. This is what would be eXpected and is reported (66) as the pattern for an endonuclease which produces a variety of oligonucleotide fragments of all different sizes. The DEAE assay procedure was also employed to study the mechanism of action of the DNase activity on denatured DNA. This procedure, as described in "Methods and Materialsfl, should distinguish between an exo- and endonucleolytic attack on denatured DNA. Assuming that the 0.1 M salt wash removes mainly mononucleotides and short oligonucleotides from the DEAE, then for an exonuclease these two washes should give Similar A260 values during the initial stages of hydrolysis. For an endonuclease the absorbency of the 0.3 M wash should be much higher than the 0.1 M wash, since mainly larger oligonucleotides are being formed. Figure 10 shows a time course of the hydrolysis of denatured DNA by the wheat DNase as measured by the DEAE assay. After an initial lag the absorbency of the 0.3 M wash was linear with time up to an A260 of about 2.0 which represents about 60% hydrolysis of 77 Figure 10: Time course of hydrolysis of denatured DNA by wheat DNase and snake venom phos- phodiesterase as measured by the DEAE assay. Samples were incubated under standard conditions with 0.5 unit of wheat DNase or in 0.05 M Tris-chloride buffer, pH 8.8, 0.03 M MgCl2 with 50 ug of snake venom phosphodiesterase. The curves for both salt washes (see "Methods and Materials") are shown: hydrolysis by the wheat DNase, 0.1 M NaCl wash, 0———O: 0.3 M NaCl wash, O———O; hydrolysis by venom phOSphodiesterase, 0.1 M NaCl wash.D-—CI: 0.3 M NaCl wash, Ih-—-II. 78 3.0J 2.4- 1.8 “ 260 1.2-' ‘ A . 0.6 5 0 - , ' , . . n 10 20 30 40 50 60 Time (Minutes) 79 the DNA. The 0.1 M wash exhibited a longer lag period and its absorbency was lower than that of the 0.3 M wash curve at all incubation times, approaching it on long incubation. Figure 10 also shows a Similar time course of hydrolysis of denatured DNA by snake venom phoSphodiesterase. As would be expected for an exonuclease the two salt washes gave almost identical curves. The production of a large proportion of oligonucleotides throughout the digestion is taken as evi- dence for the endonucleolytic cleavage of denatured DNA by the wheat enzyme. To further substantiate this a large scale digest of denatured DNA was chromatographed on a DEAE-Sephadex A-25 column (3 x 67 cm) as described in "Methods and Materials." The digest, containing 500 mg of denatured DNA in 500 ml of 0.05 M sodium acetate buffer, pH 5.0, 1.mM in zinc acetate, . 1 mM in dithiothreitol, was incubated with 100 units of wheat.DNase at 37° for 5 hours. The digestion was stopped by cooling in ice water and 28 ml of 0.1 M EDTA was added to give a final concentration of about 5 mM EDTA. This addi- tion of EDTA was necessary to prevent the precipitation of zinc hydroxide when the solution was neutralized. To the solution was added 23.2 ml of 2 M triethylammonium bicarbon- ate and the pH was adjusted to 8.0 with 2 N NHuOH. The digest was then applied to the DEAE-Sephadex column at a flow rate of 1 ml per minute. The column was extensively washed with 0.14 M triethylammonium bicarbonate at pH 8.0. A linear gradient from 0.14 to 0.3 M triethylammonium bicarbonate 80 was applied to the column, the total volume of the gradient was 4 liters. A second linear gradient from 0.3 to 0.46 M triethylammonium bicarbonate was then applied with a total volume of 8 liters. At the end of this gradient, the column was washed with 1 M triethylammonium bicarbonate. Figure 11 shows the elution pattern from the DEAE-Sephadex column. Peaks Ia' Ib and Ic are mononucleotides and described in "Methods and Materials." Peak II contains a mixture of at least 8 dinucleotides which were separated on paper chroma- tography in the 2 dimensional solvent system as described in ”Methods and Materials." The peaks following peak II are assumed to be longer oligonucleotides in the order tri-, tetra-, pentanucleotide etc., as previously reported for this system (70). Assay of the digest chromatographed here revealed that the digestion had proceeded to about 75% con- version of the denatured DNA to lanthanum nitrate-acid soluble material. A similar SXperiment in which the digest was chromatographed on DEAE-Sephadex after only 1 hour's incubation gave a similar elution pattern. However, the proportion of material in the higher oligonucleotide peaks was much greater. No P1 or nucleosides were observed in either the 1 or the 5 hour digestion of denatured DNA. Thus the wheat DNase produced, as would be expected of an endo- nuclease, a variety of oligonucleotides of varying chain length in the hydrolysis of denatured DNA. 81 .haobapooamoa mace can mz4c confidence 0H can pH scum .mzoo new mass eosaoeSoo mH seem .eoeooaaoo caos as om haopmadwoaaam mo mnoapomam one mesa edpflmpmdnoa a no one can Spas ooadupaama mes opaaaa won as H no open soda 4 .maopdd m mo oasaob Hmpop m :a m4me z w:.o op m.o aoam unoaomam amoada enooom m an oosoHHoa was many .mhopaa : mes paedemaw can no caaaob Hmpop can ”sadaoo on» on peaaaam mes m4me z m.o op :a.o soak paoaomaw Hammad 4 .m4ma z ea.o mo Ronda m and: eczema mes sasaoo on» use SSSHoo on» on omaaaam was =.mamaaopmz one mooSpozs ad confluence mm .omozn amass mo nudes ooa and: Ame oonv 42m uoHSpSSSU mo pmowao ado: m 4 .m me be “messy opmaonacoap seasosscaanpcaap z ea.o spas scenes one ooxoma mes mmi4 Novenammlmdma mo .80 no N m .sasaoo 4 .ommzn uses: an 429 poaaumaoo no mdmhfioaphs on» wadadc dcofidoaa mepapooaoaa Iowaao one Iosoa mo sasaoo mmi4 Noemaaomlm4mn a Scam savanna Soapsam ”Ha enemas 82 avsm w I'---—>Vfi“\\ (w) norqelquaouoo svsm amaaaz Soapocam co: omm com omm HHH oa.cv D CON HH oma 00H b 3 om LL To ro.H as ea as... ho m .o.oa vo.om O9Zv 83 Determination of_§hoSphate Position in Mononucleotides Obtained fropgDenatured DNA Hydrolysis The following eXperiment was performed to determine whether the mononucleotides produced on hydrolysis of dena- tured DNA were the 3'- or 5'-isomers. Samples containing either 10 mg of material from the dAMP or 3 mg of material from the dCMP: dTMP peak eluted from the DEAE-Sephadex column (see next section) were prepared in 0.08 M Tris- chloride buffer, pH 8.2, 0.008 M in MgClz. Aliquots of these solutions were treated with approximately 5 units of either 1:}. pp}; alkaline phoSphatase, snake venom 5'-nucleotidase or rye grass 3'-nucleotidase for 90 minutes at 37°. The samples were then assayed for P1 as described in "Methods and Materials" for the 3'-nucleotidase assay. Standard 3'- and 5'-AMP (ribo-) were also used as controls. Table XII shows the data from this experiment. The values are expressed as the percentage of P1 released by alkaline phoSphatase assum- ing that the enzyme would cleave all the phoSphomonoester bonds present regardless of their position in the mononucleo- tide. Thus 5'-nucleotidase was observed to release essen- tially the same amount of P1, if not more, from the mononucleo- tides of the DNA digest as was released by alkaline phOSpha- tase. The 3'-nucleotidase released only about 1% of the P1 from dAMP and about 9% from the dCMP: dTMP mixture. Both nucleotidases were found to release about 1.6% of the P1 from the opposite nucleotide. This probably was caused by slight contamination of these enzymes by non-Specific phOSphatase 84 Table XII Identification of phoSphate position in mononucleotides from the hydrolysis of denatured DNA Samples of the dAMP and dCMP: dTMP mixture separated from a 5 hour digest of denatured DNA with wheat DNase were treated with 5 units of either alkaline phOSphatase, 3'- nucleotidase, or 5'-nucleotidase at 37° for 90 minutes. The samples were then assayed for P as described in "Methods and Materials.“ The values for the percent of P liberated by each nucleotidase are based on 100% for alkaline phoSpha- tase, assuming that this enzyme hydrolyzed all of the phos- phomonoester bonds. P_i Percent of P1 released by alkaline phosPhatase Substrate 5'-Nucleotidase 3'-Nucleotidase dAMP 118 1.1 dCMP:dTMP 99 8.7 Known 5'nAMP 107 1.6 Known 3'qAMP 1.6 102 85 activity. Approximately 9% of the Pi was released from the dCMP: dTMP by 3'-nucleotidase, however, 5'-nucleotidase released 99% of the P1. The cause of the apparent discrep- ancy between these two figures is not clear. It is apparent that at least the majority, if not all. of the mononucleo- tides bear a 5'-phoSphoryl group. It was not possible to examine the deoxyguanylate because of the relatively low level of production of this mononucleotide during the hydrolysis. Thus, the wheat DNase appears to produce 5'- mononucleotides on hydrolysis of denatured and it is tacitly assumed that the oligonucleotides bear 5'-termini as well. Mononucleotide Production from Denatured DNA at Various Stages of Digestion AS mentioned previously it was observed on chromatog- raphy of the digestion products from denatured DNA hydrolysis that a very small amount of deoxyguanylate was present in the digests. This observation prompted the following SXperiment to determine the relative proportion of the 4 mononucleotides at different stages of digestion. Solutions containing 40 mg of denatured DNA in 0.05 M sodium acetate buffer, pH 4.5, 1 mM in zinc acetate, 1 mM in dithiothreitol were incubated with 40 units of wheat DNase at 37° for 15. 30, 60 and 300 minutes. At the end of the incubation period each solution was cooled in ice-water and 2.5 m1 of 0.1 M EDTA was added. The solution was allowed to stand for 5 minutes at 4° and 1.3 m1 of 2 M triethylammonium bicarbonate was added and the pH was adjusted to 8.0 with 2 N NHMOH. This material was then 86 applied to a DEAE-Sephadex column, 2.2 x 15 cm, and a batch- wise treatment with 0.18 M triethylammonium bicarbonate was performed as described in “Methods and Materials." Table XIII contains the data from a typical experiment. Since dCMP and dTMP appear in the same peak it was necessary to determine the concentration of these compounds by a Spectro- photometric analysis of the binary mixture as described in "Methods and Materials.” At all periods of digestion inves- tigated dAMP was the most prevalent mononucleotide. The level of dAMP was always about 2-fold greater than any of the other 3 deoxynucleotides. The level of dAMP was increased throughout the digestion while the levels of dTMP and dCMP remained essentially constant at all early stages of digestion. In contrast, the level of dGMP decreased from about 24% of the total mononucleotide fraction at 15 minutes to only 5% at 60 minutes and to only about 2% after 300 minutes. It should be noted that these values are the percentage of the total mononucleotide fraction represented by each mononucleotide. Thus, while dGMP decreased in per- cent of the total mononucleotide fraction the actual number of umoles of all mononucleotides, including dGMP, increased at all periods of digestion. Since the recovery of known dGMP solutions from the DEAE-Sephadex column is approximately 100 percent (see “Methods and Materials") it is felt that this low level of dGMP is real and not an artifact of the separation procedure. Furthermore, on total base analysis of all the peaks obtained from the DEAE-Sephadex column .429 UmHfipdflmd 0» mhmhmh dzadm m mm, mm m: a.mm o.~s com m ca mm Ho m.m m.sm on Na NH em on e.m m.mm on em ma em or m.a m.aa ma mace mace mzae mz4o mooapooaoaSonoa mm c4zmo mo SoapSQSosd aposooaa Hmpop mammaoaoha mo mopssdz mo paooaom peooaom mopapooaoSSoSoa Have» no pnooaom mm Goapnndhpmdd oddPOOHoaz 7. iii: hr 14H 8 .ooaaoa ofiap.£omo pm pmosuoaa evapooaoaa some no moaoan map so momma was mcpapooaoasoaoa How menace owSHSooaom =.mHmdaopmz one meonpoz: ad confluence mm m4me z ma.o and: mnaaaoo 0:» no unmapmonp omdslaopmn m an consumaom cams mopapooaoasonoa was .mdma z H.o Spas commas one: massaoo on» use .80 ma N N.N .mmi4.wopm£9omtm4mm ho massaoo on pedamam Soap macs moaaadm one .m on dopmsncm mes ma one .eoopm mes Am4mav ensconamoan ESHSoaamHmapodap z N no He m.H can 499m 2 H.o mo Ha m.~ one wSaHooo an popoaaaaop was Soapomoa one .mmpssda oom one om .om .ma non ohm pm ommzm pubs: mo nudes o: Spas oopprosa ohms HopaoHSpoanpdo ad :8 a .opdpood ends a« SE a m.: ma .Homman oudpoom aaadom z no.0 SH 429 UoHSpSSoU no ma 0: waaaampSoo maoapaaom 42a ooHSpmSoe mo Sodpmowao on» ad mowopm maoaas> pm Soapomaa evapooaosSoSoa on» no Soapamoaaoo HHHM manna 88 described previously it was possible to account for approxi- mately 100 percent of the deoxyguanylate residues in the original denatured DNA. Thus it appears that the remaining deoxyguanylate residues are contained in the longer oligo- nucleotides. Activity Towards rRNA A time course of rRNA hydrolysis by the wheat RNase is shown in Figure 8. After an initial lag the reaction proceeds in a linear manner. In order to gain insight into the mode of action of the wheat RNase on rRNA a gel filtration experiment similar to that described above for denatured DNA was performed. A column, 1.5 x 25 cm, of Bio-Gel P-6 was packed and washed as described for the P-100 column. It was necessary to use P-6 Bio-Gel for this eXperiment instead of P-100 because of the lower molecular weight of the rRNA. The rRNA was incu- bated in 0.05 M sodium acetate buffer, pH 4.5, 1 mM in d1- thiothreitol, 1 mM in zinc acetate, with 0.6 units of enzyme for various periods of time. At each time interval an 0.4 ml sample was removed and heated to 100° for 3 minutes to inactivate the enzyme. This sample was then chromatographed on the P-6 column. The elution pattern at various stages of digestion is shown in Figure 12b. This is the type of pat- tern that one would expect from an exonuclease and was, indeed, the type of pattern observed with venom phoSphodi- esterase on denatured DNA (see Figure 9c). The elution Figure 12: a. 89 Chromatography on P-6 of a mixture of rRNA and mononucleotide, AMP. Chromatography of P-6 of rRNA at various stages in its digestion by wheat RNase. Incubations were per- formed under standard conditions with 0.6 units of enzyme. Aliquots (0.4 ml) were removed at the times indicated and placed in a boiling water bath for 3 minutes. The samples were then cooled and chro- matographed as above. The elution patterns Shown are for incubation periods of a) 0 minutes: b) 15 minutes; 0) 30 minutes; d) 60 minutes. The arrow indicates the position of the known mononucleotide, AMP. 90 0.8 '- i/ A rRNA 260 2’ 0.4 ‘ AMP ./ 4///’.\\\f/ l J 4 l 0 5 10' 15 20 Volume (ml) 0.8 .. a \i A260 0014' ;_ C, Volume (m1) 91 profile of rRNA and mononucleotide (AMP) standards are shown in Figure 12a. Thus it would appear that the wheat RNase activity acts predominately in an exonucleolytic manner on rRNA. Effect of Zinc Ion on rRNA Hydrolysis The following SXperiment was performed to determine the relative percent of lanthanum nitrate-acid soluble material which was dialyzable. Ribosomal RNA (2 ml) was hydrolyzed under normal assay conditions with 2.5 units of wheat RNase for 30 minutes in a total volume of 3 ml. The reaction was terminated by the addition of 3 ml of cold lanthanum nitrate-RC1 reagent. The mixture was allowed to sit at 4° for 10 minutes and then was centrifuged for 20 minutes at 4°. To the clear supernatant fluid containing the lanthanum nitrate-acid soluble digestion products was added 1.0 m1 of 0.1 M EDTA and the pH was adjusted to approx- imately 6 with 2 N NH40H. .A sample (5 ml) of the neutralized supernatant fluid was placed in "small pore" dialysis tubing (#23) and dialyzed against 30 ml of distilled water at 4° for 5 hours. .A control containing the 4 known deoxymononucleo- tides which had been taken through the lanthanum nitrate-H01 treatment was also dialyzed to determine when dialysis equi- librium had been achieved. The A260 of the dialysis fluid was determined and the percentage of the lanthanum nitrate- acid soluble products which were dialyzable was calculated. The results of this experiment were found to be different in the presence and absence of zinc. These results are shown 92 in Table XIV. In the absence of Zn'"+ approximately 50% of the lanthanum nitrate-acid soluble material was dialyzable. In the presence of 1 mM ZnI+, however, 80% of the lanthanum nitrate-acid soluble material produced in the same period of incubation was dialyzable. Because of the inhibitory effect of ZnI+, this sample reached only about 85% of the level of hydrolysis obtained without Zn++. Thus it would appear that while a lower level of hydrolysis is obtained in the presence of ZnI+, a larger percentage of the hydrol- ysis products are dialyzable and presumably are mononucleo- tidGSO Identification of Mononuglpotides Produced from rRNA Digestion A total of 10 mg of rRNA was digested with 10 units of wheat RNase at 37° for 5 hours. The mononucleotides were separated from the digestion mixture on DEAE-Sephadex by a batch-wise elution with 0.18 M triethylammonium bicarbonate as described in "Methods and Materials." All 4 ribonucleo- tides were present in about the same amounts in the digest. The mononucleotides were then treated with 5'- and 3'-nuc- leotidase as described previously for the mononucleotides from the DNA digest. Aliquots of the nucleotidase treated material were spotted on Whatman 3 mm paper and chromato- graphed in solvent I as described in "Methods and Materials." No conversion of any of the mononucleotides to the corres- ponding nucleosides was observed with either 5'- or 3'- nucleotidase as determined by their position on the chromato- 93 Table XIV Effect of Zn'l'+ on the hydrolysis of rRNA by wheat RNase Samples of rRNA (2 mg) were incubated in a volume of 3 ml with 2.5 units of wheat RNase for 30 minutes at pH 4.5 in the presence and absence of 1 mM Zn++. The reaction was terminated by the addition of 3 ml of cold lanthanum nitrate- HCl reagent. The mixture was allowed to stand at 4° for 10 minutes and then was centrifuged at 4° for 20 minutes at 1,100 x g. To the supernatant fluid was added 1 ml of 0.1 M EDTA and the pH was adjusted to 6. Samples (5 ml) were placed in "Small poreu dialysis tubing and dialyzed against 30 ml of distilled water for 5 hours at 4°. The A260 of the dia- lysis fluid was determined. Molariiy Percent of hydrolysis Percent of products of Zn without Zn++ dialyzable Controla --- 100 O 100 50 1 mM 85 80 aSolution containing the 4 known deoxymononucleotides. 94 gram. Treatment of the mononucleotides with alkaline phOSpha- tase and chromatography in this solvent system revealed a conversion of the purine mononucleotides to nucleosides but was without effect on the pyrimidine mononucleotides. These results suggested that the RNase forms 2',3'-cyclic mono- nucleotides as hydrolysis products, and then converts the cyclic purine nucleotides to the 2'-mononucleotides. EXperi- ments were performed using the homo-polymer oligo-uridylate as substrate to see if cyclic-UMP could be identified as the mononucleotide produced. A.solution containing 2.5 mg of oligo-uridylate in 0.25 ml of 0.05 M sodium acetate buffer, pH 5, 2 mM in cysteine was incubated with 2 units of RNase at 37°. At zero, 15, 30 and 60 minutes an aliquot (50 ul) was removed and placed in a boiling water bath for 3 minutes to inactivate the enzyme. The sample was then Spotted on DEAE-cellulose chromatography paper (DE-81) along with appropriate standards and chromatographed in 0.1 M ammonium formate solvent for 4 hours. At all stages of incubation the predominate mononucleotide component had an Rf value of 0.55. The Rf values in this solvent system for known 2',3'- cyclic-UMP, 2'-UMP and 3'-UMP were 0.54, 0.29 and 0.28 reSpectively. Thus it was tentatively concluded that the enzyme produced predominately 2',3'-cyclic-UMP upon diges- tion of oligo-uridylate. A small amount of material was also present at a position approximately that of 2'- or 3'-UMP. As a further verification of the production of cyclic- uridylate, a sample of oligo-uridylate was hydrolyzed as 95 described above for a total of 120 minutes with 2 units of wheat RNase. The digestion mixture was Split into 2 por- tions. One portion was taken to dryness in a rotary flash evaporator. The material was then dissolved in 20 pl of 80% acetic acid and allowed to stand at room temperature (about 25°) for 2 hours. The acetic acid digest was then flashed to dryness and suspended in 20 ul of water and taken to dryness. This process was repeated 3 times. The sample was finally dissolved in 20 ul of 95% ethanol and flashed to dryness to remove any residual traces of acetic acid. The material was dissolved in 10 ul of water and Spotted on DEAE-cellulose paper. This hydrolysis condi- tion, 80% acetic acid at room temperature for 2 hours, should have been sufficient to convert the 2',3'-cyclic- UMP to a mixture of 2'- and 3'-UMP but should not have hydrolyzed any oligonucleotides such as UpU or UpUp etc.1+ As controls, both known 2',3'—cyclic-UMP and oligo-UMP were hydrolyzed with 80% acetic acid and subsequently treated in the same manner as the enzyme digest. The 120 minute digest which was not hydrolyzed with 80% acetic acid showed a pre- dominate Spot at the 2',3'-cyclic-UMP position as expected. In the corresponding sample which was subject to 80% acetic acid hydrolysis, the 2',3'-cyclic-UMP spot was essentially absent and the bulk of material now chromatographed in the same position as 2'- and 3'-UMP. The known 2',3'-cyclic-UMP standard which was hydrolyzed with 80% acetic acid had like- “F. M. Rottman, personal communication. 96 wise been converted to 2'- and 3'-UMP. The chromatographic pattern of oligo-uridylate which had been subjected to 80% acetic acid hydrolysis was essentially the same as the pat- tern of unhydrolyzed material. This indicates that the oligonucleotide material was not affected by 80% acetic acid hydrolysis. DISCUSSION The wheat DNase under investigation has been purified approximately 830-fold with a recovery of about 12% of the total DNase activity present in the crude homogenate. Some enzyme preparations have been obtained which were approxi- mately 1000-fold purified with a recovery of about 20% of the total activity. The assays of the ammonium sulfate fraction were always quite low because of retention of ammonium sulfate which is inhibitory to the enzyme activities. Assays of the ethanol fraction were also poorly reproducible. This fraction routinely gave high values and it was found that the addition of a small amount of ethanol to the puri- fied enzyme caused a slight but significant increase in activity. The crude homogenate contains an appreciable level of activity towards 5'4AMP and bis-p-nitrophenyl phOSphate. Both of these activities are essentially completely removed by the heat treatment at 75°. Thus while the specific activ- ity of the DNase activity decreased during this purification step, this step was necessary for the removal of at least two of the major contaminating enzyme activities from the enzyme preparation. Chromatography on the phoSphocellulose column removed about 85% of the protein in the pass through and wash fractions. However, these fractions exhibited only slight enzyme activity. The recovery of DNase activity from the 97 98 phOSphocellulose column was about 50% of that applied to the column. In some preparations recoveries as high as 70% have been obtained at this step while the recovery of protein was always about the same. The use of the combination column of phoSphocellulose and P-30 was a very beneficial procedure. The enzyme activities were obtained in a volume only one twelfth of that applied to the column, an increase in the total units of all three activities was observed, and the material was completely free of salt. The latter eliminates the need for the dialysis of the phosPhocellulose fraction, a procedure which resulted in a net loss in enzyme activity. Further concentration of the P-30 fraction was most rapidly achieved by flash evaporation of the pooled fractions at 28° and subsequent dialysis of the concentrate to bring the buffer concentration down to 0.05 M. Dialysis against car- bowax and lyophilization were also attempted. Dialysis against 30% carbowax in Buffer B was an effective means of concentration, however, the procedure was slower and material from the carbowax which dialyzed into the enzyme solution could not be removed by subsequent dialysis against Buffer B alone. This material strongly interfered with the Lowry protein determination. Lyophilization repeatedly gave poor recoveries of enzyme activity. It was necessary to carry the enzyme preparation through the heat step within about 12 hours after the initial homo- genization. The activity in the cruder fractions was very instable, however, these fractions could be stabilized for 99 several days by making them 3 M in urea. This treatment appeared to prevent the aggregation and precipitation of the wheat proteins as is consistent with the findings of Wu‘gp 2;. (43) on the effect of 3 M urea on wheat proteins. This procedure was not routinely employed, however, because of problems caused by the presence of urea in subsequent puri- fication steps. The point of sulfhydryl addition to the enzyme prep- aration was found to be quite critical. The sulfhydryl com- pound was routinely added to the enzyme preparation after the ethanol fractionation and before the heat treatment. Addition of sulfhydryl to any step prior to the ethanol fractionation resulted in a complete loss of enzyme activity within a few hours at 4°. This was presumably caused by the activation of some sulfhydryl requiring protease(s) in the cruder fractions which was finally removed by the ethanol fractionation. After the heat step the three enzyme activ- ities showed an absolute requirement for the presence of a sulfhydryl compound at pH 4.5. The enzyme preparation exhibits no detectable hydrol- ysis of 5'-AMP, bis-p-nitrophenyl phosphate, 5'-p-nitro- phenyl thymidylate, or 5'-p-nitrophenyl adenylate. A very low level of hydrolysis of p-nitrophenyl phoSphate and of 3'-p-nitropheny1 thymidylate was observed. These activities were assayed with a 10-fold greater level of enzyme and for 24 hours at 37° which is a 144-fold excess of time over the normal DNase assay conditions. Thus it would appear that 100 the purified wheat enzyme preparation contains little, if any, contaminating enzyme activities. The lanthanum nitrateaacid soluble product assay was found to be a relatively rapid and reproducible assay for the hydrolysis of both denatured and native DNA and of rRNA. The fact that the rates of hydrolysis of both denatured DNA and RNA were not proportional to enzyme concentration at high and low concentrations was somewhat of a problem. This is similar to the results obtained by Curtiss, Burdon, and Smellie (71) with the rat liver DNase and to the results obtained with other nucleases. This problem could be effec- tively overcome, however, by always choosing an aliquot of enzyme which gave values in the range of 1.0 to 2.0 A260 units which was the most nearly linear region of the curve. Values in this region were relatively proportional to each other and were found to be quite reproducible. The DEAE- cellulose assay is rather advantageous because it can dis- tinguish between exo- and endonuclease activities. The assay is fairly rapid and quite reproducible. However, because this assay is dependent on the binding of oligonuc- leotides to the resin, any appreciable change in the pH or ionic strength of the sample will greatly effect the results. For this reason the assay can only be used for a given, well defined set of assay conditions. The ability of the enzyme preparation to hydrolyze both denatured DNA and RNA is not surprising by itself, since many purified nucleases exhibit no Specificity towards the 101 sugar moiety of the substrate and thus hydrolyze both DNA and RNA. The associated 3'-nucleotidase activity could also simply be an additional activity of a non-Specific nuclease. However, SXperiments on the mode of action of the wheat enzyme on denatured DNA and rRNA indicate that the hydroly- sis proceeds by different mechanisms for these two substrates. The mononucleotides produced on hydrolysis of denatured DNA are exclusively 5'-mononucleotides, while those produced on hydrolysis of rRNA appear to be mainly the 2',3'-cyclic com- pounds. The mononucleotides isolated from a digest of dena- tured DNA were subjected to treatment with alkaline phOSpha- tase, snake venom 5'-nucleotidase, and rye grass 3'-nucleo- tidase. The 5'-nucleotidase released essentially the same amount, if not more, of P1 from the mononucleotides as did alkaline phoSphatase. The 3'-nucleotidase released only a small amount of P1 from the same mononucleotides. Thus the mononucleotides produced on digestion of denatured DNA by wheat DNase are predominately, if not exclusively, 5'-mono- nucleotides. The mononucleotides produced from rRNA diges- tion were found to be resistant to both 3'- and 5'-nucleo- tidase. Chromatography on DEAE-cellulose paper in 0.1 M ammonium formate of a digest of oligo-uridylate showed that 2',3'-cyclic-UMP was produced by the wheat RNase. This was further substantiated by conversion of the cyclic-UMP to a mixture of 2'- and 3'-UMP on hydrolysis with 80% acetic acid at room temperature. Under the hydrolysis conditions employed, the oligo-uridylate showed no detectable hydrolysis. 102 Thus the wheat RNase forms 2',3'-cyclic-UMP on hydrolysis of oligo-uridylate and it is tacitly assumed that the mononuc- leotides produced from rRNA are also the 2',3'-cyclic com- pounds. The observation that alkaline phOSphatase, which does not hydrolyze 2',3'-cyclic mononucleotides, caused con- version of the purine nucleotides to the correSponding nucleosides is of interest. It is possible that the wheat RNase forms 2',3'-cyclic mononucleotides initially and then in a second reaction converts the purine nucleotides to the currSSponding 2'-mononucleotides. These would then be hydrolyzed by alkaline phoSphatase as was observed. If, indeed, this were the case the 3'-nucleotidase activity of wheat may simply represent the second stage of this reac- tion in which the 3'-phoSphoester bond is cleaved. Since in the 3'-mononucleotide the phosphoryl group is not attached to the 2' position the phosphoryl group would be removed instead of transfered to the 2' position as would occur with the 2',3'-cyclic mononucleotide. This type of activity has been reported by Mcleod and Huang (72) for a ribonuclease from mouse lymphosarcoma. It also appears that while the wheat enzyme attacks denatured DNA.primarily, if not exclusively, in an endonuc- leolytic manner, the mode of attack on rRNA is primarily exonucleolytic. The results of the gel filtration elution patterns on P-100 indicate a predominately endonucleolytic attack on denatured DNA. The pattern obtained was that of an endonuclease which produces a variety of oligonucleotides 103 of various sizes. This was further substantiated by the results of the DEAE assay which distinguishes between endo- and exonuclease type activities. The curve for the 0.3 M NaCl wash (oligonucleotides) was much higher than that of the 0.1 M NaCl wash (mononucleotides) at all periods of incubation. When snake venom phOSphodiesterase, a known exonuclease, was assayed by this procedure the two curves coincided throughout the incubation as would be expected since only mononucleotides were being produced by this enzyme. The distribution of oliognucleotides on DEAE- Sephadex chromatography of the digestion mixtures also indicates that a wide variety of oligonucleotides are pro- duced from denatured DNA by the wheat DNase. This plus the fact that at earlier periods of incubation a larger propor- tion of the material was present in the higher olignonucleo- tide peaks is taken as further proof of the endonucleolytic action of wheat DNase on denatured DNA. The gel filtration elution patterns of digests of rRNA indicate that RNA hydrolysis in the presence of Zn++ proceeds primarily in an exonucleolytic manner. With this gel filtration procedure even very low levels of endonuc- lease activity would cause a detectable Spreading of the RNA peak before the mononucleotide peak became very evident. Even after considerable hydrolysis of the RNA had occurred there was still a good separation between the RNA and mono- nucleotide peaks. This indicates that few, if any, small oligonucleotides were produced. The dialysis eXperiment 104 also substantiates the exonucleolytic action of the wheat RNase. In the presence of 1 mM Zn+* about 80% of the lan- thanum nitrate-acid soluble products from rRNA hydrolysis were dialyzable in 5 hours at 4°. When this same eXperi- ment was performed in the absence of Zn++ only 50% of the lanthanum nitrate-acid soluble material was dialyzable in the same period of time. Since Zn++ inhibits the RNase to a certain extent (about 15%) the digestion in the presence of zinc had only proceeded to about 85% of that observed in the absence of zinc, however, 30% more of the products were found to be dialyzable in the zinc digest. Thus it seems likely that Zn++ may play a role in determining the mode of attack of wheat RNase on rRNA. A similar effect of Zn++ was observed by Walters and Loring (23) with the mung bean RNase. It is also possible that the purified RNase contains a low level of a contaminating endo-ribonuclease activity which is inhibited by Zn++. Thus in the presence of Zn++ only the exonuclease activity is observed while in the absence of Zn++ both endo- and exonuclease activities are observed. The difference in the mode of action alone might sug- gest that at least two distinct enzymes are present in the preparation. But the fact that all attempts to separate the DNase and RNase activities and the associated 3'-nucleotidase activity have been uniformly unsuccessful, together with the extreme similarity in the properties of the three activities, as described below, lead us to suspect that the three enzyme 105 activities reside in a single entity. This close association of DNase, RNase and 3'-nucleotidase activities appears to be similar to that observed with several other plant nucleases. The three associated activities of mung bean sprouts have been extensively studied (20-24), as have the activities of germinating barley (17, 29), ryegrass (18), and rice bran (25). This association may also exist for the enzymes from soybean (19) and the fraction III nuclease of germinating garlic (26). Walters and Loring (23) have concluded that the mung bean DNase and RNase-3'-nucleotidase activities reside in different proteins, since the DNase activity alone was lost on standing in the cold. It is also possible that this represents the differential alteration of the structure of a protein with two different active Sites. Work in this 5 laboratory on muskmelon seed extracts has produced an enzyme fraction, purified several hundred fold, which also possesses DNase, RNase and 3'-nucleotidase activities. The three wheat enzyme activities cochromatographed on phoSphocellulose at pH 4.5 regardless of whether a salt or pH gradient was employed for the elution of the protein. Thus the three activities remained inseparable when two different elution parameters were applied to the same column. The three wheat enzyme activities also cochromatographed on DEAE- cellulose at pH 8, on ECTEOLA-cellulose at pH 6.5, and on passage through either P-60 or P-30 gel filtration columns. 5A. B. Adams, L. D. Muschek, and J. L. Fairley, unpub- lished observations. 106 The elution pattern from the DEAE-cellulose column at pH 8 showed a rather long tail of enzyme activity on the higher salt Side of the peak. The ratios of the three activities were essentially constant throughout this tail as well as throughout the main body of the peak itself. The behavior of the three activities subjected to a batch treatment with a total of 8 different ion-exchange resins and 2 adsorbents at pH 4.5, 6.5, and 8 was also identical. The three activ- ities either did not bind to the resin or bound to relatively the same degree at each pH employed. Disc electrophoresis of the purified nuclease on 10% polyacrylamide gel at both pH 9.5 and 8.3 produced a number of protein bands, however, the three enzyme activities were located only in one of these. The ratios of the three activities throughout this band were constant. Similarly the ratio of the DNase to RNase and DNase to 3'-nucleotidase activity remained essen- tially constant from the ethanol fractionation step through- out the remainder of the purification procedure. These ratios were also constant throughout the various chromato- graphic and electrophoresis procedures described above. The fact that these ratios remain so constant throughout so many different procedures is rather strong evidence that the three enzyme activities reside in a single unit of some sort. Further evidence for the assignment of the three enzyme activities to a single entity may be summarized as follows: (a) All three enzyme activities have similar pH Optima (within about 0.5 pH units). (b) The three activities 107 are inactivated at approximately the same rate by sulfhydryl compounds at pH 8. (c) The presence of sulfhydryl compounds is essential for maintaining the three activities at pH 4.5. (d) At pH 4.5. Zn++ is essential for stabilization of the three activities. (e) Each of the three activities exhibits similar stability on storage at 4° and at -20° in the pres- ence of Zn++ and sulfhydryl compounds. (f) All three activ- ities Show similar rates of heat inactivation. (g) The three activities all Showed an increase in activity in the range of 650 to 70°. (h) Treatment with N-bromosuccinimide completely destroyed all three activities, while several other common amino acid modifying reagents caused no inacti- vation of any of the activities. (1) The three activities were effected, at least qualitatively, to the same extent by the addition of divalent metal cations, although the quanti- tative effects were different in some cases. Thus MN++, Co++, Ni++, and Cd++ were inhibitory to all activities to about the same degree. (j) The DNase and RNase activities were inhibited in a similar manner by sodium fluoride and P1. (k) Both the DNase and RNase activities showed the same unusual type of response to EDTA. Evidence for the presence of more than one protein includes the apparent activation only of the RNase activity by Mg*+, Ca++, alkylating agents and arsenite. Since the degree of binding of metal cations by the three substrates is undoubtedly different it is felt that the oa++ and Mg++ activation of the RNase activity, as well as the quantitative 108 differences observed with other metal cations may be due to effects of the metal ions on the substrate and not on the enzyme itself. These differences may accordingly not be significant. The activation of wheat RNase by alkylating agents and arsenite suggest at least two possibilities: (a) Two separate enzymes are responsible for RNase and DNase- 3'-nucleotidase activity. (b) One enzyme was reSponsible for all three activities, but the alkylating agents affected only a site absolutely essential for RNase activity. This latter possibility may also eXplain the difference in the extent of inhibition of the DNase and RNase activities by EDTA, assuming that EDTA affects the RNase site to a greater extent. A.similar difference in the degree of EDTA inhibi- tion was observed with the mung bean RNase and 3'-nuc1eoti- dase activities (2“). The unusual shape of the EDTA inhibi- tion curve suggests that the EDTA may be doing more than simple metal ion removal from the enzyme. Below 10"6 M EDTA little or no inhibition of the activities was observed. In the range from about 10'5 to 10'2 M EDTA the degree of inhi- bition remained constant for both the DNase and RNase activ- ities. This represented approximately a thousand fold increase in the EDTA concentration before the second phase of inhibition occurred. While this may simply be titration of the metal attached to the enzyme protein the biphasic nature of the curve suggests that EDTA may be reacting with at least two different groups in the protein or with metals bound to different degrees. What the exact nature of the 109 EDTA effect(s) might be is not clear at present. The three enzyme activities exhibited very similar heat stabilities. Thus the temperature values at which the activities were reduced to 50% of the unheated control after 8 minutes were 76°, 75° and 75° for the DNase, RNase. and 3'-nucleotidase activities respectively. Above 85° all three enzyme activities were completely destroyed. The three activities also showed rather similar stability to heating at 70° for various periods of time. The activity was reduced to 50% of the control after heating at 70° for 25, 26, and 31 minutes for the DNase, RNase, and 3'-nuc1eo- tidase activities reSpectively. The Q10 values in the tem- perature range from 27° to 47° were also determined. The RNase exhibited the highest Q10 values of 4.2 to “.5 while the DNase and 3'-nucleotidase exhibited Q10 that were some- what lower, near 3.1 and 2.1 reSpectively. The high value for the RNase activity may reflect an effect of temperature on the secondary structure of rRNA and may not be an effect on the enzyme itself. This may also be true for the Q10 value for the DNase activity on denatured DNA. The observation that a higher percentage of the three activities remained after heating at 70° then remained after heating at 65° for the same period of time is of interest. This may be similar to the heat reactivation effect observed with the associated DNase and RNase activities of chicken pancreas (45). Again this is strong circumstantial evidence that the three activities reside in a single entity. 110 The effect of Zn++ and sulfhydryl compounds are also interesting aspects of this enzyme preparation. Although Zn++ was somewhat inhibitory to the purified enzyme activ- ities, the presence of Zn++ at a concentration of at least 10‘“ M was an absolute requirement for the stabilization of the activities at pH 4.5. This suggests that Zn?“+ may be essential for stabilization of the proper tertiary or quaternary structure of the protein. Such a role for Zn++ has been demonstrated in several enzymes such as horse liver alcohol dehydrogenase (46), g, subtillis a-amylase (#7), and g, ggli,alkaline phoSphatase (48, #9). In these enzymes the Zn++ is essential for either the proper associa- tion of subunits to form the active enzyme or for the main- tenancy of the proper conformation of the active enzyme. The observation that Zn++ has an effect on the distribution of products produced during the hydrolysis of RNA also sug- gests that the metal may play a role in determining the Specificity of, at least, the RNase activity. The absolute :requirement for a sulfhydryl compound for maintenance of tactivity at pH 4.5 and the complete destruction of all enzyme activity by the same sulfhydryl compounds at pH 8 appears at first to be contradictory. This appears to be a general Sulfhydryl effect since essentially the results were obtained regardless of whether the sulfhydryl source was dithiothreitol, cY'Steine, or mercaptoethanol, provided that the total sulf- l'lydryl concentration was the same. A similar effect of sulf- hydryl compounds at pH 4.5 and 7. 5 was observed by Schuster 111 (18) with the associated DNase, RNase, and 3'—nucleotidase activities of rye grass. Since disulfide bonds are not reduced by sulfhydryl compounds to any appreciable extent at pH h.5, the possibility exists that the enzyme contains a sulfhydryl group and a disulfide bond, both of which are essential for normal enzyme function. This would be simi- lar to the enzyme papain, which has been shown to contain one sulfhydryl group which is essential and three disulfide bonds (50). The possibility cannot be ruled out that at pH 8 a significant proportion of the sulfhydryl compound is auto-oxidized and that the observed inactivation of the three activities is due to reaction of the oxidized material with the essential sulfhydryl group of the protein. In this case one would not need to postulate the presence of an essential disulfide bond. This seems unlikely in that treat- ment of the enzyme at pH 8 with alkylating agents caused no loss of enzyme activity. If the sulfhydryl was being oxi- dized by the media at pH 8 it should also be alkylated by the alkylating agents at that pH. One possible explaination might be that a conformational change occurs in the protein between pH h.5 and 8 which causes the sulfhydryl group to become ”shielded“ and inaccessible to the alkylating agents, but somehow, still accessible for reaction with the oxidized compounds in the media. The similarity in the rates of inactivation of the three enzyme activities by sulfhydryl compounds at pH 8 is another good circumstantial indication that the three activ- 112 ities are associated in some manner. At pH 8 the amino acid modifying reagent, N-bromo- succinimide, is capable of reacting with a variety of dif- ferent amino acid residues in a protein (51). It is, therefore, not possible to assess which amino acid residues were modified by this reagent. However, it seems important that all three enzyme activities were destroyed to the same extent in this process. The wheat DNase activity appears to be highly prefer- ential for denatured DNA- While native DNA was hydrolyzed by the enzyme, the initial rate was only about 10% of that observed with denatured DNA. As the digestion of native DNA proceeded the rate fell to only 1.3% of that observed with denatured DNA in the same period of incubation. It is not felt that the enzyme activity itself decreased during this time period since the rate of hydrolysis of denatured DNA did not decrease. Since this experiment was performed on commercial salmon sperm DNA it is quite possible that the low level of activity observed on native DNA simply repre- sented the hydrolysis of single-stranded regions in the native DNA preparation or of contaminating RNA. If this were the case then one would eXpect that the rate of hydrolysis should decrease with time as this material is removed from the native DNA preparation. This was, indeed, what was observed. This is similar to the results obtained by Linn and Lehman (32) with the endonuclease from N, crassa which also is highly preferential for denatured DNA. 113 A good separation of the 4 deoxymononucleotides was effected on DEAE-Sephadex employing a gradient of triethyl- ammonium bicarbonate (TEAB). The separation of dAMP and dGMP was complete. The dTMP and dCMP appeared in the same peak but the relative amounts of these two nucleotides can be determined either by spectrophotometric analysis of the binary mixture or by subsequent chromatography of this material on Dowex-I-Chloride by the method of Volkin g£_g;. (53) which uses dilute HCl as solvent and effects a good separation of the pyrimidine nucleotides. The same separa- tion of mononucleotides could be effected in a batch-wise treatment of the DEAE-Sephadex with 0.18 M TEAB. This sys- tem has the added advantage that the solvent is volatile and can be completely removed by repeated flash evaporation. The final product can thus be obtained in an essentially salt free form. This separation system was used to study the production of mononucleotides at various stages of the digestion of denatured DNA. At all periods of hydrolysis investigated dAMP was the predominate mononucleotide and was present in at least a 2-fold greater amount than any of the other 3 deoxymononucleotides. The production of dTMP remained relatively constant during the hydrolysis. The rate of release of dCMP was constant at least through the first 60 minutes of incubation but had almost tripled by (the end of 300 minutes. After 15 minutes incubation dGMP accounted for about zuz of the total mononucleotides, how- ever, at the end of 60 minutes the dGMP fraction was only 11b 5% of the total mononucleotides and after 300 minutes it was only about 2% of the total. Thus it would appear that the rate of release of dGMP from denatured DNA decreases appre- ciably as the hydrolysis proceeds. The recovery of the 4 known deoxymononucleotides is essentially quantitative from the DEAE-Sephadex column. Therefore, it is felt that the F low level of deoxyguanylate observed is real and is not an “5 artifact of the separation procedure. Furthermore, by total ' H analysis of all the oligonucleotide peaks from the DEAE- Sephadex column. it was possible to account for 100% of the I i deoxyguanylate residues that were present in the original DNA. Thus the oligonucleotides which are present at later stages of digestion appear to be rich in deoxyguanylate residues. The formation of high levels of dAMP and low levels of dGMP suggests that the wheat DNase possess some degree of base specificity. It would appear that bonds involving deoxyadenylate residues are preferentially attacked, while those involving deoxyguanylate residues are relatively resistant. It is not possible at present to say anything further about the base Specificity of this enzyme. In light (Of the apparent effect of an+ on the products formed on laydrolysis of rRNA, it should be mentioned that these diges— 1:ions of denatured DNA.were also performed in the presence c>f 1 mM Zn++. What effect the presence or absence of Zn++ kLas on the distribution of mononucleotides that are produced 1N8 not known. The production of mononucleotides by the wheat nticlease is similar to that observed with the N, crassa 115 endonuclease (32) except that the fungal enzyme produces large amounts of dGMP and very little dCMP. The possibility exists that these two enzymes could be used to complement each other in base sequence determinations on deoxyoligo- nucleotides and on denatured DNA. The DNase activity from soybean sprouts (19). rice bran (25), and from the diges- tive Juice of larvae of the silkworm, Bombyx 222$.(52) also have been reported to produce low levels of dGMP relative to the levels of the other three deoxymononucleotides. (The exact nature of the association of DNase, RNase, and 3'-nucleotidase activities cannot be ascertained at present. The following possibilities seem most likely: (a) The three enzyme activities are the property of a single protein, with one or more active sites. (b) The protein is a very stable complex of two or more distinct enzymes. (c) Three distinct, separate enzyme proteins are present in the purified preparation. Several lines of evidence can be interpreted as indicating the presence of subunits in the enzyme. These include the Zn++ stabilization of the enzyme activity, the disappearance of the active protein band on electrophoresis of enzyme treated with sulfhydryl compounds and 8 M urea, and the lack of proportionality of the rate of hydrolysis to enzyme concentration in the lanthanum-acid soluble product assay. It seems likely that the Zn++ effect on the enzyme is at least in part due to some stabilization (of the prOper tertiary and quaternary structure of the enzyme. {Phe lack of proportionality of the rate of hydrolysis to I r,“ ~_ a w 9.“??‘5 a"! c? ' S53 ‘Rimi (IT'CJTJJ 5.7.1 I 3 Vi ‘_-. "Hm-l. A _. . .”_ :5 ._ r- “”9 i1 116 enzyme concentration may indicate the presence of some type of association-d1sassociation phenomenom.. At very low enZyme concentrations the activity may be low because of disassociation of the active enzyme, while at high enzyme concentrations the activity is enhanced because of associa- tion of active enzyme molecules. The addition of bovine , h serum albumin did not effect the non-linearity of the assay. E1) Thus this does not appear to be Just a general protein E H effect and may represent the Specific disassociation of the F enzyme protein. The simplest explaination of the electro- phoresis patterns is that in the presence of a sulfhydryl compound and 8 M urea at pH 8 the protein is dissociated into subunits which have an electrophoretic mobility quite different from that of the intact native protein. Since after this treatment the enzyme is completely inactive it is not possible to tell whether the subunits are part of a single enzyme protein (a) or whether they are separate enzyme proteins which are only active when associated in a complex (b). Several lines of evidence also indicate that more than one active site may be present in the enzyme. The activation of only the RNase activity by Ca”, Mg”. and alkylating agents, and the greater degree of inhibition of RNase by EDTA are suggestive of more than one active site, however. other eXplainations of these results are also pos- Sible. The fact that denatured DNA inhibits the RNase activity to a greater extent (about 65%) than rRNA inhibits the DNase activity (about 42%) is interesting. Since these 117 two values are both near 50% inhibition it could be postu- lated that a single site is involved in the hydrolysis of both denatured DNA and rRNA and that the two substrates are competing for that site. The possibility of two sites cannot be ruled out, however, since it is known that many specific nucleases bind and are inhibited by nucleic acids which they cannot hydrolyze. Thus two sites could be present in the E); protein with one site reSponsible for the hydrolysis of each E H substrate but both capable of binding ENA.and RNA. The dif— é ference in the percent inhibition observed with the two sub- strates might only represent a difference in the relative degree of binding of each nucleic acid by the individual sites. Finally, the apparent difference in the mode of action of the DNase and RNase activities is strongly sugges- tive, although certainly not definite, evidence for the presence of more than one active site. If three separate enzymes are reSponsible for the three enzyme activities (c) then these three enzyme proteins would have to be extremely similar in amino acid composition and sequence in order to diSplay the similarity of properties exhibited by these three activities. If, indeed, this were the case then these proteins might represent an example of a series of duplications of a single gene at some time in the evolution of plants and the subsequent independent course of random mutation of these genes. At present it is impossible to conclude definitely which of the three possible models 118 for the associated wheat activities is correct. A final decision on the nature of the association of the three activ— ities must await large-scale preparations making possible a variety of physical chemical experiments. The apparent association of DNase, RNase, and 3'-nuc- leotidase activities that has been observed in a variety of plants may simply represent an artifact peculiar to plant tissues. One might expect that a similar phenomenom would occur in closely related plant Species such as wheat, barley, and rye grass, all of which are monocots. The presence of the same association of enzyme activities in dicots such as mung bean, muskmelon, and soybean as well, suggest that this association may be a general one throughout the plant king- dom. Many of the nucleases which have been isolated from bacterial, mold, and animal sources have been shown to possess both DNase and RNase activities. Furthermore, many of these purified enzymes still contain some phOSphomono- esterase activity. Others have only been tested for nucleo- tidase activity using d-glycerol phoSphate or p-nitrophenyl phoSphate, substrates which may not be hydrolyzed at an appreciable rate by specific nucleotidases. A careful study of these enzymes might reveal that the association of DNase, RNase, and 3'-nucleotidase activities is more wideSpread and not simply peculiar to plant tissues. The possible biological significance of such a complex of related enzyme activities is intriguing. The relative abundance of these three associated enzymes in a variety of 119 germinating, rapidly growing seedlings might suggest a pos- sible role for these enzymes in DNA replication or repair. It is possible to postulate a role for a single-strand endo- nuclease in DNA repair and related processes such as recom- bination and replication. As Lehman (15) has suggested, such enzymes might be capable of "seeking out" and attacking 'h ‘unordered regions in the DNA double-helix where damage to t J the DNA had occurred. They might also be capable of cleav- i .6 ing single-stranded fragments of DNA which might occur in the overlap regions during recombination. The DNA joining i i enzymes (polynucleotide ligase) which have been reported to date from.§, 22$; (54, 55) and from Tu-infected E, 32;; (56) all have at least one requirement in common. This require- :ment is for the presence of a free 3'-hydroxyl group on the adjacent nucleotide residue at the site of phosphodiester 'bond formation. The presence of a 3'-nucleotidase activity closely associated with a repair enzyme would be a means of insuring that the 3'-hydroxyl position would be free and capable of participating ill the formation of a phoSphodi- ester bond. This might eXplain the role of such a 3'-nuc- leotidase activity in DNA repair. At present it is not lanown whether the wheat 3'-nucleotidase activity is capable Of removing the phosphate group from 3'—deoxymononucleotides Ifor-from the 3'-terminus of oligonucleotides or DNA. Two e1'lzymes which remove 3'-phoSphoryl groups in DNA have been reported by Becker and Hurwitz (57). One of these enzymes, 3'-«1eoxynucleotidase, is Specific for the 3'-ph08phate ends 120 in DNA and 3'-mononucleotides. The other activity, g, 32;; C phOSphatase, is non-Specific at the mononucleotide level and will dephosphorylate both 3'- and 5'-mononucleotides but reacts with only 3'-phosphate ends in DNA and RNA. What role the associated wheat RNase might play in the repair process, if any, is not clear at present. Stable .DNA-RNA complexes have been isolated from plant cells (58-60), animal cells (61-63), and from some bacteria (64). These complexes do not appear to be artifacts of the DNA extrac- tion procedures employed. The RNA in these complexes incor- ‘porates labeled uracil at a rate several times faster than the total cellular RNA. The role of this DNA-RNA complex in the cell is not known. The possibility exists that the associated RNase and DNase activities may participate in reactions involving such DNA-RNA complexes. -fikfifilfl Ii- Jams}; if. .‘ 33.5.2111 . .; ’m‘mm .9 _._J..__».z_u.a;. SUMMARY A nuclease has been purified from germinating wheat seedlings to an extent of over 830-fold with a recovery of 1%.“ about 12% of the total DNase activity present in the crude 1~; homogenate. The purified preparation hydrolyzes denatured ; 4”! DNA, rRNA and the 3'-phosphoester linkage of 3'qAMP at similar rates. The preparation exhibits no appreciable _] 5'-nucleotidase, phosphodiesterase, or phoSphomonoesterase activity. The DNase, RNase, and 3'-nucleotidase activities have remained associated throughout a variety of purifica- tion procedures, including chromatography on several ion- exchange resins, gel filtration, and polyacrylamide disc electrophoresis at pH 9.5 and 8.3. The ratios of the three esctivities remain essentially constant throughout the last four steps in the purification procedure as well as through- crut all of the various procedures mentioned above. The ‘blxree activities exhibit a great degree of similarity with reSpect to the following properties: (a) pH optima, (b) reQuirement for Zn” and sulfhydryl compounds at pH 4.5. (C3) rate of destruction of activity by sulfhydryl compounds at pH 8, (d) stability to storage at both 4° and -200, (6) effect of temperature and duration of heating, (f) reacti- vation on heating between 65° and 70°. (g) effect of N- 121 122 bromosuccinimide, (h) effect of metal cations, (1) effect of inhibitors and EDTA. It is tentatively concluded that the three associated enzyme activities are either the properties of a single protein or of a very stable complex of two or more proteins. The possibility that three separate enzymes are reSponsible for the three activities seems unlikely but cannot be definitely ruled out at present. A final decision on the nature of the association of the three activities must await large-scale preparations making possible a vari- ety of physical chemical eXperiments. The DNase activity is highly preferential for dena- ‘tured DNA. Although native DNA was hydrolyzed by the enzyme, 'the rate was only about 1.3% of that observed on denatured DNA. The possibility exists that this low level of hydrol- ;ysis of native DNA represents simply the hydrolysis of dena- ‘bured regions in the native DNA preparation or of contamin- eating RNA. The hydrolysis of denatured DNA by the wheat IJNase was found to be endonucleolytic in manner by a number <>f criteria. The mononucleotides and presumably the oligo- rrucleotides produced bear 5'-phosphoryl groups. .At all Stages of digestion investigated, dAMP was the predominate mononucleotide component. The level of dAMP was always atncut 2-fold greater than any of the other three deoxymono- nucleotides. The dAMP level also increased as the digestion PIVDceeded. The level of dTMP and dCMP were relatively con- staunt throughout the early stages of digestion. .A very low l(“Tel of dGMP was observed throughout the digestion and this 123 level decreased as a percentage of the mononucleotide frac- tion as the digestion proceeded. Essentially all of the deoxyguanylate residues could be accounted for in the oligo- nucleotide fractions. Thus it would appear that the wheat DNase exhibits a relative degree of base Specificity with bonds involving deoxyadenylate residues being preferentially cleaved and bonds involving deoxyguanylate residues being :relatively resistant. The mode of action of the wheat RNase on rRNA was feund to be exonucleolytic and the mononucleotides produced appear to be the 2',3'-cyclic compounds. 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