Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

An investigation of the properties of DNase II isolated from bovine intestinal mucosa and the nature… Keys, David Stephen 1978

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1978_A1 K48.pdf [ 9.49MB ]
Metadata
JSON: 831-1.0094635.json
JSON-LD: 831-1.0094635-ld.json
RDF/XML (Pretty): 831-1.0094635-rdf.xml
RDF/JSON: 831-1.0094635-rdf.json
Turtle: 831-1.0094635-turtle.txt
N-Triples: 831-1.0094635-rdf-ntriples.txt
Original Record: 831-1.0094635-source.json
Full Text
831-1.0094635-fulltext.txt
Citation
831-1.0094635.ris

Full Text

AN INVESTIGATION OP THE PROPERTIES OF DNase II ISOLATED FROM BOVINE INTESTINAL MUCOSA. AND THE NATURE OF ITS REACTION WITH DNA by DAVID STEPHEN KEYS B.Sc, University of Toronto, 1971 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department of Biochemistry We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1978 © David Stephen Keys, 1978 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f Biochemistry The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2 0 7 5 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V 6 T 1W5 D a t e i i ABSTRACT Isolation of DNase I I from bovine small intestine by chromatography on DEAE cellulose of a 105,000 xg supernatant solution prepared from an homogenate of the mucosa i n Krebs Ringer phosphate buffer appeared to yield two a c t i v i t i e s , a major act i v i t y which was eluted from the column with 20 mM phosphate buffer and a minor act i v i t y which was eluted with a potassium chloride gradient. The two DNase II ac t i v i t i e s differed i n their response to increasing ionic strength, pH, sulfate ion concentration and temperature for the hydrolysis of DNA. The major acti v i t y degraded native DNA more rapidly than denatured DBA whereas the minor act i v i t y degraded both at the same rate. Previous investigators have reported the presence of two DNases l i s with different properties i n other tissues. In bovine intestinal DNase I I , the minor a c t i v i t y , upon rechromatography on DEAE cellulose, eluted i n the same position as the major DNase II and i t was concluded; that the appearance of the minor DNase I I ac t i v i t y was an artifa c t of the chromatography. It i s l i k e l y that a small quantity of DNase II was bound to endogenous DNA on the DEAE cellulose column i n the 20 mM phosphate buffer and later eluted from the column along with some of the DNA. with the potassium chloride gradient. DNA present i n the minor DNase II preparation probably caused the apparent differences i n properties of the two DNase l i s by interfering i n the enzymic reactions. Intestinal DNase II was p a r t i a l l y purified by ion exchange chromatography and gel f i l t r a t i o n and had properties similar to DNase l i s from other tissues. The enzyme hydrolysed calf thymus DNA endonucleolyti-i i i c a l l y at acid pH i n the absence of a divalent metal ion to oligonucleo-tides with 3*-phosphate and 5'-hydroxyl terminals. The activation energy for the reaction was 19 kcal/mole; that for denaturation of DNase II i t s e l f , k3 kcal/mole. Michaelis-Menton kinetics were observed for the reaction of DNase II with Escherichia c o l i DNA—the Michaelis constant was 2.k2 x ICT? M DNA-phosphate. The molecular weight of DNase II was estimated to be Ul,000 by gel f i l t r a t i o n on Sephadex G100. The early stages of the digestion of DNA by DNase II were investigated by labelling the reaction products with ^ 2p a t their 5 * -terminals using polynucleotide kinase and [ft -^PjATP and at their 3»-terminals using terminal deoxynucleotidyl transferase and The mode of cleavage of native DNA by DNase II was determined by comparing the polynucleotide-catalysed incorporation of 32p from - 3 ^ P ] A T P into native and denatured DNase II reaction products. Since single-strand cleavage of DNA by DNase II released 5'-hydroxyl terminals that were inaccessible to polynucleotide kinase as long as the DNase II reaction products remained double-stranded, incorporation of -^P into native products was proportional to the number of double-strand cleavages while incorporation of ^ P into denatured products was proportional to the number of double-strand cleavages plus single-strand cleavages. It was found that DNase II degraded native DNA primarily by a double-strand cleavage mechanism. After DNase II catalysed hydrolysis of DNA each of the four bases present i n DNA was found at the 5'- and 3*-terminals of the reaction products. Thus DNase II did not have an exclusive preference for one or two bases at either terminal, and l i k e l y cleaved a large number of i v different base sequences i n the DNA.. The most susceptible internucleotide linkage was GpG; the most resistant, CpT. The base s p e c i f i c i t y at the 5*-terminal changed during the reaction, especially i n the i n i t i a l and terminal phases. In the i n i t i a l phase the proportion of guanine was elevated and the order of cytosine and adenine was reversed compared to later stages i n the reaction. These changes could reflect the presence of a preferred sequence that was selectively degraded and exhausted during the i n i t i a l phase of the reaction. Different proportions of terminal bases i n cleavage products of DNA from diverse species indicated that susceptible sequences occurred with different frequencies i n the various DNAs. V TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES ix LIST OF FIGURES x ACKNOWLEDGEMENTS xiv INTRODUCTION 1 The biological function of DNase II 3 Distribution i n different tissues ••••• 3 Intracellular localization It-Correlation of DNase II activity levels with the rate of c e l l proliferation • ••••• 5 DNase II activity in malignant tissues •*•• 5 DNase II acti v i t y levels i n relation to c e l l cycle • • 7 Possible biological roles for DNase II 7 The enzymology of DNase II 3 The reaction of DNase II with DNA 8 I n i t i a l phase of the reaction 9 Middle and terminal phases of the reaction ...... 11 The structure of DNase II Ik Physical and chemical properties Ik Catalytic properties 16 The use of DNase II for the study of the structure of DNA and chromatin • • 19 Investigation of the primary structure of DNA ... 19 Investigation of the structure of chromatin 20 Intestinal DNase II •• 21 The present investigation ................................ 22 MATERIALS AND METHODS 26 MATERIALS 26 v i Page METHODS 35 Measurement of DNase ac t i v i t y 35 Acid-soluble oligonucleotide assay .............. 35 DNA-agar gel assay •••• kl Hypercnromicity assay ••••••• ••••• • kk H^-DNA assay kj Measurement of the ac t i v i t i e s of other nucleases • 51 DNase I 51 Alkaline phosphatase • 51 Phosphodiesterase I ••••• 52 5'-nucleotidase 52 Acid phosphatase 52 Phosphodiesterase II • 53 Acid BNase 5k Alkaline BNase • 5k Measurement of protein • • 55 Measurement of DNA •••• •• 55 Measurement of phosphate 55 Measurement of proteolytic a c t i v i t y 56 Measurement of radioactive compounds ..*........... 58 Preparation of ^ -thymidine labelled DNA from the mucosa of rat small intestine • 60 Characterization of the DNA preparations from rat intestine and ca l f thymus •••••• 60 Preparation of L f - 3 2 ? ] ATP from ^ P labelled inorganic phosphate • 66 Determination of the specific radioactivity of ft - ^ p l ATP 69 RESULTS AND DISCUSSION 73 A Isolation of DNase II activity from bovine intestinal mucosa 73 Preparation of a crude extract containing DNase II activity from the mucosa of bovine small intestine ••»••••••••••••••••• 73 Chromatography of the crude DNase II preparation on DEAE cellulose ................... 76 v i i Page Some factors affecting the s t a b i l i t y of DNase I I , 82 Discussion 86 B Investigation of the properties of two DNase II a c i v i -t i e s isolated from bovine intestinal mucosa 87 pH optima ...... •••• 87 Degradation of native and denatured DNA ••• 87 Effects of ionic strength 91 Effects of sulfate, an inhibitor of DNase II .... 91 Activation energies for the hydrolysis of DNA ... 9U Activation energy for the denaturation of DNase II $6 Presence of endogenous DNA i n the second DNase II preparation 99 Rechromatography of the two DNase II a c t i v i t i e s on DEAE cellulose •• 105 Chromatography of the f i r s t DNase II ac t i v i t y on DNA cellulose • 107 Chromatography of the two DNase II a c t i v i t i e s on Sephadex G100 108 Discussion •••••• I l l C Par t i a l purification of bovine intestinal DBase II .... 117 Electrophoresis of the p a r t i a l l y purified DNase II preparation i n polyacrylamide gels ..... 122 Measurement of contaminating nucleases i n the DNase II preparation ..................... 126 Further purification of DNase II 128 Discussion 131 D Some properties of intestinal DNase II 132 pH optimum •• •• •••••••••• •• 132 Effect of magnesium ion 13? Effect of ionic strength •••• ••••••• 138 Effect of sulfate ion 138 Activation energy for hydrolysis of DNA ......... 138 Activation energy for denaturation of DNase II .. ikl Molecular weight of DNase U ikl Discussion • •••• 1**2 E The reaction of DNase II with DNA and other substrates. 1^ 3 The Michaelis constants, and the maximum velocities for the reaction with DNA and polydAT 1^ 3 Degradation of native and denatured DNA 150 v i i i Page Digestion of polydAT, polydA and polydT 153 Mode of cleavage of native DNA. 153 The DNase II reaction ••••• 153 The polynucleotide kinase reaction .............. l6l Cleavage of calf thymus DNA , 163 Degradation of DNA from other sources ••••••••••• 167 Effect of 1 mM sulfate on the mode of cleavage of DNA from Escherichia c o l i ••••• 1&9 Effect of 10 mM EDTA on the mode of cleavage of calf thymus DNA •••• 169 Characterization of the products and substrates of the DNase H reaction by electrophoresis on polyacrylamide gels 17^ Base spe c i f i c i t y of cleavage of DNA by DNase II l8l The base s p e c i f i c i t y at the 5*-terminals 182 The DNase II and polynucleotide kinase reactions. 182 Separation of the 5'-32p oligonucleotide products of the polynucleotide kinase reaction from unreacted [r-32 PlATP Degradation of the 5»-32p oligonucleotides to 5»-32p nucleotides lBU Separation of the 5»-32p nucleotides 136 The base sp e c i f i c i t y at the 3'-terminals 192 The DNase II reaction 195 The acid phosphatase reaction 195 The terminal transferase reaction • 195 Separation of the oligonucleotide products of the terminal transferase reaction from 32P-labelled inorganic phosphate 19$ Degradation of the 3*- P oligonucleotides to 3'-32p nucleotides 199 Separation of the S'-^P nucleotides 202 Discussion 20lf SUMMARY 207 BIBLIOGRAPHY • •• 213 ix LIST OF TABLES Table Page I Chemicals used i n the investigation 27 II Chromatographic media used i n the investigation 31 III Enzymes and animals used i n the investigation 33 17 Ultraviolet and phosphate analyses of DNA isolated from rat small intestine and of calf thymus DNA (Sigma type I) 6k V Preparation of a crude extract containing DNase II activ i t y from the mucosa of bovine small intestine... 75 VI Rate of release of acid-soluble oligonucleotides from native and denatured DNA by DNase I I . 90 VII Effect of treatment of the second DNase II prepara-tion with protamine sulfate ic4 VIII Partial purification of intestinal DNase II 123 IX Other nucleases present i n the DNase II preparation.. 127 X Effect of further purification on ac t i v i t i e s of other nucleases present i n the DNase II preparation.. 130 XI Proportion of cleavages of DNA by DNase II that were double-strand cleavages............. 166 XII Mode of cleavage of calf thymus DNA by DNase II 168 XIII Mode of cleavage of DNA from different species by DNase II 177 XIV Calculation of the ^ P incorporated into each nuceo-tide at the 5'-terminal as a percent of the t o t a l 32p incorporation 187 XV Base sp e c i f i c i t y at the 5'-terminals for cleavage of DNA from different sources by DNase II 191 XVI Base speci f i c i t y at the 3*-terminals for the cleavage of DNA. from different sources by DNase II X LIST OF FIGURES Figure Page 1. Release of acid-soluble oligonucleotides from c a l f thymus DNA by DNase II 37 2. Effect of different concentrations of DNase II on the release of acid-soluble oligonucleotides from calf thymus DNA kO 3. The linear relationship between the logarithm of the DNase II concentration and the diameter of the zone of clearing determined by the DNA-agar gel assay....... 3^ k. Increase i n absorbance, hyperchromicity, of a DNA solution upon treatment with DNase II 5. Effect of different concentrations of DNase II on the hyperchromicity of a DNA solution 9^ 6. Rate of release of t r i t i a t e d acid-soluble oligonucleo-tides from sonicated %-DNA from Escherichia c o l i by DNase II 50 7. Effect of different amounts of trypsin on the release of acid-soluble fragments from casein 57 8. Thermal denaturation curve for DNA isolated from rat intestinal mucosa.... 62 9. Thermal denaturation curve for calf thymus DNA (Sigma type I) 63 10. Schematic representation of the reactions involved i n the synthethis of fa-32?] ATP 67 11. Chromatography of the [l-^P J ATP preparation on DEAE cellulose. 68 12. Incorporation of ^ P into oligodT(PT)9 using polynu-cleotide kinase and tT-32p) A T p 72 13. Chromatography on DEAE cellulose of a crude extract prepared from bovine intestinal mucosa 78 Ik, Rechromatography of the f i r s t peak of DNase II a c t i -v i t y from F i g . 13 on DEAE cellulose 80 x i Figure Page 15. Rechromatography of the second peak of DNase II a c t i -v i t y from Fig. 13 on DEAE cellulose 8l 16. S t a b i l i t y of intestinal DNase II 8k 17. pH optima of the DNase II activities 89 18. Comparison of the f i r s t and second DNase II a c t i v i t i e s at different ionic strengths.,.. 92 19. Effect of sulfate on the f i r s t and second DNase II act i v i t i e s 93 20. Arrhenlus plot showing the temperature dependence of the rate of hydrolysis of calf thymus DNA by DNase I I . 95 21. Temperature dependence of the rate of denaturation of DNase II 98 22. Release of acid-soluble oligonucleotides by the second DNase II i n the presence and absence of added substrate 102 23. Rechromatography of the second DNase I I a c t i v i t y on DEAE cellulose 106 2k. Chromatography of the f i r s t DNase II a c t i v i t y on DNA cellulose 109 25. Chromatography of the second DNase II act i v i t y on Sephadex 0100....... 110 26. Estimation of the molecular weight of the f i r s t DBase II by gel f i l t r a t i o n on Sephadex G100. 112 27. Chromatography on DEAE celluolose of a 105,000xg supernatant solution perpared from bovine intestinal mucosa. • 118 28. Chromatography on CM cellulose of the pooled DNase II act i v i t y from the DEAE cellulose column 120 29. Chromatography on Sephadex G100 of the pooled DNase II ac t i v i t y from the CM cellulose column 121 30. Densitometer tracing of a polyacrylamide gel showing the electrophoretic separation of the proteins present i n the DNase II preparation 125 x i i Figure Page 31. Effect of pH on the rate of release of acid-soluble oligonucleotides from DNA by DNase II 13*+ 32. pH optimum of intestinal DNase II 136 33* Effect of magnesium ions on DNase II activity 137 34. Effect of millimolar concentrations of sulfate ion on DNase II ac t i v i t y IhO 35* The rate and extent of the reaction of DNase II with different concentrations of calf thymus DNA.. l*+5 36. Lineweaver-Burke plot for the reaction of DNase II with sonicated ^H-DNA from Escherichia c o l i lk8 37. Lime weaver-Burke p l o t ^ o r the reaction of DNase II with polydAdT-methyl-3!! l*+9 38. Comparison of the reaction of DNase II with native and denatured calf thymus DNA 152 39. Reaction of DNase II with polydAT, polydA and polydT.. 155 1+0. Determination of the mode of cleavage of DNA by DNase II 157 1+1. Digestion of DNA from various sources by DNase II 160 1+2. Polynucleotide kinase catalysed incorporation of -^P into products of the reaction of DNase II with native calf thymus DNA 165 1+3. Incorporation of ^ P by polynucleotide kinase into products of the reaction of DNase II with DNA from salmon testes, rat intestinal mucosa, and bacterio-phage 3. ••••• 171 kk. Effect of 1 mM sulfate on the mode of cleavage of DNA from Escherichia c o l i by DNase II 173 1+5. Effect of 10 mM EDTA on the mode of cleavage of ca l f thymus DNA by DNase II 176 1+6. Electrophoresis of products and substrates of the DNase II reaction on polyacrylamide gels • 180 x i i i Figure Page 1*7. Scheme for determination of the base speci f i c i t y at the 5'-terminals of products of cleavage of DNA by DNase II 183 U8. Separation of 5*-^2P oligonucleotides from unreacted [?_32pJ ATP by chromatography of the polynucleotide kinase reaction mixture on Sephadex 0100 • 185 U9. Base spe c i f i c i t y at the 5'-terminals for cleavage of native calf thymus DNA by DNase II 189 50. Scheme for the determination of the base s p e c i f i c i t y at the 3'-terminals for products of cleavage of DNA by DNase I I 19^  51. Terminal dycocynucleotidyl transferase catalysed incor-poration of 32p ^ 0 products of the reaction of DNase II with DNA from various sources 197 52. Separation of 3*-3% oligonucleotides from ^ P labelled inorganic phosphate by chromatography of the base and alkaline phosphatase treated terminal transferase reaction mixture on Sephadex 0^0.. 201 xiv ACKNOWLEDGEMENTS I would l i k e to thank Dr. S. H. Zbarsky for his advice and encouragement during my stay i n his laboratory. Support of the Medical Research Council of Canada i n the form of Studentships for the period 1971-1975 and of H, R. MacMillan Family Fellowships for the period 1975-1977 are gratefully acknowledged. INTRODUCTION According to international recommendations on Enzyme Nomenclature (197:2), deoxyribonuclease II (EC 3.1.^.6), abbr., DNase II and also known as acid DNase, i s defined as a phosphodiester hydrol ase that degrades deoxyribonucleic acid to 3'-phosphate terminated oligonucleotides: 2 The cl a s s i f i c a t i o n of animal DNases into DNase I- and DNase II-type enzymes was introduced by Cunningham and Laskowski (1953) i n order to compare enzymes distributed i n different tissues with their prototypes pancreatic DNase I (Kunitz, 1950) and spleen or thymic DNase II (Maver and Greco, 19kQ). Pancreatic DNase I has an optimum pH at about 7> requires magnesium ions for a c t i v i t y and i s inhibited by citrate (Kunitz, 1950), whereas DNase II has a pH optimum at about 5 and i s inhibited by magnesium ions (Maver and Greco, I9U8). Thus DNase II i s an endonuclease which i s specific for DNA and hydrolyses i t to 3*-phosphate terminated oligonucleotides optimally at acid pH and without requiring a divalent metal ion. Cordonnier and Bernardi (1968) found that DNase II activity from a variety of different animal sources was associated with protein molecules similar i n sedi-mentation coefficients, modes of action on native DNA, chromatographic behavior, and i n enzymatic properties. DNase II has been investigated with different goals i n mind (Laskowski, 1961, 1967; Bernardi, 1968, 1971). DNase II has been studied with the purpose of elucidating i t s biological function. Since comparative DNase II acti v i t i e s of many f e t a l and adult tissue correspond i n a general way with the capacity of those tissues for proliferation or regeneration (Allfrey and Mirsky, 1952), and since DNase II activity increases at that time i n the c e l l cycle when DNA synthesis i s taking place (Slor et a l . , 1973), DNase II may have an important biological function, the nature of which has not been firmly established. Physical, chemical and enzymatic studies of DNase II have dealt with the characteri-zation of the enzyme as a protein.and with the kinetics and speci f i c i t y 3 of the enzymic reaction with DNA. DNase II has also been investigated i n order to determine whether i t could be used as a tool for probing the structure of DNA and chromatin. The biological function of DNase II Distribution i n different tissues A DNase II has been found i n the cells of a number of animal tissues and species (Cordonnier and Bernardi, 1968; Bernardi, 1968; Laskowski, 1961, 1967; Private de Garilhe, 1967; Lehman, 1967). Cordonnier and Bernardi (1968) compared the chromatographic and enzymatic properties, the sedimentation coefficients, and the mechanism of action on native DNA exhibited by p a r t i a l l y purified DNase II preparations obtained from 15 different animal sources. They concluded that the DNase II activity was associated with protein molecules endowed with very similar physical, chemical, and enzymatic properties. They also found that DNase II preparations from b u l l seminal plasma, hog serum, and l i v e r and chicken erythrocytes, showed two peaks of activity upon chromatography on DEAE cellulose. A major component was not retained i n low ionic strength buffer, and a minor one was eluted by a pH-molarity gradient. When Yamanaka et a l . (197*0 chromatographed DNase II activity from human gastric mucosa or cervix uteri on a phosphocellulose column, two peaks of DNase II acti v i t y appeared. The two ac t i v i t i e s had similar properties, but different isoelectric points. Zbllner et a l . (197*0 discovered two DNase II acti v i t i e s i n extracts of human lymphocytes; one activity was similar to spleen DNase II, the other had a different electrophoretic k mobility, and showed no preference for native over denatured DNA, Intracellular localization DNase II is associated with the lysosomes i n rat l i v e r , with the related "droplets" from rat kidney, and with the related secretion granules i n rabbit leucocytes (de Duve et a l . , 1962). The distribution of DNase II i n mouse l i v e r and pancreas, i n rat brain, and i n calf thymus suggest that DNase II i s also lysosomal i n these tissues (de Duve et a l . , 1962). Lesca (1968) found a DNase II activity i n the nuclear fraction of mouse l i v e r cells and, since no cytochrome oxidase ac t i v i t y was found i n the nuclear fraction, he concluded that the nuclear DNase II act i v i t y was not due to contamination of the nuclear preparation with lighter organelles. Shor and Lev (1971) found DNase II activity i n purified calf thymus nuclei. During purification of the nuclei the activity ratio of DNase II to acid phosphatase, a lysosomal enzyme (de Duve et a l . , 1962), increased 30-40 fold indicating that the DNase II activity found i n the nuclei was not due to lysosomal contamination. SI or (1973) purified -labelled DNase II from HeLa S3 lysosomes, and added i t to cel l s during isolation of their nuclei. He found no specific binding of lysosomal DNase II to the nuclear fraction and concluded that the DNase II acti v i t y observed i n isolated nuclei represented an in t r i n s i c activity that might be involved i n nuclear DNA metabolism. 5 Correlation of DNase II act i v i t y levels with rate of c e l l proliferation A l l f r e y and Mirsky (1952) found that adult cells which do not divide, eg. heart, brain and red c e l l s , had low DNase II levels whereas tissues capable of high mitotic a c t i v i t y , eg. spleen, intestinal mucosa, and l i v e r , had high DNase II levels. Organs capable of p a r t i a l regeneration l i k e the kidney had intermediate DNase II concentrations. Fetal calf kidney was more than twice as active i n DNase II as calf kidney; f e t a l l i v e r had nearly 3 times the DNase II concentration of calf l i v e r . Also the DNase II activity was higher i n regenerating l i v e r than i n non-regenerating l i v e r . Cordonnier and Bernardi (1968) confirmed that the highest DNase II levels were found i n lymphatic and tumoral tissues wMch are capable of high rates of c e l l proliferation, and the lowest ones i n cells l i k e sperm cells and erythrocytes that do not reproduce themselves. The specific a c t i v i t y of DNase II i n chicken brain (minus cerebellum) increases up to the 16th day of embryonic age and decreases after hatching (Shrivastaw et a l . , 1975). A similar pattern i n DNase II activity has been reported i n developing rat (Sung, 1968) and human (Rao-Subba, 1973) brains. DNase II activity i n malignant tissues A four fold rise i n DNase II activity levels i n leukemia leukocytes relative to normal leukocytes was observed by Slor (1970a). Escheribach (1971) found that acute bacterial and virus diseases caused an increase 6 i n t h e DNase I I a c t i v i t y i n t h e c y t o p l a s m o f l e u k o c y t e s i n c h i l d r e n . I n c h i l d r e n w i t h a c u t e l e u k e m i a t h e DNase I I a c t i v i t y ! was d e c r e a s e d , b u t a f t e r t h e r a p e u t i c t r e a t m e n t , t h e number o f l e u k o c y t e s d e c r e a s e d and t h e DNase I I a c t i v i t y i n c r e a s e d . T a p e r e t a l . (1971a) i n v e s t i g a t e d t h e DNase I I l e v e l s i n b e n i g n and m a l i g n a n t tumors o f t h e human c e n t r a l n e r v o u s s y s t e m u s i n g a h i s t o -c h e m i c a l t e c h n i q u e . * I n t h e b e n i g n t umors , t h e DNase I I a c t i v i t y was s i m i l a r t o t h a t o f t h o s e n o r m a l t i s s u e s f r o m w h i c h t h e tumors o r i g i n a t e d ; b u t i n t h e m a l i g n a n t t umors no DNase I I a c t i v i t y r e a p p e a r e d . T a p e r e t a l . (1971b) a l s o measured DNase I I a c t i v i t y h i s t o c h e m i c a l l y i n t h e r a t l i v e r parenchyma d u r i n g N - n i t r o s o m o p h o l i n e - i n d u c e d c a r c i n o g e n e s i s . The DNase I I a c t i v i t y was c o n s i d e r a b l y d e c r e a s e d i n f o c a l a r e a s and l a t e r i n h y p e r -p l a s t i c n o d u l e s , b u t was n o r m a l i n t h e s u r r o u n d i n g l i v e r parenchyma. DNase I I d e f i c i e n c y appeared a t t h e 38th t o 59th day o f c a r c i n o g e n e s i s , and p r e c e d e d b y a p p r o x i m a t e l y 56*?75 days t h e m o r p h o l o g i c a l s i g n s o f c a n c e r . I n t h e n e c r o b i o t i c c e l l s o f m a l i g n a n t h e p a t o c e l l u l a r t u m o r s , a r e a p p e a r a n c e o f DNase I I a c t i v i t y was o b s e r v e d . B h a t t a c h a y a e t a l . (1977) f o u n d a s p e c i f i c p r o t e i n i n h i b i t o r o f DNase I I i n mouse n e u r o b l a s t o m a c e l l s . A marked d e c r e a s e i n t h e number o f a d h e r i n g v i a b l e c e l l s , as e v i d e n c e d b y t r y p a n b l u e s t a i n i n g , 2k h o u r s a f t e r p l a t i n g c o i n c i d e d w i t h maximal DNase I I a c t i v i t y . W i t h t h e a p p e a r -ance o f t h e DNase I I i n h i b i t o r t h e DNase I I a c t i v i t y d i m i n i s h e d a n d , a t t h e same t i m e , t h e number o f a d h e r i n g v i a b l e c e l l s i n c r e a s e d . The r e s u l t s s u g g e s t t h a t a l t h o u g h t h e amount o f DNase I I p r e s e n t i n ^ S i m i l a r r e s u l t s t o t h o s e o b t a i n e d f o r DNase I I were f o u n d f o r DNase I and a c i d and a l k a l i n e KNases. 7 cancerous tissues may be high as expected i n correlation with the rapid rate of proliferation of cancer c e l l s , the DNase II act i v i t y i n malignant tissues may be low i n vivo due to the presence of a DNase II-specific inhibitor. A low level of DNase II activity may f a c i l i t a t e the incorporation or replication of abnormal nucleic acids that are able to induce malignant transformation. (Taper et a l . , 1971b) DNase II activity levels i n relation to c e l l cycle Slor et a l . (1973) studied DNase II act i v i t y i n relation to the c e l l cycle i n synchronized HeLa S3 c e l l s . A two to seven fold increase i n DNase II activity occurred at those times when DNA synthesis was taking place, and the peaks of DNase II act i v i t y coincided with the peaks of DNA synthesis. The increase i n DNase II activity was probably due to de novo enzyme synthesis since no increase i n DNase II activity occurred when puromycin was added. Acid phosphatase, a marker for lysosomal enzyme (de Duve et a l . , 1962), did not show an induction similar to that observed for DNase II i n relation to the c e l l cycle. DNase II was assayed under conditions i n which any DNase II-speciflc inhibitor present would not be detected. (Lesca, 1976) Possible biological roles for DNase II I Since lysosomes appear to contain a l l the enzyme needed to degrade nucleic acids to nucleosides, and since DNase II activity has been found primarily i n the lysosomes, a l i k e l y biological role for DNase II i s a degradative one (Bernardi, 1971)> probably performed i n concert with 8 other lysosomal hydrolases, and associated with the many functions of lysosomes (Holtzman, 1976; Dingle and F e l l , 1969, 1973). DNase II might be involved i n the protection of the genetic s t a b i l i t y of normal cells against transforming nucleic acids (Taper et a l . , 1971ab). Spleen DNase II was found to be much more effective i n inactivating transforming DNA from Haemophilus influenzae than DNase I, endonucleose I from Escherichia c o l i , or sonication (Bernardi and Bach, 1968). DNase II may have a role i n DNA metabolism i n relation to c e l l division. Indirect support for this hypothesis comes from the correlation between DNase-levels and rate of DNA synthesis i n vivo; the appearance, specifically i n response to v i r a l infection, of a large number and variety of DNases, and from the studies of DNA replication i n vi t r o (Lehman, 1967; Lesca, 1971). The enzymology of DNase II The reaction of DNase II with DNA Three different phases may be distinguished i n the degradation of native DNA by DNase II (Bernardi, 1968), The i n i t i a l phase i s defined as the phase i n which the macro-molecular and biological properties of DNA are modified, but i n which no change occurs i n the spectral properties of the DNA and no acid-soluble oligonucleotides are formed. . . . The middle phase i s characterized by an .increase i n the absorbance of ultraviolet radiation, maximally at 260nm , of the DNA solution being 9 digested with DNase II . Oligonucleotides which are soluble i n an acidic solution such as 2% perchloric acid and which absorb ultraviolet radiation at 260 nm are also released from DNA i n this phase. The terminal phase shows a slow further increase i n the absorbance of ultraviolet radiation at 260 nm of the DNA solution and a slow further release of acid-soluble oligonucleotides absorbing ultraviolet radiation at 260 iim. The i n i t i a l phase of the reaction Studies done on the i n i t i a l phase of the DNase II reaction with DNA have demonstrated that DNase II can cleave either one strand, or both strands of native DNA i n each encounter. Oth et a l . (1958) compared the rates of decrease of viscosity of DNA observed upon reaction of the DNA with DNase I and DNase II. The logarithm of the specific viscosity of DNA versus time was plotted during degradation by the DNases at pH 5*5 under conditions on similar a c t i v i t y . Under the action of DNase I the logarithm of the viscosity of the DNA decreased slowly and non-linearly i n the i n i t i a l stages of the reaction, but with DNase II the viscosity of the DNA decreased linearly and at the same rapid rate i n the i n i t i a l stages of the reaction as during the remainder of the reaction. Oth et a l . (1958) suggested that since DNase I had been shown to cleave native DNA by random single strand scissions and since the DNase II reaction with DNA had a different viscosity curve than the DNase I reaction with DNA, DNase II might cleave both strands of the DNA simultaneously. Using electr onmicroscopy MacHattie et a l . (1963) observed a random distribution 10 of duplex fragments upon reaction of DNase II with DNA from bacterio-phage T5. Using a light scattering technique Bernardi and Sadron (I96U) followed the molecular weight decrease that occurred upon degradation of DNA with DNase II. They showed that DNase II could cleave either one or both strands of native DNA i n each encounter, and estimated that the percent of t o t a l breaks which were double-strand breaks was 67$. Young and Slnshelmer (1975) > using ultracentrifugatlon at neutral and alkaline pHs, demonstrated that DNase II degraded DNA from bacteriophage A by double-strand cleavage, and also showed that one such cleavage per DNA-molecule destroyed the i n f e c t i v i t y of the DNA. An average of four phosphodiester bonds could be hydrolysed by single-strand scissions by DNase I i n a DNA molecule before the i n f e c t i v i t y of the DNA was l o s t . Using the same ultracentrifugation technique i n neutral and alkaline solutions, estimates of the number of double-strand cleavages as a percent of the t o t a l number of cleavages of DNA by DNase II ranged from 3% (Sicard et a l . , 1973) to Q0% (Bernardi and Bach, 1968$ Kopecka et a l . , 1973). Oshima and Price (197k) have suggested that a possible explanation for this range may be the presence or absence i n the DNase II reaction mixture of divalent anions. They found that sulphate, malate, oxalate, citrate, and EDTA at low concentrations caused a 20$ increase i n DNase II a c t i v i t y . The percent of t o t a l breaks due to double-strand cleavage was found to decline from Q0% i n the absence of sulphate to k% i n the presence of concentrations of sulphate that activated DNase II . Tsubota et a l . (197*0 examined the i n i t i a l kinetics of DNase II from human gastric mucosa using as substrate twisted circular duplex DNA from bacteriophage A . 11 The hydrolysis products were treated with ATP dependent DNase from Micrococcus lutaus. This enzyme hydrolyses double-stranded DNA. i n the linear form to acid-soluble oligonucleotides, but does not at-tack circular duplex molecules (Takagi et a l . , 1972; Friedman et a l . , 1972). Sucrose density gradient sedimentation of the products revealed that gastric DNase II produced both open circular DNA and linear DNA at a very early stage of hydrolysis at 35° C. However, at 0° C, the gastric enzyme converted twisted circular duplex DNA to the open c i r -cular form by introducing single-strand scissions. These results sug-gest DNase II cleaves both strands of DNA at or near opposing nucleo-tides on adjacent strands not simultaneously, but by f i r s t making a scission i n one of the strands, and then, i n a secong reaction, making a second scission i n the other strand of the DNA at or near the same level as for the f i r s t scission. These two reactions are coupled and occur i n rapid succession at 37° C, but are uncoupled at 0° C (Tsubota et a l . , 197*0. The middle and terminal phases of the reaction The kinetics of the middle and terminal phases of degradation of c alf thymus DNA by hog spleen DNase II were investigated i n detail by Soave and coworkers (1973). During the middle phase the ultraviolet absorbance, the acid s o l u b i l i t y and the reciprocal average degree of polymerization, Pn - 1* of the oligonucleotides obtained by DNase II d i -* The average degree of polymerization, Pn was taken as the absorbance at 271 nm of the nucleotides plus terminal nucleosides produced by digestion of oligonuceotides with spleen exonuclease divided by the absorbance at 271 nm of the terminal nucleosides alone. 12 gestion increased linearly with time. When a DNA solution was digested with DNase II at 22° C, an increase i n absorbance, a hyperchromicity, at 260 nm of the DNA solu-tion and an absorbance at 260 nm due to acid-soluble oligonucleotide release f i r s t became apparent at a Pn" 1 value corresponding to a chain length of 100 nucleotides, thus defining i n terms of average length of oligonucleotide, the limit between the i n i t i a l and middle phases of the reaction,. Since the Pn" 1 value of the i n i t i a l DNA was very close to zero, i t was concluded that i n i t i a l l y a "preferred" class of nucleo-tide sequences, corresponding to about 8$ of a l l susceptible sequences, was s p l i t , possibly by the double-strand cleavage mechanism, at a faster rate than the sequences s p l i t during the middle and terminal phases of the reaction (Soave et a l . , 1973). Melting curves were done on DNase II digests containing oligonucleotides of average length between 16 and 46 nucleotides. The melting temperatures of the oligonucleotides, and the resulting hyperchronicities at 260 nm, decreased with the decreasing average size of the fragments. A comparison of the melting curves obtained for the starting DNA §xid for DNA. degraded to a Pn of 46 showed that the enzyme degradation caused a destabilization and a lowering of the Tm before any hyper-chromicity was apparent. The t o t a l hyperchromic shift (enzymatic plus heating) of the oligonucleotides was found to be equal to a constant value for a l l samples, indicating that the hyperchromatic shift caused by the enzyme was essentially due to the melting of the fragments released, and that the contribution of phosphodlester bond cleavage was negligible i n the range explored. The beginning of the terminal phase I 13 i s characterized by a sudden decrease i n the reaction rate due to the progressive melting of double-stranded DNA fragments which takes place when the average size of the oligonucleotides reaches a threshold range of values. The single-stranded fragments so originated, though s t i l l containing a large number of susceptible sequences, are poor substrates for the enzyme which shows a marked preference for native over denatured DNA (Soave et a l . , 1973). The preceding argument may not be entirely correct, however, as the results that Soave et a l . (1973) reported may be due more to the method of measurement than to an actual abrupt slow-down i n reaction rate. Since hyperchromicity was found-.to be primarily due to melting of double-stranded DNA fragments, and since at the start of the terminal phase most of the DNA. digest is single-stranded and acid-soluble, further cleavage by the enzyme would not be reflected by as much hyperchromicity or acid-soluble oligonucleotide liberation as was observed previously. That the reaction does slow down was shown by chromatography of DNase II digests of DNA on DEAE cellulose columns i n the presence of urea. Even after prolonged digestion with large amounts of enzyme most of the digestion mixture was composed of oligo-nucleotides with chain lengths greaterfcthan 5 nucleotides long.(Soave et a l . , 1973). It is l i k e l y that the decrease i n the rate of single-strand cleavage by DNase II i n the terminal phase is due to the pro-duction of progressively more resistant substrates as smaller and small-er DNA fragments are formed (Laskowski, 1967). Ik The structure of DNase II Physical and Chemical Properties DNase II acti v i t i e s have been isolated from many different tissues and appear to have similar properties to those of spleen DNase II (Cor-donnier and Bernardi, 1968). Spleen DNase II i s a basic globular pro-t e i n having a molecular weight of about 38,000, and containing a carbo-hydrate moiety, and an even number of amino acid residues for a l l the amino acids which were present at low levels.(Bernardi et a l . , 1965). Because of this latter finding and because what appeared to be monomer and dimer subunits were observed on sedimentation analysis of DNase II in naturing and denaturing solvents, Bernardi (1965) suggested that DNase II was composed of two probably identical subunits. This subunit structure was consistent with the double-strand cleavage mechanism for DNase II since one subunit could cleave one strand of the DNA at the same time as the other subunit cleaved the apposing strand (Bernardi, 1968). However, Townend and Bernardi (1971) later found that the ZImonomers" that were previously observed i n denaturing solvents (Bernardi, 1965) were of a molecular weight that was very close to that of the native enzyme molecule. Furthermore, the enzyme molecule could not be disso-ciated into subunits under a variety of conditions which are extremely effective with other proteins. Oshima and Price (1973) found that iodo-acetate alkylated a single histidine residue i n DNase II with complete destruction of enzymatic a c t i v i t y . This i s more compatible with a mono-15 mer structure for the enzyme,3or at least with a single active s i t e . Lesca (1976) has found that beef l i v e r DNase II is a complex of molecular weight 45,000 comprised of a catalytic subunit of molecular weight 26,500 and an inhibitor subunit of molecular weight 21,500. He found that the inhibitor subunit bound strongly and specifically to DNase II bound covalently to a Sepharose 4B column and could only be eluted by a solution of guanidine hydrochloride. Slor (1974) showed that the DNase II specific inhibitor i n crude and p a r t i a l l y purified extracts of mouse l i v e r , spleen and kidney could be selectively dena-tured by heating for 30 minutes at 50° C, or by lowering the pH of the solution to 2.5. SUch treatments did not denature the DNase II a c t i v i -ty and highly purified DNase II contained no inhibitor (Slor, 1974). Since highly purified DNase l i s from different tissues appear to have molecular weights of from 38,000 to 45,000 (Bernardi, I968, 1971; Dula-ney and Touster, 1972; Oshima and P r i c i , 1973), and have usually been prepared with procedures involving acidification to pH 2.5, i t remains an open question whether these purified DNase l i s contain an inhibitor subunit complexed to a catalytic subunit, or whether the inhibitor pro-t e i n has been inadvertently removed or inactivated during the purifica-t i o n procedures. A possible reason that a DNase II specific inhibitor has not been found by many laboratories i s that, at the high DNA concen-trations used for the DNase II assays based on hyperchromicity of a DNA solution or acid-soluble oligonucleotide release, the inhibitor protein i s competitively displaced from the catalytic subunit by the DNA. (Lesca, 1976). In order to be able to follow the DNase II reaction at the low con-16 centrations of DNA. that are required to observe the effects of the i n h i -bitor i t i s necessary to use radioactive DNA (Lesca, 1969, 1976; Slor, 197*0. Catalytic properties DNase II activity i s influenced by pH, ionic strength, mono- and divalent cations and anions, substrate concentration, and the presence of foreign proteins (Laskowski, 1961, 1967; Bernardi, 1968, 1971). The enzyme may be affected directly through an influence on i t s struc-ture or catalytic groups, indirectly by stabilization or destabilization of the double-stranded structure of the DNA substrate, or not at a l l , the apparent effect being due to an artifact of the assay procedure used. For example, when DNase II activity i s assayed by measuring the release of acid-soluble oligonucleotides the optimal DNA concentration i s 0.4 mg/ml (Bernardi and Griff e , 196*+) and higher subtrate concentrations appear to be inhibitory (Oth et a l . , 1958; Bernardi, 1965; Rosenbluth and Sung, 1969). This i s so because an increased DNA concentration re-sults i n fewer breaks per unit length of DNA and decreases the e f f i c i e n -cy of acid-soluble oligonucleotide release. I f a more direct method of estimating enzyme activity i s used, such as the determination of phos-phatase-sensitive phosphate, i t can be shown that "inhibition" by high substrate concentration i s an artifact of the assay procedure measuring the release of acid-soluble oligonucleotides (Rosenbluth and Sung, I969). Magnesium ion at a concentration of ImM activates DNase II indirectly due to stabilization of the double-stranded structure of DNA, the pre-17 ferred substrate for DNase II over denatured DNA. Higher concentrations of magnesium ion inhibit DNase II directly. Complications arise when DNA preparations used for assay of DNase II contain endogenous magnesium ion and when DNase II preparations of different purity are compared since the inhibitory effects of magnesium ion increase with the increasing puri-t y of the DNase II preparation (Cordonnier and Bernardi, 1968). Because their preparation of DNase II from hog spleenccatalysed the slow hydrolysis of bis-(p-nitrophenyl) phosphate and the p-nitrophenyles-ters of deoxyribonucleoside 3*-phosphate, but not those of deoxyribonu-cleoside 5*-phosphate, Bernardi and Griffe (1964) concluded that this "phosphodiesterase" activity of DNase II was an intr i n s i c property of the enzyme molecule. Hodes et a l . (1967) prepared DNase II from mouse l i v e r and found the the purified enzyme was free of nonspecific phospho-diesterase a c t i v i t y . Swenson and Hodes (1969) separated the phosphodies-terase and DNase II acti v i t i e s of bovine spleen by heating the crude pre-paration prior to chromatographic purification. Although Sicard et a l . (1970) could not separate the phosphodiesterase and DNase II acti v i t i e s of a purified preparation of hog spleen DNase I I , Slor (1970b) found that DNA did not inhibit the nonspecific phosphodiesterase ac t i v i t y of hog spleen DNase I I , and bis(p-nitrophenyl) phosphate did not inhibit the DNase II activity. He concluded that the nonspecific phosphodiester-ase ac t i v i t y observed with the highly purified DNase II of Bernardi and Griffe (1964) was probably a contaminant. Lesca (1976) was able to show that the nonspecific phosphodiester-ase was a contaminant of beef l i v e r DNase II by separation of the phos-phodiesterase (MW 59*000) from DNase II using a f f i n i t y chromatography on 18 DNase II-succinylaminooctyl-Sepharase kB• When purified DNase II ( i e : the catalytic subunit) was assayed alone, Michaelis-Menten kinetics were observed (Lesca, 1976)• A sigmoid-shaped curve of velocity versus substrate concentration was observed for the enzyme i n the presence of inhibitor at pH 5.0. The enzyme-inhibitor interaction disappeared progressively with a small pH shift from 5.0 to 5.57. L i t t l e work has been done on the active site of DNase I I . Melzer (1969) suggested that the following amino acids might be involved i n the enzyme's activity: tryptophan since N-bromosuccinimide inhibited the enzyme, methionine and/or histidine since iodoacetate and hydrogen per-oxide were inhibitory whereas beta-butyrolacetone was not, ruling out the involvement of cysteine. Since diisopropylfluorophosphate was r e l a -t i v e l y ineffective as an inhibitor, i t was concluded that serine r e s i -dues were probably not very important to the enzymatic a c t i v i t y . Oshima and Price (1973) found that DNase II could be inactivated completely by iodoacetate whereas iodoacetamide was without effect, and that the loss of enzyme activity paralleled the incorporation of one carboxymethyl group per enzyme molecule. The inactivated protein was separated from active enzyme by chromatography on phosphocellulose, and contained one residue of 3-carboxymethylhistidine as the sole pro-duct of the iodoacetate reaction. DNA at a concentration of 0.5 mg per ml protected the enzyme from inactivation by iodoacetate suggesting that the active site of the enzyme contains an essential histidine (Oshima and Price, 197*0. 19 The use of DNase II for the study of the structure  of DNA and chromatin Investigation of the primary structure of DNA DNase II enzymes s p l i t short nucleotide sequences i n DNA, and can be used to assess the frequency of these sequences i n a given DNA (Ber-nardi, 1973). The number of sequences s p l i t i s very large, i n the range of 20-50% of a l l sequences, and the susceptible sequences probably over-lap to some extent (Bernardi, 1973). Since the frequency of nucleosides at the 5 ' - and 3 ' - terminal positions of fragments formed by the action of DNase II on DNA changes with the extent of degradation (Vaneko and Laskowski, Sr., 1962), there may be one or more "preferred" sequences that are s p l i t at faster rate than the remaining sequences, and progres-sively exhausted i n the i n i t i a l phase of the reaction (Soave et a l . , 1973). The s p e c i f i c i t y of DNase II has been investigated primarily i n the middle and terminal phases of the reaction. DNase II recognizes a large number of different sequences i n these phases, and has not been used i n sequencing DNA since the DNA fragments produced would be too small and too variable i n sequence. Although DNase II may be more specific i n the i n i t i a l phases of the degradation of DNA, the base sp e c i f i c i t y of DNase II i n the i n i t i a l phase of the reaction has not been extensively investigated due to the technical d i f f i c u l t i e s of determining relative-l y small quantities of terminal nucleosides i n DNA fragments of length greater than 100 base pairs (Vaneko and Laskowski, Sr., 1962). It has 20 recently become feasible to determine the terminal nucleotides of long oligonucleotides because of the development of methods of radioactively labelling the termini of long DNA fragments and of analysing the nucleo-tide sequences (see Methods i n Enzymology 2£, 1974). Investigation of the structure of chromatin Digestion with micrococcal nuclease (EC 3.1.^ .7) of DNA i n isolated nuclei has revealed the existence of a periodic structure in chromation, consisting of nucleosomes, each one containing about 200 base pairs of DNA. (Noll, 197^). A different type of periodicity, based on a 10 nucleotide repeating unit size, has been found i n each strand of DNA i n chromatin using DNase I (EC 3.1.^ .5) (Noll, 1974b). Yaneva and Dessev (1977) studied the action of DNase I I on DNA i n chromatin, and found that the formation of acid-soluble products followed a two-phase kinetic curve. At the end of the more rapid phase about 25$ of the DNA was degraded. Early in the degradation DNA was converted into double-strandfd fragments, whose sizes were multiples of about 180 base pairs. As the degradation proceeded these fragments were reduced in size. The fragments contained single-strand nicks and, under denaturing conditions the DNA was resolved into discrete single-stranded fractions, which were exact multiples of a ten-nucleotide length and formed a pattern very similar to that observed with DNase I . A l t e n b u r g e r et a l . (1976) used DNase I I as a probe into the con-formational states of chromatin . Upon digestion of mouse l i v e r chromatin 21 with spleen DNase II a shift i n the products from a 200 to a 100 base pair repeat pattern occurred.after prior condensation of the chromatin by divalent or monovalent cations. Intestinal DNase II In rat l i v e r about 0,7% of the t o t a l number of cel l s i s newly f. formed i n one day, whereas i n rat small intestine about k3% of the t o t a l c e l l population i s newly formed per day. (Stevens et a l . , 1953). In intestine new epithelial cells continuously arise by mitosis i n the crypts of Lieberkuhn, move towards the lumen of the intestine along the walls of the v i l l i and are f i n a l l y extruded from the tips of the v i l l i into the lumen about 2k hours after mitosis. (Wilson, 1962). Desquamated epithelial cells i n the lumen of the.intestine are respon-sible for many of the enzymes found free i n the intestinal t r a c t , and intestinal intracellular enzymes such as DNase II may, after autolysis, be involved i n degrading dietary nucleic acids i n the small intestine prior to absorption as the nucleoside and the free base components. (Wilson, I962). Thus DNase II may have an additional digestive function in the small intestine. DNase II may also have a role i n maintaining the highly prolifera-t i v e , but differentiated state of the normal intestinal mucosa since A l l f r e y and Mirsky (1952) found a correlation between the higher rate of desquamation and the higher DNase II activity i n adult mucosa as opposed to lower rates for both i n f e t a l intestinal mucosa. Lieberman 22 et a l . (1971) separated crypt and v i l l u s c e l l s of rat small intestine by a crude scraping method; found that DNase II activity was associated with the crypt c e l l preparation, and suggested that DNase II plays a role i n the metabolism of the actively dividing crypt cells rather than a digestive one. Stewart and Zbarsky (1963) reported the loss of nucleic acids, particularly DNA, from mucosal scrapings or slicessfrom rat intestine during incubation of the preparations i n vitro i n Krebs Ringer phosphate buffer, pH 7«>8. A crude cell-free extract of the mucosal tissue i n Krebs^ Ringer phosphate buffer was found to have high levels of DNase activity (Lee and Zbarsky, 1967). Two DNases were subsequently demon-strated to be present i n cell-free extracts of rat intestinal mucosa (Lee, Lawrence and Zbarsky, 1972). One DNase had optimum activity at neutral pH and required magnesium ion for acti v i t y , and thus was a DNase I (Kunitz, 1950). The second enzyme had an acidic pH optimum and showed no requirement for magnesium ion and thus was a DNase II (Maver and Greco, 1948). The present investigation This research was begun i n order to investigate the properties of intestinal DNase II and the nature of the enzymiclreaction with DNA. DNase II was f i r s t isolated from extracts of rat intestinal mucosa. However, the amount of DNase II obtained after three purification steps was too l i t t l e to be used for further purification and then for enzymatic 23 studies. In order to obtain larger amounts of enzyme, DNase II was isolated from bovine intestinal mucosa. Upon chromatography of a crude extract of intestinal mucosa on a DEAE cellulose column, two peaks of DNase II activity were observed. The f i r s t DNase II had properties similar to those of DNase II enzymes which have been isolated from many different tissues and purified and characterized to various degrees, (Laskowski, 196l> 1967} Bernardi, 1968, 1971). The second DNase II acti v i t y was similar to other DNase II acti v i t i e s which have been isolated from only a few tissues and studied only to a small extent, (Cordonnier and Bernardi, 1968"; Yamanka et a l . , 197k; Zollner et a l . , 197^ ). DNase II activity has been found associated with lysozomes (de Duve et a l . , 1962), and presumably with a degradative function (Bernardi, 1971)» and with nuclei (Lesca, 1968; Slor and Lev, 1971} Slor, 1973) and a possible role i n DNA metabolism i n relation to c e l l division (Slor et a l . , 1973). The DNase II activities associated with separate organelles and with diverse biological functions could be due to distinct enzymes with different properties. The properties of the two intestinal DNase II acti v i t i e s were com-pared i n order to determine whether the acti v i t i e s were due to Ithe same or different enzymes. Different amounts of inhibition of the two DNase II act i v i t i e s by ionic strength and by sulphate, an inhibitor of DNase I I , were observed. The f i r s t DNase II degraded Lnatlve DNA at a more rapid rate than denatured DNA whereas the second activity degraded both native and denatured DNA at the same rate. The activation energies for the hydrolysis of DNA by the two DNase II activities were also different. 2k Further investigation revealed, however, that the appearance of a second DNase H acti v i t y was probably due to the binding of a small amount of DNase II to DNA which had been bound to the DEAE cellulose column. The observation of different properties for the two DNase II acti v i t i e s was l i k e l y due to the presence of DNA i n the second DNase II preparation. DNase II enzymes from other tissues have been investigated i n order to evaluate their usefulness as tools for sequencing DNA. Vaneko and Laskowski, Sr. (1962) have presented evidence that the base speci f i c i t y of DNase II cleavage of DNA changes as the reaction progresses. Bernardi et al.--(l973) analysed products from the middle and terminal phases of the DNase II reaction and found that the base speci f i c i t y did not vary much i n these phases. DNase II recognizes a large number of nucleotide sequences i n DNA, but there may be a preferred class of sequences that is exhausted i n the i n i t i a l phase of the reaction (Soave et a l . , 1973). The reaction of intestinal DNase II with DNA was studied i n order to determine whether the enzyme had a different base speci f i c i t y than DNase l i s from other tissues, whether the base speci f i c i t y changed as the reaction progressed, and whether there was any preferred class of sequences that were cleaved i n the i n i t i a l phase of the reaction. Intestinal DNase II was found to have a similar base sp e c i f i c i t y as spleen DNase I I . The base sp e c i f i c i t y changed as the reaction progressed, primarily i n the i n i t i a l and terminal phases of the reaction. No evidence was obtained for a greatly increased speci f i c i t y of cleavage of DNA by DNase II i n the early stages of the reaction. The studies of the base spe c i f i c i t y of the cleavage of DNA by DNase II suggest that the enzyme 25 recognizes a large number of nucleotide sequences i n DNA. and that some sequences are preferred over others, particularly GpG i n the i n i t i a l stages of the reaction. Intestinal DNase IT was investigated i n order to compare i t s enzymic properties with those of DNase II enzymes from other tissues. The chromatographic behavior, pH optimum, activity i n the presence of different ions, molecular weight, and mode of action on DNA of intes-t i n a l DNase II were similar to the respective properties of DNase II enzymes isolated from other tissues. DNase II was also studied i n v i t r o i n order to provide a basis for future research into the biological role of DNase II i n the meta-bolism of intestinal mucosa, a differentiated yet rapidly proliferating tissue, i n vivo. 26 MATERIALS AND METHODS Materials Chemicals which were used i n this investigation are l i s t e d i n Table Ij chromatographic media, i n Table IIj enzymes, and animals, in Table III. TABLE I CHEMICALS USED IN THE INVESTIGATION Material Abbreviation Manufacturer/Supplier Comments diisopropylfluorophos-f ate pFP Aldrich Chemical Co., Milwaukee, Wis. nerve poison since inhibits acetyl-cholinesterase (Aldridge, 1953) antidote: atropine sulfate, 2 mg i.m. q 15 min. phenylmethylsulfonyl fluoride acrylamide N, N* methylene bisacry-1amide riboflavin ammonium persulfate N, N, N*, N' tetrame-thylethylene diamine B-alanine PMS bis TEMED Sigma Chemical Co., St. Louis, Mo. Eastman Kodak Co., Rochester, N. Y. E. C. Apparatus Co., Philadelphia, Pa. Matheson, Coleman and Bell, Norwood, Ohio does not inhibit acetylcholinester-ase (Fahiheza£ Gold; 1973) but less effective in inhibiting proteases due to low solubility in aq. solution - J acrylamide and bis were recrystallized from chloroform and aetone, respec-tively, as described by Loening (1967) Coomassie B r i l l i a n t Blue protamine sulfate Schwartz-Mann, Orangeburg, N. Y. agar Difco Laboratories, Detroit, Mich. Material Abbreviation 2 , 5 S d i p h e n y l o x a z o l e PPO p-bisC2-(4-methyl-5- dimethyl phenyl oxazolyl)] POPOP benzene bovine serum albumin BSA fraction v tr i s (hydroxymethyl) t r i s amino-methane dithiothreitol DDT the 4 major 2'-deoxy- 3*-dNMP's ribonucleoside-3'-phos-phates calf thymus DNA type I salmon DNA type III Escherichia c o l i DNA type VIII bacteriophage ^ DNA X DNA TABLE I—Continued Manufacturer/Supplier Comments Kent Laboratories, Vancouver, B. C. Packard InstrumentsCo., La Grange, 111. Armour Pharmaceutical Co., Chicago, i l l . Sigma Chemical Co., St. Louis, MO. oo The DNA's were dissolved in a lOmM sodium chloride solution by s t i r r i n g at 4° C over a one to two day period. The calf thymus DNA was dissolved at a concentration of 4 mg per ml so that the same solution of DNA could be used for 3 different DNase II assays (see Methods). The other DNA's were dissol-ved at a concentration of o.4 mg per ml so that a ten-fold dilution would give a solution of about the optimal absorbance for measurement of DNase II activity by the hyperchromiaity assay (see Methods) it Material TABLE I—Continued Abbrev i a t ion Manuf acturer/Supplier Comments polydeoxyadenylate- polydAT (deoxy)thymidylate yeast RNA adenosine-3'-phosphate 3'-AMP p-nitrophenyl thymidine- P-NC>20pT 5'-phosphate the 4 major 2'-deoxy- 5'-dNMP's ribonucleoside-5'-phos-phates disodium p-nitrophenyl- p-NO20P phosphate p-nitromethylthymidine- p-N02<8Tp 3'-phosphate Miles Laboratories, Elkhart, Ind. Calbiochem, La J o l l a , C a l i f . to Raylo Chemicals Ltd. Edmonton, Al t a . polydeoxyadenylate polydA P. L. Biochemicals Inc. Milwaukee, Wis. oligo(deoxythymidyl-a t e ) ^ N o r i t A o l i g o ( p T ) 1 Q Fischer S c i e n t i f i c Co. Triethylammonium b i -carbonnate TEAB Prepared by bubbling carbon diox-ide through a 1 molar s o l u t i o n of r e d i s t i l l e d triethylamine u n t i l the pH decreased to 7.5 (Porath, 1955) Material TABLE I—Continued Abbrev iat ion Manuf acturer/Supplier Comments Casein (Technical grade) thymidineCmethyl-3Hj Nutritional Biochemical Corp., Cleveland, Ohio New England Nuclear, Boston, Mass. Contained at least 85% thymidine- H as determined by thin layer chroma-tography on MN 300 cellulose plates run in d i s t i l l e d water according to Randerath and Randerath (1967) (thy-mine, Rf, o.65; thymidine Rf, 0.80) sonicated H DNA from ° H DNA Escherichia c o l i B [C8-3HJ polydeoxy- polydA - 3 H adenylic acid 3 3 methyl H polythy- polydT- H midylate Miles Laboratories Elkhart, Ind. Specific A c t i v i t i e s ; H DNA polydA-3H polydT- g polydAT- H 127 uCi/umole phosphate 31.4 55.7 19.8 " o polydeoxyadenylate- polydAT- H thymidylate (methyl-3H) tK-32P labelled adeno- [<X-32p] ATP sine triphosphate y - 3 2 p labelled adeno- llf-32pli ATP sine triphosphate 32 P labelled inorganic phosphate 32 Pi New England Nuclear or Amersham/Searle Specific activities of from 2 to 20 Ci/umole TABLE II CHROMATOGRAPHIC MEDIA USED IN THE INVESTIGATION Materials Manufacturer/Supplier Comments MN 300scellulose plates (20 x 20 cm) polyethyleneimine impreg-nated MN 300 c e l l u l o s e plates (20 x 20 cm x 0.1mm) Whatman Chromatography Products. DE 81 c e l l u l o s e paper and ion exchange c e l -luloses DEAE 22, 32, CM 22, 32, and phosphocellulose < p l l > Sephadex G25, G50 and G100 f i n e , sulfopropyl (SP) Sephadex G50 and G100 and s u l f o e t h y l (SE) Sephadex C25 Schleicher and Schwell Inc. Keene, N. H. Brinkman Instruments (Canada) Ltd. Toronto, Ont. Mandel S c i e n t i f i c Co. Ltd. Montreal, Quebec Pharmacia (Canada) Ltd. Dorval Quebec When samples with Rf's greater than 0.7 were to be chromatographed, the plates were prerun with d i s t i l l e d wa-ter to remove a yellowish material which accumulated on the plates du-r i n g storage and which ran near the solvent f r o n t . DEAE and CM c e l l u l o s e were precycled with 0.5N NaOH and 0.5N HC1 according to the manufacturer's i n s t r u c t i o n s and were e q u i l i b r a t e d with the s t a r -t i n g buffer (with respect to both pH and conductivity) before use. The Sephadex media were swollen i n s t a r t i n g buffer f o r the recommended times and the ion exchange Sephadex-es were e q u i l i b r a t e d with s t a r t i n g b u f f e r before use. co t-1 Molecular weight c a l i b r a t i o n k i t : RNase A, MW 13,700 chymotripsinoglo A, MW 25,000 ovalbumen, MW 45,000 aldolase, MW 158,000 Materials TABLE II—Continued Manufacturer/Supplier Comments blue dextran, MW 2 x 10 hydroxyapatite Munktell 410 c e l l u l o s e Pharmacia (Canada) Ltd. Dorval, Quebec Bio-Rad Laboratories, Richmond, C a l i f . DNA c e l l u l o s e " DNA c e l l u l o s e was prepared with native and with denatured c a l f thymus DNA and Munktell 410 c e l l u l o s e by the method of Alberts and Herrick (1971). TABLE I I I ENZYMES AND ANIMALS USED IN THE INVESTIGATION M a t e r i a l s M a n u f a c t u r e r / S u p p l i e r Comments t r y p s i n DNase I (EC 3.1.4.5) f r o m b o v i n e pancreas DNase I I (EC 3.1.4.6) fr o m hog s p l e e n a l k a l i n e phosphatase (EC 3.1.3.1) fr o m E s c h e r i c h i a c o l i a c i d p h o s p h a t a s e (EC 3.1.3.2) fr o m p o t a t o p h o s p h o d i e s t e r a s e I (EC 3.1.4.1) from C r o t a l u s adamentus venom Schwartz-Mann Orangeburg, N. Y. P. L. B i o c h e m i c a l s Milwaukee, Wis. S p e c i f i c A c t i v i t y 52,000 u n i t s / m g by t h e method o f K u n i t z (1950) 52,800 u n i t s / m g 47 u n i t s / m g 67 u n i t s / m g co CO W o r t h i n g t o n p r o d u c t p u r - 1 chased from ICN Canada L t d . M o n t r e a l , Quebec ,06 u n i t s / m g I n a c t i v a t i o n o f c o n t a m i n a t i n g 5 ' n u c l e -o t i d e s was done by t h e method o f S u l -k o w s k i and L a s k o w s k i ( 1 9 7 1 ) . A f t e r t r e a t m e n t 96% o f t h e o r i g i n a l phos-p h o d i e s t e r a s e a c t i v i t y and 3.3% o f t h e 5' n u c l e o t i d a s e a c t i v i t y r e m a i n e d . p h o s p h o d i e s t e r a s e I I (EC 3.1.4.18) from s p l e e n 0.32 u n i t s / m g w i t h t h e a s s a y u s i n g p-N0 2#Tp 22 u n i t s / m g w i t h t h e a s s a y m e a s u r i n g t h e c o n v e r s i o n o f 3'-phosphate o l i g o n u c l e -o t i d e s t o 3*-phosphate n u c l e o t i d e s . TABLE Ill—Continued Materials Manufacturer/Supplier Comments RNase A (EC 2.7.7.16) from bovine pancreas polynucleotide kinase (EC 2.7.1.78) from T4 x«-l infected E. c o l i B terminal transferase (EC 2.7.7.31) from calf thymus Sigma Chemical Co. St. Louis, Mo. P. L. Biochemicals Milwaukee, Wis. Miles Laboratories Elkhart, Ind. 76.5 units/mg 8,700 units/mg where one unit catalyses the transfer of 1 mole of phosphate from ATP to polynucleotide in 30 min. at 37°C 2,500 units/mg where one unit catalyses the incorporation of 1 mole of dAMP into acid insoluble material in 1 hour at 37°C male Wistar rats Animal unit of the Uni- The rats' weight was 180-200 g after 24 versity of British Columbia, hours of starvation. cattle Intercontinental packers 8950 Shaunessy, Vancouver Small intestines were removed from cat-tl e about 10-15 minutes after slaughter and were tied off, placed in an ice-wa-ter bath and transported to the univer-sity 35 Methods Measurement of DNase II activity Acid Soluble oligonucleotide assay This assay was used to determine DNase II activity by measuring the absorbance at 260 nm of the acid-soluble oligonucleotides liberated from DNA upon digestion with the enzyme. (Bernardi, I96U). DNase II was incubated at 37°C with UOO ugm of calf thymus DNA i n 120 mM sodium acetate buffer pH 5.0 containing 8 mM ethylenediamininetetracetate (EDTA) i n a t o t a l volume of 1.0 ml. After periods of time which ranged from 5 to 30 minutes, depending on the activity of the enzyme, 0.5 ml of cold 6% perchloric acid was added to stop the reaction. The reaction mixture was chilled i n an ice-water bath for 10 minutes and then centri-fuged at 12,000 xg for 10 minutes i n a Sorvall RC-2B refrigerated centri-fuge. The absorbance at 260 nm of the supernatant solution was measured with respect to a substrate blank i n which the DNA had been incubated without enzyme, the perchloric acid added, and then the enzyme added. The readings were also corrected for dilution with perchloric acid. As Illustrated i n F i g . 1, the absorbance at 260 nm of the acid-soluble oligonucleotides released increased linearly with time between the absorbance values of 0.8 and 3.5. When lower concentrations of enzyme were used, there was an i n i t i a l lag phase i n which the increase i n absorbance was not linear with time. This lag phase was due to the 36 Fig. 1 Release of acid-soluble oligonucleotides from calf thymus DNA by DNase II. DNase II was incubated at 37°C with 400 ugm DNA in 1.0 ml of 120 mM sodium acetate buffer, pH 5.0, containing 8 mM EDTA. The absorbance at 260 nm of the oligonucleotides which were soluble in 2% perchloric acid was measured with respect to a substrate blank solution in which the enzyme had been added after the perchloric acid. 38 hydrolysis of DNA. hy DNase II i n i t i a l l y to fragments which were not acid-soluble (Roseribluth and Sung, I969). The effect of enzyme concentration upon the release of acid-soluble oligonucleotides from DNA i s shown.in Fig. 2. The absorbance at 260 nm of the oligonucleotides released increased linearly between the absorbance values of 0.8 and 3.5 as the enzyme concentration was increased. In this linear range the acid-soluble oligonucleotide assay can be used to measure DNase II activity i n both crude and purified preparations of DNase II because the acid precipitation procedure c l a r i f i e s murky solutions so that the absorbance can be read without interference. The assay has therefore been used to • measure DNase II activity at different stages of the purification procedure i n order to estimate the extent of purification and to compare the properties of different DNase II preparations. One unit of DNase II activity i s defined as the amount of enzyme that liberates acid-soluble oligonucleotides with an absorbance of 1.0 at 260 nm i n 1 minute under the conditions of this assay. The specific a c t i v i t y was obtained by dividing the enzymic activity by the milligrams of protein present i n the same volume of solution. 39 F i g . 2 Effect of different concentrations of DNase II on the release'; of acid-soluble oligonucleotides from calf thymus DNA. DNase II was incubated for 20 minutes at 37°C with kOO ugm DNA i n 1.0 ml of 120 mM sodium acetate buffer, pH 5.0, containing 8 mM EDTA and the absorbance at 260 nm of the acid-soluble oligonucleotides released was measured. kl DNA-agar gel assay This assay i s a modification of the assay used by Jarvis and Lawrence (1969) to measure DNase I acti v i t y . The diameter of the zone of clearing produced by the action of DNase II on DNA embedded i n agar gel was measured after the unhydrolysed DNA had been precipitated with hydrochloric acid. Agar, 200 mg, was dissolved in 15 ml of 150 mM sodium acetate buffer, pH 5*0, containing 10 mM EDTA by heating the mixture u n t i l the boiling temperature was just reached. The solution was allowed to cool to about 6o°C, and 5 ml of a solution containing 20 mg calf thymus DNA i n 10 mM NaCl were added. Three m i l l i l i t r e portions of the resulting solution were pipetted onto microscope slides over areas of 2 by 3 inches bordered by cellulose tape. After the gels had set, holes 2 mm i n diameter were punched (with a gel puncher from Biorad), and k u l portions of the DNase II containing solutions were added to the wells. The slides were placed i n a covered plastic box containing a moist paper towel, incubated at 37°C overnight, and then immersed i n 1 N HC1 for a few minutes. In Fi g . 3 i t may be seen that the diameters of the zones of clearing, (minus the diameters of the holes) were proportional to the logarithms of the enzyme concentrations. Since a large number of solutions could be assayed for DNase II simultaneously, this method was used to measure DNase II i n fractions from column chromatography. Because the diameter of the zone of clearing varied as the logarithm of the DNase II concentration, a small error i n the deter-mination of a diameter resulted i n a larger relative error i n the calcu-lat i o n of the corresponding DNase II concentration. Therefore, the F i g . 3 The l i n e a r r e l a t i o n s h i p between the logar i thm o f the DNase II concentra t ion and the diameter o f the zone o f c l e a r i n g determined by the DNA agar ge l assay. D i f f e ren t d i l u t i o n s o f the same s o l u t i o n conta in ing DNase II a c t i v i t y were incubated at 37*C overnight with 0.1% c a l f thymus DNA embedded i n 1% agar ge l i n sodium acetate b u f f e r , pH 5.0 conta in ing EDTA. The unhydrolysed DNA was p rec ip i t a t ed with 1 N HCl and the diameters of the zones o f c l e a r i n g (minus the diameters o f the wel ls i n which the enzyme so lu t ions had been placed) were found t o be l i n e a r with the logarithms of the DNase II concentrat ions . Th is assay i s a mod i f i ca t ion o f the assay used by J a r v i s and Lawrence (1969) t o measure DNase I a c t i v i t y . E E CD C \_ a O M — O <D C o N O -4—» E a 02 0.5 1 2 3 4 5 6 7 8 9 1 0 L o g ( r e l a t i v e D N a s e I c o n c e n t r a t i o n ) kk DNase II activity could not be measured as accurately with this assay as i t could be with the acid-soluble oligonucleotide assay i n which there was a linear relationship between the absorbance of the acid-soluble oligonucleotides liberated and the DNase II concentration. Hyperchromicity assay The increase i n absorbance, the hyperchromicity, of a DNA. solution-upon digestion with DNase II was measured by a modification of the method used by Kunitz (1950) to determine DNase I act i v i t y . The enzyme was incubated with i*Ougm calf thymus DNA i n 1.0 ml of 100 or 150 mM sodium acetate buffer containing 10 mM EDTA. The solutions were maintained at 37 °C i n cuvettes placed i n the thermostated com-partment of a Gilford model 2000 or a Cary model 15 spectrophotometer. F i g . k i s a tracing of a plot of the increase i n absorbance at 260 nm versus time obtained from the Gilford spectrophotometer, with respect to the absorbance of a solution containing only buffer and substrate. The same i n i t i a l lag phase observed using low enzyme concentrations i n the acid-soluble oligonucleotide assay was also seen here, presumably for the same reason. In Fig. 5 a linear plot was obtained when the slopes from the linear portions of the curves i n F i g . k were plotted against the different amounts of enzyme that were used. The advantage of this assay was that each determination of DNase II activity resulted i n a curve of hyperchromicity versus time instead of the single values that were obtained for the two previous assays. The disadvantage was F i g . k Increase i n absorbance of a DNA. solution upon treatment with DNase II. DNase II was reacted at 37°C with kO ugm DNA, i n 1.0 ml of 150 mM sodium acetate buffer pH 5.0, containing 10 mM EDTA. The reaction was continuously monitored at 260 nm with respect to a control solution which contained no enzyme and which was set to read zero absorbance at 260 nm. •p-h7 that the assay could not he used with crude enzyme preparations due to the formation of a precipitate which interfered with the measurement of the absorbance of the DNA solution. %-DNA assay The acid-soluble radioactive oligonucleotides released from sonicated %-DNA from Escherichia c o l i by digestion with DNase II were measured by a modification of the method of Lesca (1976). DNase II was incubated with %-DNA at 37° C i n 1.0 ml of 150 mM sodium acetate buffer, pH 5.0, containing 10 mM EDTA. At various times 0.1 ml aliquots were removed and added to a mixture of 0.2 ml of 6% HClOi,. and 10 u l of bovine serum albumin solution (50 mg/ml). The mixtures were allowed to stand for 10 minutes at 0°C and then centrifuged at 12,100 xg for 10 minutes. Aliquots (200 ul) of the supernatant solutions were added to 10 ml portions of a napthalene fluor (see: Measurement of radioactive compounds), and the solutions were counted i n the 3H channel of a Packard Model 25^ 3 l i q u i d s c i n t i l l a t i o n counter. In Fig. 6 the counts per minute of acid-soluble radioactivity released from %-DNA by DNase II action i s plotted as a function of time. Although the curve was linear for less than five minutes, there was no i n i t i a l lag phase present when either sonicated 3H -DNA from E. c o l i or polydAdT-methyl- 3H was used. This simplified the determination of i n i t i a l velocities. Fig, 5 Effect of different concentrations of DNase II on the hyperchromicity of a DNA. solution. Points on this curve correspond to slopes taken from the linear portions of curves similar to that shown i n F i g . k. The assays were done under the conditions described i n F i g . k for different amounts of enzyme. V o l u m e of e n z y m e s o l u t i o n (jul.) 50 T i m e ( m i n . ) F i g . 6 Rate of release of t r i t i a t e d acid-soluble oligonucleotides from sonicated 3H-DNA from Escherichia c o l i DNA. The reaction was done at 37*C i n 150 mM sodium acetate buffer, pH 5.0, containing 10 mM EDTA. The oligo-nucleotides that were soluble i n k% perchloric acid were counted i n a napthalene based li q u i d s c i n t i l l a t i o n (fluid. 51 Measurement of the acti v i t i e s of other nucleases DNase I DNase I activity was measured by a modification of the method of Kunitz (1950). The increase i n absorbance at 260 nm of a solution containing kO ug calf thymus DNA. i n 1,0 ml of 0.1 M TrisHCl, pH 7.2, 10 mM MnClg upon treatment with enzyme solution was measured at 37° C using a Gilford model 2000 spectrophotometer. One unit of DNase I activity was defined as the amount of enzyme that coused an increase i n absorbance of 1.0 x 10"^  per minute at 260 nm. Alkaline phosphatase Solutions containing alkaline phosphatase were incubated at 37°C with 1 mM p-nitrophenylphosphate i n 0.6 M TrisHCl, pH 8.0, and the absorbance at kOO nm of the p-nitrophenol released was recorded as a function of time. (Torriani, 1966). One .Unit of enzyme activity was defined as the amount of enzyme that released 1 umole of p-nitrophenol per;:minute under,the above assay conditions. The molar absorbance (£) for p-nitrophenol was taken to be 17 x 10^  (Hynie and Zbarsky, 1970). A similar definition for units of enzyme activity was used for phospho-diesterase I and II and acid phosphatase. 52 Phosphodiesterase I Incubation was carried out at 37°C with O.k mM p-nitrophenyl-thymidine-5 ,-phosphate i n 33 mM TrisHCl, pH 8.9, containing 0.5 mM CaCl2. The absorbance at kOO nm of the p-nitrophenol released was recorded as a function of time (Hynie and Zbarsky, 1970). 5' nucleotidase Contaminating 5' nucleotidase present i n venom phosphodiesterase from Worthington was measured before and after inactivation by the method of Sulkowski and Laskowski (1971). Solutions containing 5' nucleotidase were incubatedi for 2h hours at 37°C with 3 mM adenosine-s' -phosphate i n a 200 mM glycine-NaOH, pH 9.0, containing 10 mM MgCl2. The reaction was stopped by the addition of an equal volume of 20% trichloroacetic acid and the phosphate that had been released was measured by the method of Ames (1966). Acid Phosphatase Solutions containing acid phosphatase were incubated . at 37°C with 1 mM p-nitrophenylphosphate i n 150 mM sodium acetate buffer, pH 5.0, containing 10 mM EDTA. After 10 minutes the reaction was stopped by the addition of 200 u l of 2 N NH^ OH and the absorbance of the solution was measured at UOO nm. A substrate blank solution was prepared by incubation 53 of the substrate, addition of the NH^ OH, and then addition of the enzyme. The absorbance at kOO nm of this solution was subtracted from that of the assay solution. Phosphodiesterase II Assay #1. Solutions containing phosphodiesterase II were incubated with 1 mM p-nitrophenylthymidine-S1-phosphate i n 0.15 M sodium acetate buffer, pH 5.0, containing 10 mM EDTA. After 10 minutes at 37 °C the reaction was stopped by the addition of 200 u l of 2 N MfyOH and the absorbance of the solution was read at 1+00 nm. The absorbance at U00 nm of a substrate blank solution which was prepared i n a similar manner as the blank solution for acid phosphatase was prepared, was subtracted from the absorbance reading taken for the enzymic reaction. Assay #2. Deoxyribooligonucleotide-phosphate, 0,8 umoles, the product of exhaustive digestion of calf thymus DNA with hog spleen DNase II (P.L. Biochemicals), was incubated with solutions containing phospho-diesterase II i n 0.//M sodium acetate buffer, pH 5.0, containing 10 mM EDTA. After 10 minutes at 37'C, an equal volume (1,0 ml) of 2.5% perchloric acid containing 0.25% uranyl acetate was added. The mixture was cooled at O^ C for 10 minutes and then centrifuged at 12,000 xg for 10 minutes at k°C. The absorbance of the supernatant solution was read at 260 nm, and the absorbance at 260 nm of a substrate blank was subtracted. (Bernardi and Bernardi, 1966). One activity unit i s the amount of enzyme that liberates mononucleotides having an absorbance of 1.0 at 260 nm per minute under the conditions of the assay. 5h Acid RNase The solution containing acid RNase was incubated with 0.4 mg yeast RNA i n UO ml of 100 mM sodium acetate buffer, pH 5.0, containing 10 mM EDTA. After 30 minutes at 37°C the reaction was stopped by the addition of 0.5 ml of 6% perchloric acid. The mixture was cooled at 0*0 for 10 minutes, and then centrifuged at 12,100 xg for 10 minutes at 4°C. The absorbance of the supernatant solution was read at 260 nm and the absorbance at 260 nm of a substrate blank was subtracted. One a c t i v i t y unit i s the amount of enzyme that liberates acid-soluble oligonucleotides having an absorbance of 1*0 at 260 nm under the con-ditions of the assay. Alkaline RNase The assay procedure was the same as that described for acid RNase except that the buffer used was 20 mM TrisHCl pH 7.5. 55 Measurement of Protein Protein content was determined by the method of Lowry et a l . (1951) using standard curves which were constructed by measuring different quantities of bovine serum albumin. Protein content was also estimated by absorbance at 280 nmj 1 unit of absorbance at 280 nm was taken to indicate a protein concentration of 1.2 mg per ml. (Kunitz, 1950) This la t t e r method was useful i n estimating the protein content of solutions containing high concentrations of Tris buffer since Tris has been found to interfere with the determination of protein by the method of Lowry et a l . (1951) (Rej and Richards, 197*0 Measurement of DNA DNA was measured by determining deoxyribose content with diphenyl-amine by a modification of the method of Dische (Burton, 1968). Calf thymus DNA (Sigma, type l ) was used as a standard. Measurement of Phosphate Inorganic and t o t a l phosphate were determined by the method of Ames (1966) using sodium phosphate solutions as standards. 56 Measurement of Proteolytic Activity Proleolytic activity was measured by the method of Kunitz (1947) as described by Laskowski (1955). A solution containing proteolytic enzymes was incubated with 0.5$ casein i n 100 mM phosphate buffer, pH 7.6, at 37*C. The reaction was stopped by the addition of trichloroacetic acid (TCA) to 1% f i n a l concentration, and the mixture was cooled at 0°C for 10 minutes and then centrifuged at 12,100 xg for 10 minutes. The extent of proteolysis was determined by reading the absorbance at 280 nm. An enzyme blank solution was prepared by incubation of the enzymic solution i n the absence of substrate, addition of the trichloroacetic acid, and then addition of the casein substrate. The absorbance at 280 nm of the enzyme blank was subtracted from that of the reaction mixture. This served as a correction for proteolysis of any endogenous protein present i n the solution containing the proteolytic enzymes. In order to obtain an estimate of the amount of proteolytic enzyme present i n a given solution, the solution was assayed for proteolytic activity by the casein digestion assay and the resulting absorbance at 280 nm was compared with absorbances at 280 nm resulting from the digestion of casein with known amounts of trypsin. In Fig. 7 the absorbance at 280 nm of acid-soluble fragments of casein released by trypsin digestion i s plotted against the quantity of trypsin used i n the assay. 57 jug. T r y p s i n F i g . 7 Effect of different amounts of trypsin on the release of acid-soluble fragments from casein. Trypsin was reacted at 37°C with 0.5$ casein i n 100 mM phosphate buffer, pH 7.6, " ^ and tfce absorbance at 280 nm of casein fragments that were soluble i n 1% trichloroacetic acid was measured. 58 Measurement of radioactive compounds 32p_labelled compounds were counted i n 10 ml of aqueous solution by Cerenkov radiation i n the tritium channel of a Packard 2k25 liquid s c i n t i l l a t i o n counter. Cerenkov radiation i s the electromagnetic radiation emitted when a charged particle passes through a dielectric medium at a greater velocity than the velocity of light i n that medium. (Parker and E l r i c k , 1970). Electronically polarized molecules, which are produced along the path of the charged particle, return to their ground state with the emission of electromagnetic radiation. When the velocity of the charged particle i s greater than that of light i n the medium, there i s a certain direction i n which the emitted electromagnetic radiation interferes constructively. Although 90$ of the 32 p^-spectrum is above the Cerenkov threshold, most of the continuous spectrum of radiation i s directionally emitted i n the ultraviolet and counting i n the 3H channel of a liquid s c i n t i l l a t i o n counter is consequently of relatively low efficiency. An efficiency of 35$ for Cerenkov radiation was estimated by ;comparing the observed counts per * minute with the manufacturers' data accompanying the 32p compounds. The counts per minute were corrected for the decay of the 32p# o Tritiated compounds were counted i n the tritium channel of the Packard 21+25 liquid s c i n t i l l a t i o n counter using > napthalene fluor that was composed of 15 gm 2,5-diphenyloxazole (PPO), 150 mg p-bls[2-(l+-methyl-5-phenyl-oxazolyl)] benzene (dimethyl POPOP), 2l+0 gm napthalene, and 1 l i t r e each of toluene, dioxane and absolute ethanol (Chiu and Sung, 1972). 59 The counts per minute were not corrected for quenching, hut a l l had similar external channel ratios. 60 Preparation of thymidine labelled DMA, from the mucosa of rat small intestine A male Wistar rat weighing 180-200 gm was starved for 24 hours and then anesthetized with ether, %-methyl thymidine (1.3 x 10^ cpm i n 200 u l of water) was injected into the t a i l vein and the rat was allowed to recover from the effects of the anesthesia. Thirty minutes after the injection the rat was stunned by a blow to the head and decapitated. Intestinal DNA was prepared by the method of Colter et a l . (1962) as modified by Meyer (1964). Prom 5 gm of fresh intestinal mucosa 31.5 mg DNA were recovered with a t o t a l radioactivity of 18.7 x 10^ (1.4$ of the injected counts) and with a specific radioactivity of 592 cpm per mg DNA (219 cpm/umole DNA-phosphate). Unlabelled rat intestinal DNA was also prepared: 22.3 mg DNA were recovered from 4 gm of wet mucosa. Characterization of the DNA preparations from rat intestine and calf thymus Thermal denaturation curves for DNA from rat intestine and calf thymus were obtained by the method of Mandal and Marmur (1968). DNA at a concentration-of 20 ug/ml was heated i n 150 mM sodium chloride-15 mM sodium citrate pH 6.5 (standard saline citrate) solution i n the thermostated chamber of a Gilford model 2000 spectrophotometer. The temperature rise i n 0 C and increase i n absorbance at 260 nm of the DNA solution with respect to a blank solution containing standard saline citrate buffer were continuously recorded versus time. The absorbance values were corrected for the thermal expansion of the solutions, and the ratio of the absorbance at 260 nm at particular temperature t to the absorbance 61 at 25° was plotted as a function of the. temperature t . Fig. 8 shows the thermal denaturation curve obtained for DNA. isolated from rat intestine. The midpoint of the transition (Tm) was at 85.7°. This corresponded to a GC content of kO% as calculated from the equation GC = (Tm - 69.3) x 2.kk (Mandel and Marmur, 1968). The increase of the relative absorbance from 1.0 at 50° (not shown) to about 1.05 at 70° indicated that there.'was a low molecular weight species i n the DNA preparation that melted i n this low temperature range. Since there was a large transition at a relatively high temperature, most of the prepa-ration consisted of native DNA molecules. Fig. 9 depicts the thermal denaturation curve obtained for DNA from calf thymus. The midpoint of the transition was 84.9° and corres-ponded to a GC content of 38$. Table IV gives the results of ultraviolet and phosphate analyses of the DNA which were carried out by the methods of Felsenfeld (1968) and Ames (1966) respectively. The values obtained for umoles nucleotide per mg DNA and umoles phosphate per mg DNA agreed f a i r l y well considering that two disparate methods of analysis were used. The extinction coefficient at 260 nm with respect to phosphate (£(P) 260) was close to 6,600, a value characteristic of many native DNA molecules, and was not over 7,200, a value indicating denaturation (Chargaff, 1955). The maximal thermal hyperchromicity at 260 nm (h 260) i s equal to maximum absorbance at 260 nm of a DNA solution after heating _ ^ absorbance at 260 nm of a DNA solution at room temperature (Mahler and Cordes, 1971). The values obtained were representative of native DNA (Mahler and Cordes, 1971). The fraction of the DNA originally T e m p e r a t u r e (*C) F i g . 8 Thermal denaturation curve for DNA isolated from rat intestinal mucosa. The DNA at a concentration of 20 ugm/ml was heated i n standard saline citrate solution and the relative absorbance of the DNA solution was corrected for thermal expansion and plotted as a function of the temperature. T e m p e r a t u r e ( # C ) Fig.,9 Thermal denaturation. curve for calf thymus DNA (Sigma type I ) . The DNA was heated i n a similar manner as for Fig. 8. TABLE IV ULTRAVIOLET AND PHOSPHATE' ANALYSES OF DNA ISOLATED FROM RAT SMALL INTESTINE AND OF CALF THYMUS UNA (SIGMA TYPE I ) E x t i n c t i o n c o e f f i c i e n t at 260mm Maximal thermal umoles umoles with respect hyperchromicity F r a c t i o n o f D N A nucleotide phosphate to phosphate at 260 mm o r i g i n a l l y i n v o l v e d D N A per mg U N A per mg DNA £(P) 260 h260 i n h e l i x Rat D N A 2.29 2.45 6,755 0.38 0.93 (u n l a b e l l e d ) CA Rat 3 H - D N A 2.54 2.70 6,685 0.38 0.94 *" C a l f thymus 2.14 2.00 6,800 0.43 1.0 D N A (Sigma type I ) 65 involved i n helix was calculated by dividing the concentration of DNA that underwent denaturation by the concentration of DNA that was present i n denatured form at 100°C (Felsenfeld, 1968). These values also indicated that the DNA was present i n the double-stranded form. 66 Preparation of [if - 3 2p]ATP from 32p labelled inorganic phosphate The prodedure of Glynn and Chappell (1964) as modified by Schendel and Wells (1973) involves the substrate level phosphorylation of ADP during the conversion of glyceraldehyde-3-phosphate to 3-phosphoglycerate by enzymes of the glycolytic pathway as illustrated i n Fig. 10. The glyceraldehyde-3-phosphate reaction i s made irreversible by oxidizing the NADH to NAD4"with thiazoylblue and phenazine methosulfate. Because the nucleotide content of commercially available glyceraldehyde-3-phosphate dehydrogerase can vary, NAD+should be added to the reaction mixture to ensure that a high yield of [% - 32p]Ajp i s consistently obtained (Martin and Voorheis, 1977). The [T-^2P]ATP was separated from reactants and byproducts by chromatography on a DEAE-cellulose column, 0.8 x 16 cm, equilibrated with 0.05 M triethylammonium bicarbonate (TEAB), pH 7.5. The column was eluted k°C with a linear gradient of 0.05 to 0.5 M TEAB, pH 7.5. One microlitre of each 2 ml fraction collected was added to 10 ml of water and counted by Cerenkov radiation. The elution profile of radioactivity from the column i s illustrated i n F i g . 11. The identity of the eluted radioactive compounds was determined by subjecting aliquots from peak fractions to thin layer chromatography on polyethyl-enemine cellulose plates run i n 1.25 M L i C l by the method of Randerath and Randerath (1967). The Rf values were: 5* AMP, 0.66; P i , 0.63; ADP, 0.53; 5* ATP, 0.22. Since 5*-AMP and Pi were not resolved, 32p labelled inorganic phosphate was determined by measuring the amount of radioactivity that would not bind to Norit A under the conditions described by Weiss et a l . (1968). The fractions containing the [r- 32P] ATP were 67 H 0 II C — H I C OH I CHgOK^ D - glyceraldehyde - 3 - phosphate V H^PO: NAD+«-glyceraldehyde-3-phosphate dehydrogenase NADH + H 0 II C .0—32PO" H C — OH I CHgOPOg 1, 3 diphosphoglycerate oxidizing dyes (thiazolyl blue and phenozine metnosulphate) 3-phosphoglycerate kinase t-^ [r- 32P] ATP H 0 II C I 0 OH CH20P03 3-phosphoglycerate F i g . 10 Schematic representation of the reactions involved i n the synthesis of [if- ^FjATT? CO o 150h CL (/) c ZD O O 0 10 20 30 Ai Fraction no. (2ml./fraction) F i g . 11 Chromatography of the ft*- 32p] A TP preparation on DEAE cellulose. The column, 0.8 x 16 cm, was eluted at k*C with a linear gradient of 6.05 to 0.5 M triehylammonium hicarbonate (TEAB), pH 7.5. One microlitre of each 2 ml fraction collected was added to 10 ml Of water and the radioactivity was measured by the Cerenkov radiation emitted. 6 9 pooled; the solution contained 0.54$ 3 2 P - ADP and 1.2$ 32P _ inorganic phosphate. The triethylammonium bicarbonate was removed by l y o p h i l i -zation of the solution. Determination of the specific radioactivity of LV- 32V\ATT? Known amounts of oligodT(pT)9 that were determined by ultraviolet absorbance were phosphorylated using [Y- ^PJATI? and polynucleotide kinase by the method of Chaconas et a l . (1975). 01igodT(pT)9 was prepared by dephosphorylation of oligo(pT)lO with alkaline phosphatase from Escherichia c o l i (Worthington). Because Weiss et a l . (1968) found that an endonuclease contaminant was present i n the alkaline phospha-tase preparation from Worthington, the alkaline phosphatase was pre-incubated at 65"C and the reaction was carried out at 65 C i n 0.6 M TrisHCl, pH 8.0, containing 10 mM MgClg i n an attempt to inactivate the contaminating enzyme. In accord with the observation of Torriani (1966), the alkaline phosphatase activity was stable under these conditions. After the reaction with alkaline phosphatase the solution containing the oligodT(pT)9 was cooled to 0°C and chromatographed on a Sephadex 650 column, 0.9 x 50 cm. The oligodT(pT)9 was eluted with 50 mM t r i e t h y l -ammonium bicarbonate, pH 7.5; the peak fractions absorbing at 266 nm were pooled and freeze-dried. The oligodT(pT)9 was dissolved i n water; o the solution was made 0.5 N i n NaOH and incubated at 37 C for 15 minutes i n order to inactivate the alkaline phosphatase (Ho and Gilham, 1973$ Delaney and Spencer, 1976). The solution was then neutralized with HCl 70 and the concentration of oligodT(pT)9 was determined using a nucleotide extinction coefficient of 8.7 x 10 at 226 nm (Cassani and Bollum, 1969). Known amounts of oligodT(pT)9 were phosphorylated using 3 2 P ] A TP and polynucleotide kinase (Chaconas et a l . , 1975)* The reaction mixture contained 6.28 or 12.56 pmoles oligodT(pT)9 and [/If- ^2P]ATP (486,000 cpn) i n 50 mM Tris HC1, pH 7.6, with 10 mM dithlothreitol, 5 mM MgCl 2 and 2 units of polynucleotide kinase from bacteriophage Tk i n a volume of 20 u l . One microlitre aliquots were removed at various times and spotted along one side of a 23 by 28 cm sheet of DE8l cellulose paper which had been prespotted twice with 50 u l of 1 mM ATP, 50 mM EDTA. The prespotting was required to prevent irreversible binding of radioactive ATP to the origin. One microlitre of the reaction mixture was taken before addition of the polynucleotide kinase and was spotted as a control. Descending paper chromatography was carried out for 2 hours at room temperature i n 0.35 M ammonium formate buffer, pH 5.5 (van de Sande et a l . , 1973). Under these conditions the Rfs of ATP, AMP and inorganic phosphate (Pi) were 0 . 4 6 , 0 . 4 8 and 0.71 respectively, and oligonucleotides of at least 10 nucleotides i n length remained at the origin (Sgaramella and Khorana, 1972). The DE8l paper was dried and the origins were cut out, placed i n 10 ml of water i n glass s c i n t i l l a t i o n vials and counted by Cerenkov radiation. After subtraction of control values of about 100 cpm for the reaction mixture without the addition of enzyme, the counts per minute for 32p incorporation at various times were multiplied by 20 to relate the 1 u l aliquots back to the original 20 u l reaction mixture. In F i g . 12 a time curve of the incorporation of 3 2 P into oligodT(pT)9 is shown. 71 For 6.28 and 12.56 pmoles, 64,000 and 126,000 cpm were incorporated respectively. Two curves of incorporation were done as a check on the reproducibility of the determination. The specific radioactivity of the - 32P]ATP was taken as the cpm incorporated over the pmoles of oligodT(pT)p present. That i s , 128,000/12.56 ~ 64,000/6 2 Q = 10,191 cpm/pmole. Assuming 35$ efficiency for measuring 32p b y cerenkov radiation, this i s equivalent to 27 Ci/umole. CO o CD -t—> Z3 c 150 100 2. 50 c D O o 0 • / • • / ' ' i i i i 0 5 . 10 15 20 Time (minutes) 25 30 F i g . 12 Incorporation of ^ P into oligodT(pT)9 using polynucleo-tide kinase and tT- ^p]ATP. Aliquots of the reaction mixture were spotted on DE8l cellulose paper. Descending paper chromatography was carried out i n 0.35 M ammonium formate buffer, pH 5.5« The paper was dried, the origins cut out, and the 32p present was determined by measuring the Cerenkov radiation emitted. 73 RESULTS AND DISCUSSION A Isolation of DNase II activity from bovine intestinal mucosa Preparation of a crude extract containing DNase II activity from the mucosa of bovine small intestine A bovine small intestine obtained from a freshly slaughtered animal and kept at 0 C, was cut into five foot lengths. Each length was thoroughly flushed with tap water and the fat and other tissues were cut off with scissors. The section of intestine was cut longi-tudinally and placed mucosal side up on a metal tray inverted over crushed ice. The mucosa was scraped off with a du l l meat cleaver and suspended i n an equal volume of Krebs Ringer phosphate buffer, pH 7.8 > to give a t o t a l volume of 5 l i t r e s . The mixture was homogenized 2.5 l i t r e s at a time for 3 minutes at the "high" setting i n a Waring Commercial Blender, cooled to 0°C i n an ice-water bath, and rehomogenized as before. After the f i r s t homogenization, clumps of cells s t i l l remained i n the mucosal mixture as could be seen by phase contrast light microscopy. After the second homogenization, many nuclei, but no intact c e l l s , were observed. The homogenate was centrifuged at 16,300 xg for 30 minutes at k°C i n a Sorvall RC 2B refrigerated centrifuge. The precipitate was resuspended i n a minimum volume of Krebs Ringer phosphate buffer, pH 7.8, and recentrifuged as before i n order to recover as much DNase II activity as possible. The two supernatant solutions were f i l t e r e d through 8-12 layers of cheese cloth to remove l i p i d material, combined,and frozen i n 7h 300 ml lots at -80°C. This relatively low speed centrifugation was done to remove some cellular debris and organelles i n order to improve the subsequent centrifugation at 105,000 xg. Table V indicates the volumes of solution and the amounts of protein and DNase II activity that were involved i n the preparation of a crude extract of bovine intestinal mucosa. The large volume of solution and the large amount of protein present made i t d i f f i c u l t to process the entire solution by ultracentrifugation or column chromatography, but attempts to concentrate the solution and remove a substantial amount of protein by acidification and/or ammonium sulfate precipitation procedures similar to those described by Bernardi et a l . (1966) resulted i n a large loss of DNase II ac t i v i t y . This may have been due to the different properties of intestinal DNase I I , or to the different number and kinds of proteins present i n the intestinal extract. A l l of the DNase II activity present i n the homogenate was recovered in the combined super-natant solution with a modest increase i n specific a c t i v i t y . That the DNase II activity was higher i n the combined supernatant solution than i t was i n the homogenate may have been due to the release of "latent" DNase II from lysozomes during the procedure. 75 TABLE V PREPARATION OF A CRUDE EXTRACT CONTAINING DNase II ACTIVITY FROM THE MUCOSA OF BOVINE SMALL INTESTINE Volume Protein DNase II S p e c i f i c A c t i v i t y Preparation (ml) (gm) Units (Units/mg protein) Homogenate 4484 159 9681 0.0606 F i r s t I6,300xg Supernatant 3560 85.0 8259 0.0967 Resuspended P r e c i p i t a t e 1730 42.4 1285 0.0303 Second l6,300xg Supernatant 925 15.3 1073 0.0703 Resuspended P r e c i p i t a t e 975 18.5 263 0.0142 Combined I6,300xg Supernatants 4480 103 10,125 0.0983 76 Chxomatography of the crude DNase II preparation on DEAE cellulose A 300 ml lot of the 16,300 xg supernatant solution was thawed, adjusted to pH 7.8 with 1 N NaOH, and centrifuged at 105,000 xg for 1 hour at k°C i n a Beckman model L centrifuge. A DEAE 22 cellulose column, 5 x kO cm, was prepared and equilibrated with 20 mM phosphate buffer, pH 7.8. The 105,000 xg supernatant solution was diluted 2^  times with d i s t i l l e d water, so that the conductivity of the sample solution was the same as the buffer with which the DEAE cellulose column was equilibrated. The sample was applied to the column, and the column was eluted at a flow rate of 2.5 ml per minute f i r s t with 20 mM phosphate buffer, pH 7.8, and later with a linear 0 to 1 M potassium chloride gradient i n the same buffer. Fractions of 20 ml were collected and were assayed for protein by absorbance at 280 nm, for DNase II by the acid-soluble oligonucleotide assay, and for concentration of potassium chloride by conductivity measurements using known amourts of potassium chloride i n the same buffer as standards. The elution profile from the DEAE cellulose column i s illustrated i n F i g . 13. There were two large peaks of absorbance at 280 nm, but the second peak was not due entirely to protein. Although the absorbance at 280 nm of the pooled fractions indicated a protein concentration of 8.5 mg per ml of solution, the protein concentration as determined by the method of Lowry et a l . (1951) was only 0.37 mg/ml. Since the 280 nm to 260 nm absorbance ratio was 0.60, i t i s suggested that nucleic acids present i n the solution could have accounted for much of the absorbance seen at 280 nm. DNase II activity was determined by the acid-soluble oligonucleotide 77 F i g . 13 Chromatography on DEAE cellulose of a crude extract prepared from "bovine intestinal mucosa. The column, 5 x ho cm, was eluted with 20 mM phosphate buffer, pH 7.8, and with a linear 0 to 1 M potassium chloride gradient i n the same buffer. Protein concentration was estimated by absorbance at 280 nm, and DNase II act i v i t y , by the acid-soluble oligonucleotide assay. 1 0 0 2 0 0 F r a c t i o n n o . ( 2 0 m l / f r a c t i o n ) D N a s e ET a c t i v i t y / u n i t s \ f r a c t i o n ] -ho M K C l 8 H.6 0 - 1 0.5 0 79 assay (see methods). Two major peaks of DNase II activity were observed; the second peak was sometimes eluted throughout the potassium chloride gradient. The f i r s t peak of DNase II activity contained 35$ and the second peak, 10$ of the DNase II units which had been applied to the column. An aliquot of the f i r s t DNase II activity was rechromatographed on a DEAE cellulose column, 2 x 18 cm. The column was eluted with 20 mM phosphate buffer, pH 7.8, and then with 0.5 M potassium chloride i n the same buffer. The elution pro f i l e from the column i s illustrated i n Fig. Ik. Of the 8.34 units of DNase II activity applied to the column 6.66 units (80$) were eluted with the 20 mM phosphate buffer. The second DNase II ac t i v i t y , 5.87 units, was dlalyzed against 20 mM phosphate buffer, pH 7.8, for 30 minutes using a Blorad "Biofibre beaker-50" to yield 1.62 DNase II units. After standing overnight at k C the solution, now containing only 0.771 units of DNase II acti v i t y , was applied to a DEAE cellulose column, 2 x 18 cm. The column was eluted with 20 mM phosphate buffer, pH 7.8, and then with 0.5 M potassium chloride in the same buffer. F i g . 15 depicts the elution profile from the column. The DNase II ac t i v i t y , 0.590 units, was eluted with the potassium chloride step. This corresponded to a yield of 77$ of the DNase II activity actually placed on the column, and to an overall yield of 10$ of the aliquot of DNase II activity taken from the second peak of the original DEAE cellulose column. F r a c t i o n n u m b e r F i g . Ik Rechromatography of the f i r s t peak of DNase II activity from Fig. 13 on DEAE cellulose. The column, 2 x 18 cm, was eluted f i r s t l y with 20 mM phosphate buffer, pH 7.8, and secondly with 0.5 M potassium chloride in the same buffer. Protein concentration was estimated by absorbance at 260 nm and DNase II activity by the acid-soluble oligonucleotide assay. 0.5 M KCl F r a c t i o n n u m b e r Rechromatography of the second peak of DNase II activity from Fig. 13 DEAE cellulose. The column 2 x 18 cm was eluted with 20 mM phosphate buffer,' pH 7.8, and then with 0.5 M potassium chloride i n the same buffer. The protein concentration was estimated by the absorbance at 280 nm and the DNase II activity i n each fraction by the acid-soluble oligonucleotide assay. 82 Some factors affecting the s t a b i l i t y of DNase II A possible reason for the low yields of DNase II activity obtained after chromatography, di a l y s i s , and storage at k°C may have been the i n -s t a b i l i t y of DNase II activity i n the 20 mM phosphate buffer used. Separate solutions containing the f i r s t and second DNase II activities respectively i n 20 mM phosphate buffer, pH 7.8, and i n the same buffer containing either 10 mM EDTA or 10 mM MgClg* were stored at k°C for 9 days. Aliquots of these solutions were taken at different times and assayed for DNase II activity by the acid-soluble oligonucleotide assay. The curves i n Fig. 16a. and b demonstrate the decrease i n activity observed for the f i r s t and second DNase II activities respectively. The addition of 10 mM MgCl2 to the phosphate buffer destabilized the DNase II activity whereas the addition of 10 mM EDTA stabilized DNase II. The second DNase II activity did not decrease as rapidly or as much i n the phosphate buffer with or without MgClg as did the f i r s t DNase II activity. This may have been due to protection of the second DNase II activity by endogenous substrate. Evidence for the presence of DNA i n preparations of the second DNase II activity w i l l be presented later. The loss of DNase II activity may have been due to proteolysis of the enzyme. The solutions that contained the f i r s t and second DNase II activities also had proteolytic activity equivalent to 2.6 and 18 ugm of trypsin per ml, respectively, as determined by the casein digestion assay. Furthermore, each ml of undiluted 105,000 xg supernatant contained proteolytic activity equivalent to 60 ugm of trypsin. In order to determine whether proteolysis of DNase II was causing 83 F i g . 16 Stability of intestinal DNase I I . DNase II activity was measured by the acid-soluble oligonucleotide assay and the units of DNase II activity per ml of enzyme solution were plotted as a function of the time that the enzyme solutions had remained at U*C. (a) and (b) refer respectively, to the f i r s t and second DNase II activities for F i g . 13. 20 mM phosphate buffer, pH 7.8 same buffer containing 10 mM MgGl, same buffer containing 10 mM EDTA D N a s e I I u n i t s o P P p P <~> P P p p ro lo ^ ^ ro co o 85 the decrease i n DNase II ac t i v i t y , 1 ml of ^ (isopropyl fluorophosphate (DFP), a protease inhibitor which irreversibly inactivates proteolytic enzymes containing serine at their active sites by alkylating that serine, was added to 260 ml of 105,000 xg supernatant solution. The proteo-l y t i c a c t i v i t y was reduced to 5$ of that originally present, DNase II was not affected by the addition of the DFP and remained stable at U*C in the solution containing DFP over a period of at least Ik days. When a 105,000 xg supernatant solution which had been treated with DFP, was chromatographed on a DEAE cellulose column under conditions described previously, 80-90$ of the applied DNase II units were eluted with the 20 mM phosphate buffer, pH 7.8, and about 10$, with the potassium chloride gradient. A similar result was obtained with untreated 105,000 xg supernatant when 10 mM EDTA was added to the buffer solutions that were used for elution of the column. The EDTA may chelate the divalent metal ions that are required for optimal activity of some proteases. It was decided to include 10 mM EDTA i n a l l buffers used i n elutions of DNase II from columns and i n concentration and dialysis of solutions containing DNase II . This was done both because of the toxic i t y of DFP and because DFP does not inactivate the zygomen precursors of proteolytic enzymes which may later be converted to active proteases. Otsuka and Price (197k) found that even after treatment of the enzyme preparation with DFP, DNase I activity was lost i n a solution that did not contain divalent metal ions. They suggested that the loss of DNase I activity was due to activation of zygomen precursors of proteolytic enzymes. 86 Discussion A procedure was described for the preparation of large amounts of mucosal extract from bovine small intestine with very l i t t l e loss of DNase II ac t i v i t y . The extract was stored at -80°C for at least two year without any further loss of DNase II activity. Two DNase II activities were separated on DEAE cellulose, and on rechromatography on DEAE cellulose, each activity was eluted i n the same position as observed originally. Although there was a problem of the low yields of DNase II acti v i t y , the results appeared to indicate that there were two different DNase II acti v i t i e s i n accord with the findings of previous investigators that two DNase II activities were present i n some tissues (CordOnnier and Bernardi, 1968; Yamanaka et a l . , 197^ J Zollner et a l . , 197^). EDTA probably stabilized DNase II by chelating metal ions which would otherwise have caused a loss of DNase II activity either directly by denaturation of the enzyme molecule, or indirectly through enhancement of the act i v i t y of proteases present i n the DNase II preparation. The latter hypothesis i s supported by experiments demonstrating the presence of proteolytic activity i n solutions containing DNase II and the s t a b i l i -zing effect of diisopropyl fluorophosphate, a protease inhibitor, on DNase II ac t i v i t y . 87 B Investigation of the properties of two DNase II activities isolated  from bovine intestinal mucosa pH optima The two DNase II activities were observed in the presence of EDTA and had acidic pH optima as shown in Fig. 17. The absorbance at 260 nm of the acid-soluble oligonucleotides released from calf thymus DNA, 400 ugm/ml, upon incubation with enzyme for 10 minutes in 120 mM sodium acetate buffer containing 8 mM EDTA, was taken as a measure of the enzymatic activity at the various pH values. The fi r s t DNase II activity had a broad acidic pH optimum centered at about pH 4.8 (Fig. 17a), whereas the second DNase II activity had a narrower pH optimum also cen-tred at pH 4.8 (Fig. 17b). Degradation of native and denatured DNA Calf thymus DNA was denatured by placing the DNA solution, 4 mg DNA per ml of 10 mM sodium chloride, into a stoppered test tube in a boiling water bath for 15 minutes and then cooling the DNA solution in an ice-water bath. DNase II activity was determined by the acid-soluble oligonucleotide assay using 400 ugm/ml of either native or denatured DNA. Table VI indicates that the first DNase II activity degraded native DNA at a rate nearly three times faster than i t degraded denatured DNA, whereas the second DNase II activity degraded both native and denatured DNA at about the same rate. 88 pH optima of the DNase II a c t i v i t i e s . The absorbance at 260 nm of the acid-soluble oligonucleotides released from calf thymus DNA (kOO ugm/ml) upon digestion with enzyme for 10 minutes i n 120 mM sodium acetate buffer containing 8 mM EDTA was taken as a measure of the enzy-matic activity at different pH values. (a) pH optimum of the f i r s t DNase II activity (b) pH optimum of the second DNase II activity 90 TABLE VI RATE OF RELEASE OF ACID-SOLUBLE OLIGONUCLEOTIDES FROM NATIVE AND DENATURED DNA BY DNase I I Increase i n absorbance at 260nm per minute per ml of enzymic s o l u t i o n Native DNA Denatured DNA F i r s t DNase I I 0.56 0.20 A c t i v i t y Second DNase I I A c t i v i t y 0.13 0.13 91 Effects of ionic strength In order to determine whether an increase i n ionic strength affected the two DNase II activities differently the enzymes were assayed i n 20 mM sodium acetate buffer, pH 5.0, containing 10 mM EDTA and different concentrations of sodium chloride. Fig, 18 compares the two DNase II acti v i t i e s i n solutions of different ionic strength, DNase II acti v i t y was measured by the acid-soluble oligonucleotide assay and expressed as a percent of the activity observed i n the buffer containing no sodium chloride. Slight increases i n activity were seen for the f i r s t DNase II at 0.1 M NaCl, and for the second DNase II at 0.05 M NaCl. The second DNase II was less active than the f i r s t DNase II i n solutions of high ionic strength. Effects of sulfate, an inhibitor of DNase II Since sulfate i s an inhibitor of DNase II from other tissues (Bernardi and Gr i f f e , 1964; Koener and Sinsheimer, 1957; Rosenbluth and Sung, 1969), the effect of different concentrations of sulfate on the two intestinal DNase II activities was measured using the acid-soluble oligonucleotide assay. As may be seen from F i g . 19, both DNase II activ i t i e s were inhibited by. low concentrations of sulfate. The second DNase II was inhibited more than the f i r s t DNase II activity at high concentrations of sulfate. The shapes of the curves for the two DNase II act i v i t i e s were different: a slight increase i n activity was observed 92 Molarity of NaCl 18 Comparison of the f i r s t and second DNase II acti v i t i e s at different ionic strengths. DNase II activity was measured .by the acid-soluble oligonucleotide assay i n 20 mM sodium acetate buffer, pH 5.0, containing 10 mM EDTA and sodium chloride to the indicated molarity. The DNase II activity was expressed as a percent of the activity observed i n the buffer containing no sodium chloride. 93 • mmm > • M M •4— 0 CO O 0 s 120 100 0 .02 .04 .06 .08 Molarity of Na2S04 F i g . 19 Effect of sulfate on the f i r s t and second DNase II a c t i v i t i e s . DNase II activity was measured by the acid-soluble oligonucleotide assay i n 20 mM sodium acetate buffer, pH 5.0, containing 10 mM EDTA and sodium sulfate to the indicated molarity. The DNase II activity was expressed as a percent of the activity observed i n the buffer containing no sodium sulfate. 9k f o r the second DNase II activity at 0.01 M sulfate concentration whereas no such increase i n activity was seen for the f i r s t DNase I I . Activation energies for the hydrolysis of DNA Different activation energies for the DNase II catalysed hydrolysis of DNA could indicate that the two DNase II activities were due to different enzyme molecules. Hydrolysis of calf thymus DNA by DNase II was measured as a function of time at different temperatures by the acid-soluble oligonucleotide assay. The slopes of the linear portions of the absorbance at 260 nm versus time curves were taken as indicative of the rate of hydrolysis of the DNA at the various temperatures. Fig. 20 demonstrates that, when the rates.of hydrolysis were plotted on a loga-rithmic scale as a function of the reciprocal of the absolute temperature, straight lines of different slopes could be drawn through the points for the f i r s t and second DNase II a c t i v i t i e s . The activation energies for the hydrolysis of native calf thymus DNA were calculated from the Arrhenius equation (Mahler and Cordes, 1971) to be 19 kcal/mole for the f i r s t DNase II and 8.1 kcal/mole for the second DNase I I . Oth et a l . (1958) found an activation energy of 23 kcal/mole for the hydrolysis of calf thymus DNA by DNase II from thymus. 3.0 3.1 32 3.3 3.A 4-'('Kx.10"3) T F i g . 20 Arrhenius plot showing the temperature dependence of the rate of hydrolysis of calf thymus DNA by DNase I I . The logarithm of the reaction rate was plotted as a function of the reciprocal of the absolute temperature. — • — plot using the f i r s t DNase II activity — A — plot using the second DNase II activity 96 Activation energy for the denaturation of DNase II In order to compare the enzyme molecules more directly, the decrease in DNase II activity which occurred after heating at various temperatures was measured. Aliquots of the f i r s t or second DNase II activity were heated at 37 > 45, or 50° C for times up to 30 minutes. The enzymic solutions were then cooled rapidly to 0°C to stop further denaturation, and the DNase II activity remaining was determined at 37°C by the acid-soluble oligonucleotide assay. The DNase II activity remaining was plotted as a function of the time that the enzyme solution had been heated at a particular temperature. Slopes of tangents drawn to these curves at zero time were taken as indicative of the i n i t i a l rates of denaturation of the enzyme molecules. Fig. 21 exhibits a plot of the logarithm of the rate of denaturation as a function of the reciprocal of the absolute temperature. The "activation energy" for denaturation was calculated from the Arrhenius equation (Mahler and Cordes, 1971) to be 43 kcal/mole for the f i r s t DNase II and 51 kcal/mole for the second DNase IIj these values f a l l within the range observed for other enzymes (Mahler and Cordes, 1971). Although the values are f a i r l y close, especially considering the different purity of the two preparations, this does not necessarily imply that the two DNase II activities are due to the same enzyme molecule since different enzymes can denature at the same rate. However, should the activation energies for denaturation have been very different this would have indicated the presence of different enzyme molecules. 97 F i g . 21 Temperature dependence of the rate of denaturation of DBase I I . The DNase II activity remaining was plotted as .v a function of the time that the enzyme solution had been heated at a particular temperature. Slopes of tangents drawn to these curves at zero time were taken as indica-tive of the i n i t i a l rates of denaturation and were plotted on a logarithmic scale as a function of the reciprocal of the absolute temperature. denaturation of the f i r s t DNase II • ac t i v i t y denaturation of the second DNase II acti v i t y 99 Presence of endogenous DNA. i n the second DNase II preparation The properties of the f i r s t and second DNase II a c t i v i t i e s described above were determined using the acid-soluble oligonucleotide assay because the second ac t i v i t y preparation contained a, large amount of material which precipitated i n the hyperchromicity assay and obscured the results. The usual blank or control f o r the acid-soluble oligo-nucleotide assay was a solution i n which DNA had been incubated for the same time and at the same temperature as for the enzymatic reaction. The perchloric acid was added to the control solution at the same time as the enzymatic reaction was stopped. An amount of enzyme solution equal to that used i n the enzymatic reaction was then added to the control solution. This solution, which was called a "substrate blank" because i t was the substrate that was incubated, now contained a l l the components that were present i n the reaction mixture except that the enzyme had not acted on the substrate. The absorbance at 2 6 0 nm found for the substrate blank solution was subtracted from the absorbance at 2 6 0 nm found for the reaction mixture. In order to determine whether there was endogenous substrate i n the enzyme preparations "enzyme blank" solutions were prepared. That i s , the enzyme solutions were incubated i n buffer for the same time and at the same temperature as for the reaction, perchloric acid was added, and then substrate (DNA) was added. Thus, the enzyme blank contained a l l the components of the reaction mixture except that i t was the enzyme and not the substrate that had been incubated alone. 100 ; For the f i r s t DNase II about the same low absorbance at 260 nm was obtained for the substrate and enzyme blank solutions. F i g . 22 demonstrates that for the second DNase II almost identical curves of absorbance at 260 nm against time were observed for the release of acid-soluble oligonucleotides i n the presence and absence of added substrate. This indicates that the second DNase II preparation contained an amount of endogenous substrate almost equal to that added i n the acid-soluble oligonucleotide assay. The second DNase II was prepared by pooling fractions from the DEAE cellulose column, and concentrating the solution by u l t r a f i l t r a t i o n through an Amicon PM 10 membrane using nitrogen at 5.0 p . s . i . pressure. Salt was removed from the solution by " d i a f i l -tration": aliquots of 20 mM phosphate buffer, pH 7.8, containing 10 mM EDTA were added sequentially to the concentrated solution containing the second DNase H and the solution was subjected to u l t r a f i l t r a t i o n u n t i l the conductivity of the solution was similar to that of the buffer which had been added. The solution containing the second DNase II was then lyophilized. In order to determine whether the endogenous substrate i n the second DNase U preparation was nucleic acid, protamine sulfate was added to the preparation. Protamine, a basic protein, binds to the negatively charged phosphate groups of nucleic acids. The resulting nucleoprotein complex i s insoluble at salt concentrations equivalent to about 0.1k M sodium chloride (Davidson, 1972). The lyophilized powder containing the second DNase II was dissolved i n water at a concentration of 25 mg/ml. To 2.0 ml of this preparation was added 0.6 ml of a 1% solution of protamine sulfate. The mixture was allowed to stand i n an ice-water bath for F i g . 22 Release of acid-soluble oligonucleotides by the second DNase II i n the presence and absence of added substrate. The reaction was carried out i n 0.15 M sodium acetate buffer, pH 5.0, containing 10 mM EDTA. kO ugm/ml calf thymus DNA present i n the reaction mixture no exogenous DNA present i n the reaction mixture < U J U 0 9 3 \® eouoqjosqv 103 10 minutes and then was centrifuged i n the cold at 12,100 xg for 10 min-utes. The supernatant solution was decanted and; the precipitate resus-pended i n 2 ml of water. Table V H demonstrates that the absorbances at 280 nm and 260 nm of the solution containing DNase II were reduced 3 and 5 fold respectively by protamine sulfate treatment. The A28o/A26o ratio was increased from 0.65 to O.89. Pure nucleic acids have an A28o/A260 ratio of 0.5; mucoproteins, 0.8, and proteins containing only amino acids, 1.75 (White, Handler and Smith, 1974). Since protamine forms an insoluble complex with nucleic acids, the formation of a pre-cipitate as well as the decrease i n the absorbances at 280 nm and 260 nm and the increase i n the A28o/A260 ratio upon treatment of the second DNase II preparation with protamine sulfate indicate the presence of nucleic acids i n the second DNase II preparation. In Table VII, i t may be seen that for the untreated DNase I I , the enzyme blank was much higher than the substrate blank, whereas after treatment with protamine sulfate the enzyme blank was only sl i g h t l y higher than the substrate blank. This indicates that i t was l i k e l y the endogenous nucleic acids i n the untreated second DNase II preparation that were responsible for the high enzyme blank. Although the second DNase I I preparation contained nucleic acids, these could have been DNA. and/or RNA. In order to determine whether the second DBase II a c t i v i t y contained DBA sp e c i f i c a l l y , the deoxyribose content was determined by the method of Dische as modified by Burton (1968). The lyophilized powder containing the second DBase II ac t i v i t y was found to contain lk% (w/w) DBA by this method. Thus, the 25 mg/ml solution of TABLE VII EFFECT OF TREATMENT OF THE SECOND DNase I I PREPARATION WITH PROTAMINE SULFATE Preparation Volume (ml) Absorbances of Solutions A280 A260 A 2 8 0 / A 2 6 0 DNase I I A c t i v i t y ( A c i d - s o l u b l e o l i g o n u c l e o t i d e assay) (0.1 ml x 15')  Total DNase I I u n i t s with respect to 1 2 1 I 2 assay substrate enzyme value blank blank A260 A 2 6 0 A 260 -second DNase I I 2.0 82.8 126.5 0.65 3.99 1.06 3.63 5.82 0.72 second DNase I I supernatant 2.2 protamine s u l f a t e resuspended pre-c i p i t a t e 2.4 24.6 27.5 0.89 3.14 0.46 1.10 0.27 1.36 0.42 4.49 3.92 1.09 0.096 105 second a c t i v i t y used for the protamine sulfate experiment, contained 3.5 mg DNA per ml. Since the 0.1 ml of enzyme solution added to each assay tube contained 0.35 mg DNA and since the amount of calf thymus DNA added to each assay was 0.U mg, the assay value for the enzyme blank for the second DNase II ac t i v i t y was of a comparable magnitude to the assay value for the reaction with calf thymus. Rechromatography of the two DNase II ac t i v i t i e s on DEAE cellulose Since i t had been found that 10 mM EDTA stabilized the two DNase I I a c t i v i t i e s , and since the presence of DNA i n the second a c t i v i t y prepa-ration could have caused apparent differences i n the properties of the two a c t i v i t i e s , each of the two DNase II ac t i v i t i e s were again rechroma-tographed separately on DEAE cellulose columns i n order to determine whether the two ac t i v i t i e s were due to chromatographically distinct species or t o an artifa c t of the chromatographic procedure. The f i r s t DNase II was eluted from the DEAE cellulose column with 20 mM phosphate buffer, pH 7.8, containing 10 mM KETA as i n the f i r s t rechromatography experiment (see F i g . Ik) except that the yield of DNase II units was now 99$. The second DNase II activity u/as rechromatographed on DEAE cellulose. The column, 2 x 18 cm, was eluted with 20 mM phosphate buffer, pH 7.8, containing 10 mM EDTA, and with 1 M potassium chloride i n the same buffer. F i g . 23 shows the elution pro f i l e from the column. Ninety percent of the applied DNase II units were eluted with the 20 mM phosphate buffer, 106 F r a c t i o n n u m b e r F i g . 23 Rechron»tography of the second DBase II ac t i v i t y on DEAE cellulose. The column, 2 x 18 cm, was eluted with 20 mM phosphate buffer, pH 7.8, containing 10 mM EDTA, and then with 1 M potassium chloride i n the same buffer. Protein was estimated by absorbance at 280 nm, nucleic acid, by absorbance at 260 nm and DNase II activity by the DHA-agar gel assay. The antilogarithm of the diameter of the zone of clearing produced by the action of DNase II on DNA (10**) gives the relative enzyme concentration i n the different fractions. 107 and 5-10$ with 1 M potassium chloride i n the same buffer. It may be recalled that i n a previous rechromatography experiment (Fig. 15) no DBase II ac t i v i t y had been eluted with the 20 mM phosphate buffer, and 77$ of the applied DBase II ac t i v i t y had been eluted with the 1 M potassium chloride step. A possible explanation for the different results obtained i n the two experiments may be deduced from the observation that, i n the f i r s t experiment, due to the absence of the st a b i l i z i n g agent EDTA i n the enzyme solution, the overall yield of DBase II ac t i v i t y from the fractions of the original chromatography to the rechromatography fractions was 10$--the DBase II a c t i v i t y that eluted with the 1 M potas-sium chloride step. In the second experiment with the inclusion of 10 mM EDTA i n a l l solutions, the overall yield of DBase II ac t i v i t y was 95-100$ and of this 5-10$, about the same overall percent as before, was eluted with the 1 M potassium chloride step. It may be that i n the f i r s t experi-ment a l l of the DBase II enzyme was denatured or degraded except for 10$ which was stabilized by binding to endogenous DBA. Most of the 10$ of the DBase II a c t i v i t y was then eluted with the 1 M potassium chloride step. Chromatography of the f i r s t DBase II activity on DBA cellulose In order to determine whether the f i r s t DBase II ac t i v i t y could be bound to DBA that was bound to cellulose and i n this way give r i s e to a "second" DBase II a c t i v i t y , a DBA cellulose column, 2 x 18 cm, was prepared by the method of Alberts and Herrick (1971) and the f i r s t DBase II a c t i v i t y was chromatographed on i t . The column was eluted with 20 mM 108 Tris HC1, pH 7.8, containing 1 mM EDTA and with a linear gradient of 0 to 1 M potassium chloride i n the same buffer. F i g . 24 demonstrates that the DNase II act i v i t y was bound to the DNA cellulose column i n the 2 0 mM Tris buffer and was eluted at the beginning of the potassium chloride gradient. Chromatography of the two DNase II ac t i v i t i e s on Sephadex G100 In order to estimate the molecular weights of the two DNase II enzymes each was chromatographed separately on a Sephadex 0100 column, 2.5 x 7 8 cm. The column was eluted at a flow rate of 17.4 ml per hour with 0.25 M Tris HC1, pH 7.5, containing 1 0 mM EDTA and k ml fractions were collected. The column was calibrated by the application, separately, of blue dextran, bovine serum albumin (MW 67,000) and chymotrypsinogen A (MW 25,000), ovalbumin (MW 45,000) and ribonuclease A (MW 13.700). Each sample was applied i n 1,0 ml of the elution buffer. Upward elution was used and 2 ml of a solution of 1 0 $ sucrose i n the elution buffer was added after the sample i n order to ensure even application of the sample to the column. The positions of elution of the above samples were determined by absorbance at 280 nm. DNase II ac t i v i t y i n column fractions was determined by the acid-soluble oligonucleotide assay. The elution prof i l e for the second DNase II ac t i v i t y i s illustrated i n Fig. 25. The positions of the maxima of the symmetrical elution peaks for the other samples and for the f i r s t DNase II act i v i t y are indicated by arrows. Multiple peaks of ac t i v i t y were observed for the second DNase II; the I. , .. 10 (xlO E i — — - — ~ ~ • n 0 10 20 30 Fract ion number (5ml/fraction) Pig. 2k Chromatography of the f i r s t DNase II activity on DNA. cellulose. The column, 2 x 18 cm, was eluted with 20 mM t r i s HCl, pH 6.8, containing 1 mM EDTA and then with 1 M potassium chloride i n the same buffer. 110 F r a c t i o n n o . U m l . / f r a c t i o n ) F i g . 25 Chromatography of the second DNase XI act i v i t y on Sephadex G100. The column, 2.5 x 78 cm, was eluted upwards with 0.25 M Tris HCl, pH 7.5, containing 10 mM EDTA. Protein was estimated by absorbance at 280 nm; the second DNase II act i v i t y , by the acid-soluble oligonucleotide assay. The arrows indicate the elution positions of blue dextran, bovine serum albumin (BSA), ovalbumin (oval), chymotryp-sinogen A and HNase A as determined by maximal absorbance at 280 nm. In a separate experiment the f i r s t DNase II was found to elute i n a single symmetrical peak with a maximum at the position indicated by the arrow. I l l peak associated with the species of lowest molecular weight coincided with the elution position of the f i r s t DNase II. This i s consistant with the second DNase II being the same molecular weight as the f i r s t DNase II and binding with progressively larger fragments of DNA to yie l d peaks of enzymatic a c t i v i t y eluting from the column i n different molecular weight ranges. From elution positions shown i n Fig. 25 the partition coefficients (Kav values) of proteins of known molecular .weight between the mobile phase, the buffer solution moving down the Sephadex column, and the entire gel phase, the liq u i d imbibed i n the gel phase plus the gel matix i t s e l f j were calculated by the method described by Fischer (1969) and ReHand (1971). These partition coefficients were plotted i n Fig. 26 against the logarithms of the molecular weights of the proteins. The partition coefficients for the f i r s t DNase II was determined and by i n -terpolation on Fig. 26 the molecular weight of the f i r s t DNase II was estimated to be 4l,0G0. As suggested previously the molecular weight of the second DNase II i s l i k e l y the same as the f i r s t DNase II. Discussion Previous investigators found two DNase II ac t i v i t i e s i n some tissues (Cordonnier and Bernardi, 1968; Yamanaka et a l . , 1974; Zollner et a l . , 1974). Since DNase II activity had been found i n calf thymus nuclei (Slor and Lev, 1971) as well as i n lysosomes (de Duve et a l . , 1962), i t was considered possible that the two DNase II ac t i v i t i e s isolated from 112 0.6 > 0 0.5 C CD 0.4 o H— CD O O 0.3 . c o • • Parti 0.2 RNase A • Ve - C h t g A • \ DNase H - Ovalbumin \ « - B S A 1 1 1 1 I I I I 2 3 U 5 6 7 8 9 1 0 3 M o l e c u l a r w e i g h t ( X10 ) F i g . 26 E s t i m a t i o n o f t h e m o l e c u l a r w e i g h t o f t h e f i r s t D N a s e I I b y g e l f i l t r a t i o n o n S e p h a d e x G100. T h e p a r t i t i o n c o e f f i c i e n t s ( K a v v a l u e s ) o f t h e p r o t e i n w h o s e e l u t i o n p o s i t i o n s a r e s h o w n i n F i g . 25 w e r e p l o t t e d a g a i n s t t h e l o g a r i t h m s o f t h e m o l e c u l a r w e i g h t s o f t h e p r o t e i n s . T h e m o l e c u l a r w e i g h t o f t h e f i r s t D N a s e I I w a s e s t i m a t e d b y i n t e r p o l a t i o n . 113 Intestine and other sources might have different intracellular locations and biological functions. We found, however, that the appearance of a second DNase II act i v i t y was an a r t i f a c t of the DEAE cellulose chromatography. Negatively charged DNA present i n the 105,000 xg supernatant solution was bound electro-s t a t i c a l l y to the positively charged DEAE cellulose column and about 10 percent of the DNase II a c t i v i t y was bound to the DNA. When the column was eluted with a potassium chloride gradient, the bound DNase II ac t i v i t y was eluted along with some, but not a l l of the DNA. The DNA may also have been degraded somewhat due to DBase I I action. Evidence that the f i r s t and second DBase II ac t i v i t i e s are not due to distinct enzyme molecules comes from the rechromatography experiment i n which 9 0 $ of the second a c t i v i t y was eluted with 20 mM phosphate buffer i n a similar manner, to the f i r s t a c t i v i t y . The earlier rechromatography experiment appeared to give contradictory results because i n the absence of EDTA only 10$ of the DBase II that was associated with DBA was stable. This DBase II was bound to the column with the DBA; the 9 0 $ of the DBase II act i v i t y that would have been eluted with the phosphate buffer had been denatured or digested by proteases and was not observed. That DBase II can bind to and be eluted from DBA which i s bound to a cellulose column was shown by the DBA cellulose chromatography experiment. The second DBase II preparation contained DBA, and several peaks of DBase II ac t i v i t y were seen when the second DBase II was chromatographed on a Sephadex G100 column. This suggests that the DBase II activity may have been bound to DBA fragments of different lengths. These fragments could 114 have been liberated by the action of DNase I I i t s e l f . The presence of DNA i n the second DNase II preparation could account for some of the apparently different properties of the f i r s t and second DNase II a c t i v i t i e s . Some of the variance i n properties may also have resulted from the different purity of the two prepa-rations—the f i r s t and second DNase II preparations had specific ac-t i v i t i e s of about 0.5 and 0.05 DNase II units per mg of protein, res-pectively. The second DNase II apparently degraded denatured DNA at the same rate as native DNA. However, i n the assay with denatured DNA, endogenous native DNA was being added along with the enzyme. I f the enzyme did degrade denatured DNA at a lower rate than native DNA, this would not be apparent because of the presence of endogenous na-tive DNA i n the reaction mixture. The second DNase II had lower activity i n solutions of high ionic strength than the f i r s t DNase II. A precipitate formed i n the pH 5 assay solution containing the second DNase U ac t i v i t y , but not in the solution containing the f i r s t DNase II activity. Due to the presence of excess DNA and/or protein i n the second DNase II prepa-ration, precipitation of a DNA-protein complex may have occurred, perhaps more readily at higher ionic strengths, and caused an apparent-l y lower a c t i v i t y for the second DNase II by removal of substrate from the assay solution. This effect would have been superimposed upon the effect of inhibition of the enzyme i t s e l f by high ionic strength which was also seen for the f i r s t DNase II act i v i t y . The second DNase II had lower a c t i v i t y than the f i r s t DNase II 115 i n solutions containing0.G3 to 0.1 M sulfate. This may have been due to the precipitation of a SNA-protein complex from the assay solution i n a similar manner as described previously at high ionic strength. The similar shape of the curves of the second DNase II ac t i v i t y as a function of increasing ionic strength and sulfate concentration indicates that a similar process might have occurred i n both experi-ments. The apparently lower activation energy for the DNA hydrolysis reaction with the second DNase II as opposed to the f i r s t DNase II may have been due to the presence of larger amounts of DNA i n the - * assay tubes for the S<c*<*d DNase II ac t i v i t y . The assay tubes for the f i r s t DNase II contained hOQ ugm DNA per ml; those for the second DNase II contained U00 ugm exogenous DNA plus 350 ugm endogenous DNA per ml. High DNA concentrations cause an apparent inhibition of DNase II because hot a l l cleavages of the DNA result i n acid-soluble oligonucleotide release (Rosenbluth and Sung, 1969)* Since the activation energies for the denaturation reactions were f a i r l y close, the f i r s t and second DNase II ac t i v i t i e s were due to enzymes that denatured i n i t i a l l y at about the same rate. Although the i n i t i a l rates of denaturation for both ac t i v i t i e s were approxi-mately similar, after a period of time the second a c t i v i t y was not denatured to the same extent as the f r i s t a c t i v i t y . This implies that either other proteins OIL the DNA i n the second DNase II prepa-ration had a stab i l i z i n g effect on a portion of the second activity. We found that the appearance of a second intestinal DNase II ac t i v i t y was an a r t i f a c t . It i s possible that the appearances of 116 second DBase II a c t i v i t i e s i n other tissues were also artifactual. Cordonnier and Bernardi (1968) used DEAE cellulose chromatography to separate the two DBase II a c t i v i t i e s existing i n some tissues and suggested that the appearance of a second DBase II act i v i t y may have been due to the presence of other proteins which may have interfered with the chromatographic behavior of the enzyme. When Zollner et a l . (1974) subjected extracts from human lymphocytes to electro-phoresis on 13.k% polyacrylamide gels containing 0.3 mg herring sperm DBA per ml, he found five bands of DBase II act i v i t y . It i s possible that these bands could be due to a single DBase II enzyme which formed complexes with other proteins or DBA during the elec-trophoresis. Yamanaka et a l . (1974) separated two DBase II a c t i v i t i e s by phosphocellulose column chromatography, by i s o l e c t r i c focusing and by polyacrylamide gel electrophoresis of extracts from human gastric mucosa and cervix ueteri. Since a l l other properties of the two DBase II a c t i v i t i e s were identical, and since the a c t i v i t i e s were not separated completely from each other, one of them may have been an a r t i f a c t . 117 C Partial purification of bovine intestinal DNase H The following procedure was routinely used to prepare i n t e s t i -nal DNase I I . The f i r s t step of the procedure, DEAE cellulose co* lumn chromatography, was done as described previously except that the buffers contained 10 mM EDTA. The f i r s t DNase II ac t i v i t y was purified further; the second DNase II act i v i t y was discarded. A 105,000 xg supernatant was prepared from a crude extract of bovine intestinal mucosa and applied to a DEAE cellulose column as described previously. The column, 5 x kO cm, was eluted at a flow rate of 2.5 ml per minute with 20 mM phosphate buffer, pH 7.8, con-taining 10 mM EDTA, and then with a linear 0 to 1 M potassium chloride gradient i n the same buffer. Fractions of 20 ml. were collected and were assayed for protein by absorbance at 280 nm, for DNase II by the DNA-agar gel assay, and for concentration of potassium chloride by conductivity measurements. F i g . 27 shows the elution profile ob-tained for the DEAE cellulose column. Three peaks of absorbance at 280 nm were seen. The concentration of DNase II was proportional to the antilogarithm (lO d) of the diameter (d) of the zone of clearing produced i n the DNA-agar gel assay. Most of the DNase II a c t i v i t y , 80$ of the applied units, was eluted with the 20 mM phosphate buffer. The fractions containing the DNase II ac t i v i t y were pooled and the enzyme solution was concentrated by u l t r a f i l t r a t i o n through an Amicon PM 10 membrane with a molecular weight cut off of 10,000. During ultra-f i l t r a t i o n aliquots of water were added u n t i l the conductivity of the enzyme solution was the same as that of the buffer used i n the next E c o 00 CN CD u £ D _Q i _ O tn _Q < Fig. 27 -46 , o d - ~ 7 U106) KCl 100 200 300 400 F r a c t i o n n u m b e r 500 5 rl.O 4 -0.8 3 -0.6 2 -0.4 1 -0.2 0 -o Chromatography oa DEAE cellulose of a 105,000 xg supernatant solution prepared from bovine intestinal mucosa. The column, 5x1+0 cm, was eluted with 20 mM phosphate buffer, pH 7.8* containing 10 mM EDTA, and.then with a linear 0 to 1 M potassium chloride gradient i n the same buffer. Fractions of 20 ml were collected. The protein concen-tration was estimated by the absorbance at 280 nm and DNase II concentration by the antilogarithm (10d) of the diameter (d) of the zone of clearing produced i n the DNA-agar gel assay. 119 purification step, 10 mM phosphate buffer, pH 6.0, containing 10 mM EDTA. The pH of the enzyme solution was adjusted to 6.0 with IN HCl. The DBase II solution was applied to a CM 22 cellulose column, 5 x 1+0 cm. The "eluted at a flow of 5 ml per minute with 10 mM phos-phate buffer, pH 6.0, containing 10 mM EDTA, and then with a linear 0 to 1 M potassium chloride gradient inithe same buffer. Fractions of 20 ml were assayed for protein, DBase II and potassium chloride concentration as described previously. The elution pro f i l e obtained for the CM cellulose column i s illustrated i n Fig. 28. Two peaks of absorbance at 280 nm were seen. The DBase II ac-t i v i t y was eluted at a concentration of about 0.25 M potassium chlo-ride. The fractions containing the enzyme ac t i v i t y were pooled and the solution was concentrated to about 50 ml by u l t r a f i l t r a t i o n . The DBase II solution was applied to a Sephadex 0100 column, 5 x 90 cm. The column was eluted at a flow rate of 1.0 ml per mi-nute with 0.25 M Tris HCl buffer, pH 7.5 > containing 10 mM EDTA, and 20 ml fractions were collected. F i g . 29 illustrates the elution prof i l e from the column. Protein was estimated by absorbance at 280 nm and DBase II a c t i v i t y , by the DNA-agar gel assay. The fractions containing the DNase II ac t i v i t y were pooled and the enzyme solution was concentrated by u l t r a f i l t r a t i o n through an Amicon PM 10 membrane. The solution was desalted by sequentially adding aliquots of 20 mM Tris HCl, pH 7.5, containing 10 mM EDTA to a concentrated solution containing the DBase II a c t i v i t y and subjecting the solution to u l -t r a f i l t r a t i o n . As this was repeated the buffer composition of the solution approached that of the added buffer asymptotically. The con-1 2 0 F r a c t i o n N u m b e r F i g . 28 Chromatography on CM cellulose of the pooled DBase I I a c t i v i t y from the DEAE cellulose co-lumn. The column, 5 x Uo cm, was eluted with .< 10 mM phosphate buffer, pH 6.0, containing 10 mM EDTA, and then with a linear gradient of 0 to 1 M potassium chloride. Fractions of 20 ml were collected. The protein and DNase II concentrations were measured as described i n Fig. 27. 121 E c o 0 0 (NJ a Q) O C a _Q t -O CO < F r a c t i o n Number F i g . 29 Chromatography ©h Sephadex 0100 of the pooled DBase I I act i v i t y from the CM cellulose column. The column, 5x90 cm, was eluted with 0.25 M Tris HCl, pH 7.5, con-taining 10 mM EDTA and 20 ml fractions were collected. Protein and DBase II concentration were measured as described i n F i g . 27. 122 centrated, desalted solution containing DNase II act i v i t y was stored o i n 1 ml aliquots at -30 C. Estimation of the y i e l d and specific a c t i v i t y of DNase II after each step i n the purification procedure are given i n Table VIII. Centrifugation of the crude extract at 105,000 xg resulted i n the loss of about 12$ of the DNase II activity; more act i v i t y was lost i f the pH was less than 7*8. The f i r s t DEAE cellulose effluent con-tained the " f i r s t DNase II ac t i v i t y " referred to previously and was processed further on CM cellulose and Sephadex columns. The second DEAE cellulose effluent contained the "second DNase II ac t i v i t y " , an artifact of the DEAE cellulose chromatography, and was discarded. The greatest loss of DNase II a c t i v i t y occurred upon chromatography on the Sephadex 6100 column. However, this loss may have been more apparent than r e a l since Intestinal phosphodiesterase II of molecular weight 150,000 - 170,000 (Flanagan and Zbarsky, 1977) was l i k e l y par-t i a l l y separated from DNase U , of molecular weight Ul,000, on the column. Since phosphodiesterase n i s active under the same condi-tions as DNase I I , i t i s l i k e l y that there would be less contribu-tio n made to the DNase II assay values by the phosphodiesterase II enzyme after the Sephadex chromatography. Overall, DNase II was purified 32 fold with respect to proteins present i n the solution, and the yield of DNase II act i v i t y was Ul$. Electrophoresis of the p a r t i a l l y purified DNase II preparation i n polyacrylamide gels In order to obtain an estimate of the number and amounts of 123 TABLE VIII PARTIAL PURIFICATION OF INTESTINAL DNase II Preparation DNase II Units Protein (mg) S p e c i f i c A c t i v i t y (Units/mg protein) % Yield j.» Units Purif: catioi Combined Supernatants 487 5310 0.0917 100 1 105,000xg Supernatant 427 4240 0.101 88 1.1 105,000xg P r e c i p i t a t e 24.3 1094 0.0222 5 0.24 F i r s t DEAE Ce l l u l o s e E f f l u e n t 348 666 0.522 71 5.7 Second DEAE Cel l u l o s e E f f l u e n t 75 1678 0.0447 15 0.49 CM Cel l u l o s e E f f l u e n t 329 307 1.07 68 12 Sephadex GlOO Ef f l u e n t 198 67 2.96 41 32 1 2 4 other proteins present as impurities, the DNase II preparation was subjected to polyacrylamide gel electrophoresis using methods des-cribed by Gabriel (1971a). The separating gel was composed of 7.5$ acrylaraide and 0.2$ bis in? a 0.37 M potassium acetate buffer pH 4.3, and the stacking gel, of 2.5$ acrylimide and 0.625$ bis i n a 0.062 M potassium acetate buffer, pH 6.7. The electrophoresis buffer was 0.035 M^-alanine - 0.014 M acetic acid, pH 4.5. About 50 u l of the DNase II solution obtained from the Sephadex G100 column containing 10$ sucrose and a drop of 0.1$ methyl green as a marker was applied to the gels which were contained i n glass tubes, 5 x 75 mm. Electrophoresis was carried out at a current of 5 milliamperes per tube for 2 hours at k°C. After electrophoresis, some of the gels were removed, stained for protein with Coomassie blue by the method of Gabriel (1971b), and scanned at 550 nm i n a Gilford spectrophotometer equipped with a linear scanning device. F i g . 30 i s a densitometer tracing of a polyacrylamide gel showing the electrophorectic separation of proteins present i n the DNase H preparation. The position of the DNase II ac t i v i t y was estimated by placing unstained gels i n contact with a DNA-agar gel plate which was prepared similarly as for the DNA-agar gel assay described i n the methods section except that no holes were punched i n the gel. After incubation overnight at 37°C, the unreacted DNA was precipitated with 1 N HCl and an oval zone of clearing was observed with the long axis being along the long axis of the gel. The centre of this zone of clearing was judged to be adjacent to the original position of the DNase II i n the polyacrylamide gel. This position i s shown i n Fig. 30 by an arrow. 125 0 1 2 3 A D i s t a n c e f r o m t o p o f g e l (cm.) F i g . 30 Densitometer tracing of a polyacrylamide gel showing the electrophoretic separation of proteins present i n the DNase II preparation. The gel was stained with Coomassie blue by the method of Gabriel (1971a) and the densitometer tracing was done at 550 nm using a Gifford spectrophotometer equipped with a gel scanning device. DNase II was assayed by placing an unstained gel i n contact with a DNA-agar gel plate pre-pared similarly as for the DNA-agar gel assay. . After incubation overnight at 37°C, the unreacted DNA was precipi-tated with 1 N HCl and an oval zone of clearing was deemed to be adjacent to the original position of the DNase II on the gel. This position i s shown by an arrow i n Fig. 30. 126 Measurement of contaminating nucleases i n the DNase II preparation Aliquots of the p a r t i a l l y purified DNase II preparation were assayed for different nucleases hy procedures described i n the Methods section. Table DC demonstrates that there was no DNase I activity present and that the amounts of alkaline and acid phosphatase, and phosphodiesterase I present were low. However, larger amounts of acid and alkaline RNase and phosphodiesterase II ac t i v i t i e s were evident i n the DNase II prepara-tion. The phosphodiesterase II act i v i t y may be lower than indicated. Assay #1 measures the hydrolysis of thymidine 3-p-nitrophenylphosphate; DNase II also degrades this substrate at a slow rate (Bernardi, 1964). Assay #2 measures the hydrolysis of 3-phosphate oligonucleotides prepared with hog spleen DNase II. The limit digest of DNA prepared with hog spleen DNase II may have contained oligonucleotides susceptible to the action of bovine intestinal DNase II because of differences i n the two enzymes with regard to base sp e c i f i c i t y and/or extent of degradation of DNA. Of the contaminating nucleases present phosphodiesterase II would have interfered the most with studies of degradation of DNA by DNase II since phosphodiesterase II acts on the same substrate as DNase II and i s active at acid pH i n the presence of EDTA (Bernardi and Bernardi, 1966), the same conditions which are optimal for DNase II act i v i t y . 127 TABLE IX OTHER NUCLEASES PRESENT IN THE DNase II PREPARATION* Units Enzyme Activity mg Protein DNase II 2.19 DNase I 0.00 -4 Alkaline phosphatase 3.5 x 10 -3 Phosphodiesterase I 9.4 x 10 _2 Acid phosphatase 2.0 x 10 Phosphodiesterase II: Assay #1 0.28 Assay »2 5.91 Acid RNase 3.77 Alkaline RNase 2.77 * Aliquots of the Dnase II preparation obtained from the Sephadex G100 column (Fig. 29) were assayed for nucleolytic ac-t i v i t i e s by procedures described in the Methods section. 128 Further purification of DNase II In order to reduce the phosphodiesterase II contamination of DNase II several different chromatographic purification methods were attempted. Hydroxyapatite chromatography by the method of Bernardi (1971b) resulted i n the absorption of the DNase II activity to the co-lumn i n 20 mM phosphate buffer, pH 6.8, but less than k% of the applied DNase II units could be eluted either with a linear gradient of 0.02 to 0.5 M phosphate buffer, pH 6.8, or, i n a separate experiment, with a l i -near [gradient of 0 to 0.5 M potassium chloride i n 20 mM phosphate buffer, pH 6.8. That a loss of DNase II activity occurs i n phosphate buffer espe-c i a l l y i n the presence of magnesium, a divalent metal ion, has been shown previously (Fig. l 6 ) . It i s possible that the low yield of DNase II ac-t i v i t y upon chromatography on hydroxyapatite was due to loss of DNase II act i v i t y inathe sphosphate buffer perhaps exacerbated by the presence of calcium, an integral part of hydroxyapatite (Bernardi, 1971b). It i s not possible with hydroxyapatite chromatography to use EDTA i n the eluting buffers to stabilize the DNase II acti v i t y since the presence i n the s o l -vent of substances such as EDTA and citrate having a stronger a f f i n i t y for calcium than phosphate can reduce the binding capacity of hydroxyapatite to zero (Bernardi, 1971b). Oshima and Price (1973) purified porcine spleen DNase II by chromatography on sulfoethyl-Sephadex and phosphocellulose. How-ever, their methods were not found to be effective for purifying intestinal DNase II since the enzyme was not retained by the columns at ionic strengths sufficient to remove any appreciable amountsof contaminating protein. The difference i n chromatographic behavior of spleen and intestinal DNase II 129 may be due to the different tissue origins or the different methods of preparation of the two enzymes, DNase II was eventually purified further by repeating the proce-dure described previously using DEAE and CM cellulose and Sephadex co-lumns of smaller size and the purified DNase II from the Sephadex G-100 column (Fig, 29) as the starting material. Since intestinal phospho-diesterase II has a molecular weight of 150,000 to 170,000 for the rat enzyme (Flanagan and Zbarsky, 1977) and intestinal DNase I I , a molecu-l a r weight of 41,000, the two enzymes should have been separated i n the original Sephadex 6100 column. In order to overcome any electrostatic interactions which may have hindered the complete separation of the two a c t i v i t i e s , the Sephadex column used i n this second purification pro-cedure was eluted with 20 mM Tris HCl, pH 7.5, containing 10 mM EDTA and 1 M potassium chloride instead of with 0.25 M Tris HCl, pH 7.5, containing 10 mM EDTA. Table X compares DNase I I , phosphodiesterase II and acid and alkaline BNase acti v i t i e s before and after further p u r i f i -cation. After purification the specific a c t i v i t i e s of DNase II and acid BNase were increased and those of phosphodiesterase II and alkaline BNase were decreased. The ratio of phosphodiesterase I I , acid BNase and alkaline BNase to DNase II had decreased 11, 3» and k f o l d respec-t i v e l y . This indicates that the phosphodiesterase II contaminant had been substantially reduced with respect to the BNase II ac t i v i t y . 130 TABLE X EFFECT OF FURTHER PURIFICATION ON THE ACTIVITIES OF OTHER NUCLEASES,::PRESENT IN DNase I I PREPARATION Enzyme A c t i v i t y Units Ratio of Nuclease mg P r o t e i n to DNase I I Before Further P u r i f i c a t i o n DNase I I 2.19 1.0 Phosphodiesterase I I 5.91 2.7 Aci d RNase 3.77 1.7 A l k a l i n e RNase 2.77 1.3 A f t e r Further P u r i f i c a t i o n DNase I I 7.12 Phosphodiesterase I I 1.73 0.24 Acid RNase 4.58 0.64 A l k a l i n e RNase 2.20 0.31 131 Discussion During the purification procedure 10 mM EDTA was included i n a l l buffers i n order to prevent loss of DNase II act i v i t y . When EDTA was omitted from the buffers used for elution of the columns very l i t t l e DNase II ac t i v i t y was recovered after the f i r s t three chromatographic purification steps. EDTA probably stabilized DNase II by chelating metal ions which would otherwise have caused a loss of DNase II activity either directly by denaturation of the enzyme molecule, or indirectly through enhancement of the act i v i t y of proteases present i n the DNase II preparation.,. Intestinal DNase II was p a r t i a l l y purified by ion exchange column chromatography and gel f i l t r a t i o n . Several different proteins other than DNase II were demonstrated i n the DNase II preparation by poly-acrylamide gel electrophoresis. The major contaminating nucleases were acid and alkaline BNase which do not act on DNA, and phosphodiesterase I I . The phosphodiesterase II act i v i t y present i n the DNase II preparation was reduced substantially by further purification. DNase II had a speci-f i c a c t i v i t y of 7.12 units per mg protein i n the most purified preparation. This represented a 78 fold purification of DNase H with respect to the original crude extract of bovine intestinal mucosa. The overall yield of DNase II units was 13$. 132 B Some properties of Intestinal DBase II pH opt imum - y~. - -The pH optimum of the "purified intestinal DNase II was deter-mined by measuring the DNase II activity as a function of time by the acid-soluble oligonucleotide assay* A l l buffers were made up to a constant conductivity of 6.5 mmhos by the addition of sodium chlo-ride and the pH was measured i n each assay tube. F i g . 31 illustrates the effect of pH on the rate of release of acid-soluble oligonucleo-tides from DNA by DNase I I . In a l l the curves an i n i t i a l lag phase i n the release of acid-soluble oligonucleotide was observed. This was l i k e l y due to additional cleavages of DNA by DNase II that did not produce DNA fragments small enough t o be acid-soluble. Cleaver and Boyer (1972) found that oligonucleotides, 17 nucleotides i n length, were 50% precipitated by 5% perchloric acid. Since the f i n a l concentration of perchloric acid i n the DNase II assay mix was 2%, i t i s l i k e l y that only small oligonucleotides less than 20 nucleo-tides i n length 5were acid-soluble to an appreciable extent and con-tributed to the absorbance at 260 nm i n the assay. Another feature of the reaction which i s shown i n F i g . 31 i s that the extent of de-gradation of DNA was lower at lower pHs. The reaction at pH 3.8 slowed down at 30 minutes, whereas the reaction at pH 5.7 showed no sign of leveling off at 30 minutes. Lowering the pH of a solution causes denaturation of calf thymus DNA; the pH at which denaturation occurs depends on the amounts and kinds of salts present and the 133 Fig. 31 Effect of pH on the rate of release of acid-soluble oligonucleotides from DNA by DNase II . The reaction was carried out with kOO ugm DBA per ml i n 120 mM sodium acetate buffer containing 8 mM EDTA. — • — pH 3.6 — A — pH 3.8 — • — pH 4.3 PH h.9 — A — pH 5.1 - e — pH 5.7 PH 5.9 0 1 0 2 0 3 0 T i m e ( m i n u t e s ) 135 length of the DNA fragments present (Beaven et a l , , 1955; Reisner and Somen, 1973), At low pH DBA fragments were probably denatured ear-l i e r i n the reaction than at high pH. Since DNase II degrades denatured DNA at a slower rate than native DNA (Table VI), the denaturation of the DNA fragments l i k e l y resulted i n a decrease i n the rate of acid-soluble oligonucleotide formation. Fig. 32 demonstrates that DNase II had a broad pH optimum centered about pH 4.8. Each point on the graph represents the slope of the linear portion of the curve for that pH from Fig. 31* By taking the pH values corresponding to half the maximum velocity, two apparent pKa values for DNase II were estimated to be 3.9 and 5*6. The values are within the ranges that would be expected for a 0- or ^ -carboxyl group of aspartic or glutamic acid and an imidizolium group of histidine respectively i n the active site of the enzyme (Mahler and Cordes, 1971; Segel, 1975). Effect of magnesium ion DNase II act i v i t y was measured i n the presence of different concentrations of magnesium chloride by the hyperchromicity assay using kO ugm calf thymus DNA i n 1.0 ml of 120 mM solium acetate buffer, pH 5.0. The slopes of the hyperchromicity at 260 nm versus time curves were plotted against the magnesium chloride concentration. F i g . 33 shows that the DNase II act i v i t y was activated sl i g h t l y by low MgCLg concen-trations, and inhibited by MgClg concentrations greater than 10 mM. F i g . 32 pH optimum of intestinal DNase II. Each point on this graph represents the slope of the linear portion of the curve for that pH from F i g . 31* 137 0 2 0 4 0 6 0 M g C l (mM.) F i g . 33 Effect of magnesium ions on DNase II act i v i t y . DNase II acti v i t y was measured by the hyperchromicity assay i n 120 mM sodium acetate buffer, pH 5.0, containing ko ugm calf thymus DHA/ml. Velocities were taken as the slopes of the linear portions of hyperchromicity curves obtained for various MgCl2 concentrations. 138 Effect of ionic strength Fig. 18 demonstrates that DNase II ("fi r s t activity") was activated on addition of NaCl to 0.1 M and inhibited by further additions of NaCl. Effect of sulfate, an inhibitor of BNase II Fig. 19 illustrates the inhibition of DNase II by sulfate concen-trations from 0.01 to 0.1 M. The effect of very low sulfate concentrations oh DNase II ac t i v i t y may be seen i n Fig. 34. DNase II act i v i t y was measured by the hyper-chromic i t y assay i n 120 mM sodium acetate buffer, pH 5.0, containing ho ugm DNA per ml. Velocities were taken as the slopes of the linear portions of hyperchromicity curves obtained for different concentrations of sodium sulfate. DNase II was activated maximally at a 1 mM concen-tration of sulfate. The addition of 10 mM EDTA also resulted i n about a 20$ increase i n DNase II ac t i v i t y . Oshima and Price (1974) found that 0.5 mM sulfate increased the ac t i v i t y of hog spleen DNase II by about 20$ corresponding to a 15 fold increase i n the number of single strand cleavages of the DNA substrate. Activation energy for hydrolysis of DNA The activation energy for the hydrolysis of calf thymus DNA by DNase II f i r s t a c t i v i t y was previously estimated to be J$ kcal/mole (Fig. 20) Fig* 3h Effect of millimolar concentrations of sulfate ion on DNase II activity. DNase II activity was measured by the hyperchromicity assay i n 120 mM sodium acetate buffer, pH 5.0, containing kO ugm calf thymus DNA/ml. Velocities were taken as the slopes of the linear portions of hyper-chromicity curves obtained for different concentrations of sodium sulfate. The DNase II activity i n the presence of 10"5 M sulfate ion was the same as that found when no sulfate ion was present. Ikl Activation energy for denaturation of DNase II The activation energy for denaturation of the DNase II molecule was estimated to be 43 kcal/mole (Fig. 21). Molecular weight of DNase II The molecular weight of DNase II was estimated to be 41,000 by gel f i l t r a t i o n on Sephadex G100 (Fig. 26). DNase II i s probably a positively charged molecule at neutral pH since i t was not retained on a positively charged DEAE cellulose column i n 20 mM phosphate buffer, pH 7*8, but was bound to a negatively charged CM cellulose column i n 20 mM phosphate buffer, pH 6.0. As w i l l be discussed i n the next section, the products of the reaction of DNase II with DNA can be labelled with 32p using polynucleo-tide kinase and [T- ^2P]ATP, separated from mononucleotides, and degraded to 5'-32p nucleotides. This implies that DNase II cleaves DNA endonucleo-l y t i c a l l y to form oligonucleotides with 5* hydroxyl and 3 ' phosphate-terminals. Further evidence for endonucleolytic cleavage of DNA by DNase II comes from the observation that DNase II was active under conditions used to measure endonucleolytic a c t i v i t y , but showed no acti v i t y under exo-nuclease assay conditions. Hog spleen DNase I I , an endonuclease, can be assayed by measuring the degradation of DNA into products soluble i n 2$ perchloric acid (Bernardi, 1968). Spleen exonuclease can be assayed by 11*2 measuring the products of DM digestion that are soluble i n 1.3$ per-chloric acid containing 0.13$ uranyl acetate (Bernardi and Bernardi, 1966). When DNA i s degraded by intestinal DNase I I , absorbancesof 0.6at 260 nm i n 2$ perchloric acid and zero i n 1«3$ perchloric acid containing 0.13$ uranylacetate were observed. This indicates that DNase i s active under conditions used to measure endonucleases, not exonucleases• Discussion The enzyme isolated from bovine intestinal mucosa has an acidic pH optimum, i s active i n the presence of EDTA and i s inhibited by mag-nesium. This i s the original definition of a DNase II that was proposed by Cunningham and Laskowski (1953). As w i l l be demonstrated i n a latte r section the enzyme also degrades DNA endonucleolytically to 3'-phosphate terminated oligonucleotides, and thus i s a DNase II by the current definition (Enzyme Nomenclature, 1972). Other properties of intestinal DNase II such as chromatographic behavior, molecular weight, inhibition by high ionic strength and by high sulfate ion concentration, are similar to those of DNase II enzymes isolated from other sources (Cordonnier and Bernardi, I968; Laskowski, 1961, 1967; Bernardi, 1968, 1971). Iks E The reaction of DNase II with DMA, and other substrates The Michaelis constants and the maximum velocities for the reaction with DNA. and polydAT A f i r s t attempt to determine the Michaelis constant, Km,for the reaction of DNase U with DNA was made by using the hyperchromicity assay to follow the DNase II reaction with different concentrations of cal f thymus DNA i n 120 mM sodium acetate buffer containing 8 mM EDTA. Fig. 35 shows that the extent of degradation as measured by the t o t a l increase of the absorbance at 260 nm over the course of the reaction was proportional to the concentrations of calf thymus DNA used: kO, 20, and 10 ugm per ml i n Fig. 35 a>> b, and c, respectively. However, the rate of degradation of DNA was the same i n a l l three reactions. This implies that the substrate was present i n a saturating concentration with respect to the enzyme. In order to have lower concentrations of DNA i n the reaction i t was necessary to use radioactively labelled DNA so that the acid-soluble oligonucleotides released could be detected. %-DNA, sonicated, from Escherichia c o l i was a good substrate with which to determine the Km for DNase II because there was no i n i t i a l lag phase i n the release of acid-soluble oligonucleotides and thus i n i t i a l velocities could be determined. H^-DNA was digested with DNase II and the acid-soluble radioactivity released was measured and plotted as a function of time, as described i n the Methods section. The i n i t i a l slopes of these curves were taken as indicative of the Ikk The rate and extent of the reaction of DBase II with different concentrations of calf thymus DBA. The increase i n absorbance, the hyperchromicity, at 260 nm, of a DBA solution upon digestion with DBase II i n 120 mM sodium acetate buffer, pH 5.0, containing 8 mM EDTA was plotted as a function of time. The rate of the reaction was taken as the slope of the linear portion of the curve, and the extent of degradation, as the t o t a l increase i n absorbanceCat 260 nm. a kO ugm DBA/ml b 20 ugm/ DBA/ml c 10 ugm DBA/ml o I n c r e a s e i n a b s o r b a n c e a t 2 6 0 nm. o 0 p P o P ^ ° ro .. . T ~ K I T p ro o co r 146 i n i t i a l velocities of the reaction at different DNA concentrations. Fig. 36 exhibits a Lineweaver-Burk plot for the reaction of DNase II with sonicated % DNA. from Escherichia c o l i . The Michaelis constant. Km, was estimated to be 2.42x 10"7 M DNA-phosphate, and the maximum velocity, 435 cpm of acid-soluble radioactivity released per minute. In reactions that were allowed to go to completion, an average of 33.^  cpm of acid-soluble radioactivity was released for each pmole of DNA-phosphate present. Thus the maximum amount of DNA digested per minute was 3^5/33.^ =13.0 pmoles DNA-phosphate. Because the DNase II preparation was not pure, the maximum velocity could not be expressed i n terms of the pure enzyme, but was, instead, expressed i n terms of the protein present i n the DNase II preparation. The maximum velocity was 44l pmoles DNA-phosphate/min/ugm protein. Fig. 37 illustrates the Lineweaver-Burk plot obtained for the reaction of DNase II with polydAT-methyl-3H. The Michaelis constant, Km, was estimated to be 2.63 x 10"^  M polydAT-phosphate, and the maximum velocity, 556 cpm of acid-soluble radioactivity per minute, or 326 pmoles polydAT-phosphate/min/ugm protein. For the reaction of DNase II with % DNA the Km was 2.42 x 10"7 M DNA-phosphate. DNase II was, therefore, saturated with substrate i n the hyperchromicity and acid-soluble oligonucleotide assays which contained 8.0 x 10"M and 8.0 x 10"^  M concentrations of DNA-phosphate, respec-t i v e l y . Pig. 36 Lineweaver-Burk plot for the reaction of DBase II with sonicated %-DNA from Escherichia c o l i . Reactions were carried out at 37°C i n 150 mM sodium acetate buffer, pH 5.0, containing 10 mM EDTA and various concentrations of 3H-DUA. The i n i t i a l slopes of curves of acid-soluble radioactivity released as a function of time were taken as indicative of the i n i t i a l velocities of the reaction at different DNA concentrations. 1/V (x103) ( c p m /min.) i ^ y _ i i i -5 0 5 10 1/S (xlO6) ( M D N A - p h o s p h a t e ) " 1 1/v (xid3) ( c p m / min.) 8 F i g . 37 1/S (x106) (M p o l y d A T - p h o s p h a t e ) " ' Lineweaver-Burk plot for the reaction of DNase II with polydAdT-methyl-3H. The experiment was carried out and the values calculated as for ^ H-DNA (Fig. 36) . 150 Degradation of native and denatured DBA. The rates of hydrolysis of native and denatured DBA by DNase II were compared. Calf thymus DBA. at a concentration of 0.4 mg per ml i n 10 mM sodium chloride was denatured by heating i n a boiling water bath for 15 minutes, and then cooling i n an ice-water bath. The reaction of DNase II with 40 ugm/ml of either native, or denatured DBA was carried out i n 100 mM sodium acetate, pH 5.0, containing 10 mM EDTA. The hyperchromicity of each solution was plotted as a function of time; the curves obtained are shown i n Fig. 38. With native DBA absorbance at 260 nm increased linearly with time at f i r s t and then gradually decreased; at this high enzyme to DBA ratio the i n i t i a l lag phase was not apparent. With denatured DBA the absorbance increased non-linearly at f i r s t and ~ then'increased linearly with time. Some double-stranded regions of the DBA were probably reformed by aggregation upon cooling of the denatrued DBA since a relatively high concentration, 0.4 mg/ml, of DBA was used. (Marmur et a l . , 1963) • It i s l i k e l y that the rate of degradation observed i n i t i a l l y with the denatured DBA was due to digestion of the small number of double-stranded regions that may have reformed i n the DBA upon cooling. The linear region of the curve probably represented the actual rate of degradation of denatured DBA. Since the slopes of the linear portions of the curves for native and denatured DBA were 0.009 and 0.0014 absorbance units at 260 nm per minute respectively, DBase II degraded native calf thymus DBA 6.4 times faster than denatured calf thymus DBA. An earlier estimate of the relative rates of degradation of native and denatured DBA by DBase II (Table VI) probably included the degradation of Fig. 33 Comparison of the reaction of DNase II with native and denatured calf thymus DNA. DNase II was reacted with ho ugm/ml of either native, or denatured DNA i n 100 mM sodium acetate buffer, pH 5.0, containing 10 mM EDTA. The hyperchromicity of each solution was plotted as a function of time. with native DNA with denatured DNA £| T i m e ( m i n u t e s ) 153 double-stranded regions iMte calculation • of the reaction rate for dena-tured DNA. Digestion of polydAT, polydA, and polydT The rates of degradation of polydAT, polydA and polydT by DNase II were compared by measuring the rate of release of acid-soluble radio-a c t i v i t y from the t r i t i a t e d compounds upon reaction with DNase II i n 150 mM sodium acetate buffer, pH 5.0, containing 10 mM EDTA by the pro-cedure used for the 3g DNA assay, described i n the Methods section. Fig. 39 il l u s t r a t e s the acid-soluble radioactivity released as a function of time for each polynucleotide. The rates of reaction of DNase II with 0.23 uM polydAT-methyl-3!!, 0.29 uM polydA-8-%, and 0.22 uM polydT-methyl- were 21+5, 26, and 15 cpm of acid-soluble radioactivity released per minute, respectively. That the rate of reaction of DNase II with polydAT was at least-10 times that with polydA or polydT i s probably due to the double h e l i c a l regions such as "hairpin loops" which can occur i n polydAT (Scheffler et a l . , 1968) and to the preference of DNase II for the double-stranded over the single-stranded configuration. Mode of cleavage of native DNA DNase II can cleave either one or both strands of native DNA. Different values for the relative numbers of single-strand and double-strand cleavages of DNA by DNase II from other tissues have been found 15V F i g . 39 Reaction of BNase II with polydAT, polydA and polydT. The acid-soluble radioactivity released from the t r i t i a t e d compounds upon reaction with DNase II at 37°C i n 150 mM sodium acetate buffer, pH 5.0, containing 10 mM EDTA, was plotted as a function of time. — • — polydAT-methyl-% — • — polydA -8 -% polydT-methyl-^H Time (min.) 156 by previous Investigators using ultracentrifugation i n neutral and alkaline media (Young and Sinsheimer, 1965; Bernardi and Bach, 1968; Kopecka et a l . , 1973; Oshima and Price, 197*0. Radioactive labelling of DNase II reaction products using poly-nucleotide kinase and {V- P^JATP i s a rapid and direct way of determining the mode of cleavage of DNA by DNase I I . Fig. kO indicates how the relative amounts of single-strand and double-strand cleavages of DNA by DNase II were determined. In a certain fragment of native DNA. DNase II made one single-strand and one double-strand cleavage. When the native digestion products from the DNase II reaction were incubated with polynucleotide kinase and j^f- ^PJATP, only the 5'-hydroxyl groups at the double-stranded ends were labelled •30 since incorporation of P into an internal 5'-hydroxyl group occurred at a much slower rate than into an external 5 1-hydroxyl group (Weiss et a l . , 1968; Oshima and Price, 197k). Conditions used for the polynucleotide kinase reaction were not favourable for the exchange reaction between 5' -phosphate terminated oligonucleotides and \jf- ^P\ATP noted by van de Sande et a l . (1973) and by Beckner and Folk (1977). Since two 5'-hydroxyl groups were liberated f o r each double-strand cleavage, half the counts per minute or pmoles incorporated was proportional to the number of double-strand cleavages. When the DNase I I digestion products were denatured before incubation 32 with polynucleotide kinase, P was incorporated into the 5*-hydroxyl ends resulting from single-strand cleavages as well as those resulting 32 from double-strand cleavages. The difference between the P incorporated into the denatured DNase II digestion products and the 32p incorporated 157-n a t i v e DNA D N a s e I I d i g e s t i o n p r o d u c t s w i t h n a t i v e d i g e s t i o n p r o d u c t s D N a s e I I P HO--OH P OH P P ^ P OH P 32p p o l y n u c l e o t i d e k i n a s e (T- ^PjATP w i t h d e n a t u r e d d i g e s t i o n p r o d u c t s 32 p — 32, — 32 P l e t n = p m o l e s ^ P i n c o r p o r a t e d l e t d « p m o l e s 32p i n c o r p o r a t e d i n t o n a t i v e d i g e s t i o n p r o d u c t s i n t o d e n a t u r e d d i g e s t i o n p r o d u c t s n o . o f d o u b l e - s t r a n d c l e a v a g e s <x ~ -n o . o f s i n g l e - s t r a n d c l e a v a g e s ^ ( d - n ) F i g . kO D e t e r m i n a t i o n o f m o d e o f c l e a v a g e o f D N A b y D N a s e I I . 158 into the native digestion products was proportional to the number of single-strand cleavages. The DNase II reaction DNase II was incubated at 37°C with ho ugm DNA. i n 1.0 ml of 100 mM sodium acetate buffer, pH 5.0, containing 10 mM EDTA. The re-action was followed by measuring the increase i n absorbance at 260 nm of the DNA solution versus time with respect to a control solution to which no enzyme had been added. Fig. hi illustrates the hyperchromi-c i t y plots obtained for the digestion of DNA from different sources by DNase I I . Duplicate DNA solutions were incubated with DNase II for 5 minutes at 37°C. The reactions were stopped by the addition of 5 0 u l of Lj3 M t r i s HCl, pH 8.0, 20 u l of 1.0 M MgCl2> and 45 u l of 1 N NaOH; the f i n a l pH was 8.0. This was done i n order to ensure that the so-lutions had an optimal pH and divalent metal ion concentration for the polynucleotide kinase reaction. The extent of degradation of the DNA was estimated from Fig. Ul, by expressing the increase i n absorbance at 260 nm after 5 minutes of reaction as a percent of the t o t a l i n -crease i n absorbance at 260 nm. The reaction of DNase II with calf thymus DNA was also carried out for 1, 10, 20, and 30 minutes at 37° C. The reaction mixtures, as well as control solutions containing DNA, were each divided into two equal portions. One portion of the solu-tion was heated i n a boiling water bath for 15 minutes, and then quickly cooled i n an ice-water bath i n order to denature the double-stranded oligonucleotides or DNA; the other remained at 0° C and con-Fig. kl Digestion of DNA from various sources by DNase II. The reaction was carried out with kO ugm/ml of DNA i n 100 mM sodium acetate buffer, pH 5.0, containing 10 mM EDTA. The hyper chromic i t y of the reaction solution was plotted as a function of time, and the extents of degradation of a particular DNA at various times were estimated by expressing the absorbances of the DNA solution at those times as percents of the maximum absorbance attained. H VO 161 tained native oligonucleotides, or DNA. The polynucleotide kinase reaction The reaction conditions were worked out with consideration of the properties of polynucleotide kinase from bacteriophage TU,(Richard-son, 1971J van de Sande et a l . , 1973; Lillehaug and Kleppe, 1975a,b; Okazaki et a l . , 1975; Lillehaug et a l . , 1976). A reducing agent, lOmM dithiothreitol was added to stabilize the polynucleotide kinase (Ri-chardson, 1971)* The reaction was carried out at a pH of 8.0 which i s within the optimum pH range for the enzyme for the phosphorylation of 5' hydroxyl end groups i n DNA and which is high enough so that the exchange reaction of 5'-phosphate end groups with ATP i s mini-mized (van de Sande et a l . , 1973). T h e j V - ^ p ] ATP was of sufficient incorporation could be rel i a b l y determined. The concentration of -"^Pj ATP was 20 mM i n order to ensure quantitative incorporation of ^ P intotte5 ! <^w^gioupsof double-stranded DNA (Lillehaug et a l . , 1976). Although this concentration i s much lower than the Km for the enzyme (Richardson, 1971)* a higher concentration was not used because of the p o s s i b i l i t y of increasing the rate of the exchange reaction (van de Sande et a l . , 1973). Only 2 units of polynucleotide kinase were used per assay because of the p o s s i b i l i t y of phosphorylation of single-strand nicks i n native DNA using larger amounts of enzyme (Lillehaug et a l . , 1976). To 100 u l of a solution containing DNase II digestion products radioactivity, at least 200 cpm per pmole, so that the extent of 2^p 162 or DNA., were added 2 u l of 0.98 mM ATP, enough ATP to give a specific radioactivity of at least 200 cpm per pmole ATP and 2 units of Tk polynucleotide kinase. The incubation was carried out at 37° C and at various times 5 u l aliquots were removed from the reaction mixture and placed on sheets of DE 81 cellulose paper, 23 x 28 cm which had been pre spotted twice with 50 u l aliquots of 1 mM ATP, 50 mM EDTA. A 5 u l aliquot of the reaction mixture was taken before the addition of the polynucleotide kinase and was spotted as a control for the possible absorption of ATP to the origin. Descending chro-matography was run for two hours at room temperature with 0.35 M ammo-nium formate buffer, pH 5.5, (van de Sande et a l . , 1973). Under these conditions the Rfs of ATP, AMP and P i were 0.46, 0.48 and 0.71 respec-t i v e l y . Oligonucleotides of at least 10 nucleotides i n length re-mained at the origin (Sgaramella and Khorana, 1972). The DE 8l cellu-lose paper was dried, and the origins were cut out and placed i n 10 ml of water i n l i q u i d s c i n t i l l a t i o n v i a l s . The radioactivity retained at the origin was estimated by measuring the Cerenkov radiation pro-duced i n the % [ channel of a l i q u i d s c i n t i l l a t i o n counter. The counts per minute were converted into pmoles of 2^p incorporated into the original amount of digestion product present i n the reaction mixture. This was done by calculating the specific radioactivity of t h e j ^ - ^ p ] ATP used, either from the manufacturer's data, or by using oligodT (pT) 9 as described i n the Methods section. 163 Cleavage of calf thymus DBA Fig. k2 depicts the ^ P radioactivity incorporated "by polynu-cleotide kinase into the native and denatured products of the diges-tion of native calf thymus DNA by DNase II as well as into native and denatured calf thymus DNA. A significant incorporation of P into native DNA indicated that there was' a substantial number of 5'-hydroxyl groups present i n the DNA before digestion with DNase I I . The higher incorporation of ^ P into denatured DNA than into native DNA demonstrated that there were single-stranded 5*-hydroxyl nicks i n the DNA prior to digestion with DNase II and that this technique of ^ P incorporation using polynucleotide kinase was able to differen-t i a t e between external and internal 5'-hydroxyl groups. The incorporation of 32p fcy polynucleotide kinase into denatured products of digestion of native DNA by DNase II was greater than for the control DNA and increased with thr duration of the DNase II re-action. This indicates that the products of the reaction of DNase II with DNA had: 5'-hydroxyltamaals. Because the 32p radioactivity i n -corporated remained at the origin i n the DE 8l paper chromatography system i n whieh AMP migrated with an Rf of 0.48, DNase II cleaved the DNA endonucleolytically to liberate oligonucleotides. Table XI illustrates the method used to calculate the percent of cleavages of DNA by DNase II that were double-strand cleavages. In order to determine the amount of ^ P incorporated into 5 1 -hydroxyl groups released from DNA by DNase II action, the pmoles of 3% i n -corporated into denatured and native DNA were subtracted from the F i g . 42 Polynucleotide kinase catalysed incorporation of -^P into products of the reaction of DNase II with native calf thymus DNA.. The DNase II reaction with 40 ugm/ml DNA was carried out for various times i n 100 mM sodium acetate buffer. pH 5.0, containing 10 mM EDTA. The reaction mix-tures, as well as control solutions containing DNA, were each divided into two equal portions. One part of the solution was denatured; the other was allowed to remain native. The 32 P radioactivity incorporated into these products was plotted as a function of the time of reaction with polynucleotide kinase. native DNA denatured DNA native 5 minute reaction products denatured 5 minute reaction products native 10 minute reaction products denatured 10 minute reaction products native 20 minute reaction products denatured 20 minute reaction products native 30 minute reaction products denatured 30 minute reaction products 165 Time (min.) TABLE XI PROPORTION OF CLEAVAGES OF DNA BY DNase I I THAT WERE DOUBLE STRAND CLEAVAGES Substrate Total P 3 2 P incorporated 3 2 P incorporated incorporated due to DNase I I p r o p o r t i o n a l to (pmoles) a c t i o n (pmoles) no. of double-strand cleavages 32 P incorporated p r o p o r t i o n a l to no. of s i n g l e -strand cleavages double-strand cleavages % of t o t a l denatured 5 minute r e a c t i o n products denatured c a l f thymus DNA 120 60 120 - 60 = 60 53.4 = 26.7 60 - 53.4 = 6.6 26.7 26.7-6.6 = 80% •oclOO ON n a t i v e 5 minute r e a c t i o n products 86.7 86.7 - 33.3 = 53.4 na t i v e c a l f thymus DNA 33.3 167 pmoles of "*"P incorporated into denatured and native products of d i -gestion of DBA by DBase I I . Because two 5'-hydroxyl groups origina-ted from each double-strand cleavage, the number of double-strand 32 cleavages was proportional to half the amount of P incorporated into native digestion products. Since was incorporated into 5 1-hydroxyls from single-strand cleavages only after denaturation of the reaction products, the number of single-strand cleavages was pro-portional to the pmoles of P incorporated into denatured products minus that incorporated into native products. The relative number of double-strand cleavages was calculated as a percent of the t o t a l num-ber of cleavages. Table XII gives values for percent double-strand cleavage calculated i n this way for 5 minute reactions of DBase II with Uo ugm/ml of ca l f thymus DBA. l n 100 mM sodium acetate buffer, pH 5.0, containing 10 mM EDTA i n 10 separate experiments. The mean value obtained was 36.1 -12.6. The relatively large background incorporation of 32 P due to nicks and ends present i n calf thymus DBA before digestion with DBase II made i t more d i f f i c u l t to measure accurately the incorporation of P into 5'-hydroxyl groups released by DBase II action and probably ac-counted for some of the v a r i a b i l i t y of the results. Degradation of DNA from other sources. In order to determine the mode of cleavage of other DNA by DNase I I , and the background incorporation, with DNA from other species, DBA from salmon testes, rat intestinal mucosa, bacteriophage A , and 168: TABLE XII MODE OF CLEAVAGE OF CALF THYMUS DNA BY DNase I I Experiment Double strand cleavages Number (% of t o t a l cleavages) 1 90 Mean =86.1 2 71 Standard D e v i a t i o n = - 12.6 3 86 C o e f f i c i e n t of 4 81 V a r i a t i o n = 14.6% 5 90 Standard D e v i a t i o n of the Mean: 6 100 Sx = - 3.98 7 63 ( b ) 9 5 % Confidence L i m i t s x = 86.1 ± 9.0 8 80 99% Confidence L i m i t s 9 , 100 x = 86.1 - 11.2 10 100 (a) Values f o r % double strand cleavages that were gre a t e r than 100, were assumed to be 100. I f t h i s was not not done, these values were r e j e c t e d by Chauvenet's c r i t e r i o n . (b) Using Student's t - t e s t (Brewer et a l . . 1974). 169 Escherichia c o l i was digested with DNase II under the same conditions used previously for the digestion of ca l f thymus DNA. Polynucleotide 32 t\ kinase catalysed incorporation of P into salmon, r a t , and A DNA and their respective reaction products formed on digestion with DNase II is illustrated i n Fig. 43. The lower incorporation of P into salmon and A DNA than into calf thymus and rat DNA indicated that there were fewer 5•-hydroxyl groups present i n salmon and X DNA. For a l l three DNA species i n Fig. kk the incorporation of ^ P into digestion pro-ducts resulting from DNase II action was substantially greater than into the corresponding DNA control. Effect of 1 mM sulfate on the mode of cleavage of DNA from Escherichia c o l i F i g . kk i l l u s t r a t e s the effect of 1 mM sulfate on the mode of cleavage of DNA from Escherichia c o l l by DNase I I . The reaction was op carried out for 5 minutes and P was incorporated as previously des-cribed for the reactions of DNase II with DNA. In the presence of sulfate 32p incorporation increased slight-l y for both native and denatured digestion products, but no large i n -crease was seen i n the incorporation of 32P into 5'-hydroxyl groups resulting from single-strand cleavages. Effect of 10 mM EDTA on the mode of cleavage of calf thymus DNA The effect of 10 mM EDTA on the mode of cleavage of ca l f thymus 170 32 Fig. 43 Incorporation of P by polynucleotide kinase into products of the reaction of DNase II with DNA from salmon testes, rat intestinal mucosa, and bacteriophage A . The DNase II reaction was carried out for 5 minutes and 32P was incorporated as described i n Fig. 42. (a) salmon DNA (b) rat DNA (c) DNA — • native DNA. — © denatured DNA —tk— native 5 minute reaction products denatured 5 minute reaction products 171 172 Fig. kk Effect of 1 mM sulfate on the mode of cleavage of DNA from Escherichia c o l i by DNase II. The DNase II reaction was carried out for 5 minutes and 32p incorporated as described i n Fig. k2. (a) no sulfate ion present (b) with 1 mM sodium sulfate present — • — native DNA — £ — denatured DNA — A — native reaction products —A— denatured reaction products 17k DNA by DNase II i s illustrated i n F i g . 45. In the presence of EDTA the 32P incorporation increased s l i g h t l y for both native and denatured reaction products. No evidence was seen for a large increase i n the number of single-strand cleavages by DNase II i n the presence of EDTA. In Table XIII are given the values for percent double-strand cleavage of DNA from different species by DNase H calculated from the incorporation curves displayed i n Figs. 43, kk, and 45. From the results shown i n Table XIII as well as from those found i n Table XII, i t appears that intestinal DNase II cleaved native DNA primarily by a double-strand cleavage mechanism. Neither the addition of 1 mM sulfate to the reaction mixture, nor the removal of the EDTA from i t seemed to have any substantial effect on the mode of cleavage of DNA by DNase I I . Characterization of the products and substrates of the DNase II reaction by electrophoresis on polyacrylamlde gels Although the size distribution of the products of the degradation of DNA by DNase II from other tissues has been investigated by chromato-graphy of reaction mixtures on DEAE cellulose columns (Bernardi, 1968; Soave et a l . , 1973), even after very prolonged incubation of DNA with DNase I I , 50$ or more of the products were unresolved by this technique. (Soave et a l . , 1973). Polyacrylamlde gel electrophoresis of the products of the reaction of DNase II with DNA and other substrates was carried out i n order to estimate the size ranges of the digestion products as well as to determine 175 F i g . h5 Effect of 10 mM EDTA on the mode of cleavage of calf thymus DBA by DBase II. The DBase II reaction was carried out for 5 minutes and 32p was incorporated as described i n Fig. 42. (a) complete DBase II reaction mixture including 10 mM EDTA (b) with omission of EDTA — • — native DBA — 9 — denatured DBA — A — native reaction products —tti denatured reaction products 177 TABLE X I I I MODE OF CLEAVAGE OF DNA FROM DIFFERENT SPECIES BY DNase I I * Double-strand Cleavages Preparation Used (percent of t o t a l cleavages) DNA from salmon t e s t e s 82 DNA from r a t i n t e s t i n a l mucosa 84 DNA from bacteriophage ^  100 DNA from E c h e r i c h i a c o l i - s u l f a t e 80 + 1 mM s u l f a t e 79 DNA from c a l f thymus + EDTA 100 - EDTA 100 DNase I I was reacted with 40 ugm/ml DNA i n 100 mM sodium acetate b u f f e r , pH 5.0, co n t a i n i n g 10 mM EDTA excepjt when the EDTA was omitted. 178 whether discrete sizes of products were formed i n the early stages of the reaction. DNA from bacteriophageX and calf thymus, and polydAT were digested with DNase II under the same conditions used to determine the mode of cleavage of DNA by DNase II . The reactions were stopped by extraction of the solutions with equal volumes of water-saturated phenol. Phenol dissolved i n the aqueous layer was removed by three extractions with ether; traces of ether were removed by bubbling nitrogen through the solution. Substrate solutions which had not been digested with DNase II were treated similarly. The samples were electrophoresed on 5% polyacrylamide gels at 5 volts/cm i n 90 mM Tris-90 mM boric acid-2.5 mM EDTA by the method described by Maniatis et a l . (1975). Since intercalation of ethidium bromide between base pairs i n double-stranded DNA causes a large increase i n the ethidium bromide fluoresence emission spectrum (LePecq and Paoletti, 1967)t treatment of the gels with ethidium bromide allowed the DNa fragments to be visualized.* The lengths of the DNA fragments were estimated by comparison of their distances of migration on the gels with those of DNA. fragments resulting from digestion of replicatlve form DNA from bacteriophage 0X Yjk by a restriction endonuclease from Haemophilis aegyptius, Hae I U . The lengths of the 0X174 DNA fragments have been determined by sequence analysis (Sanger et a l . , 1977)* As illustrated i n F i g . 46 , continuous fluorescent patterns resulting from oligonucleo-*Thanks are due to Anne Lui who performed this experiment. 179 F i g . k6 Electrophoresis of products and substrates of the DNase II reaction on polyacrylamlde gels. The sample applied at the extreme l e f t was a digest of implicative form DNA from bacteriophage 0K 17h by a restriction endonuclease from Haemophilis aegyptius, Hae III. From top to bottom the bands shown represented double-stranded DNA fragments of 1346, 1073, 873, 606, 310, 278, 271, 23k, 19k, 118 and 72 base pairs. (Sanger et a l . , 1977). The other samples applied were 10, 20, and 30 u l portions of 5 minute DNase II digests of polydAT and DNA from bacteriophage A and calf thymus as well as 30 u l portions of the unreacted substrates. ISO 181 tides ranging from less than 100 to more than 1,300 base pairs. < i n length were seen for 5 minute DNase II digests of polydAT and DNA from bacteriophage X and calf thymus. Fluorescent bands with positions on the gel comparable to those of species of high molecular weight were seen for polydAT and DNA from bacteriophage A and Escherichia c o l i . Electrophoresis of DNA from calf thymus and salmon testes resulted i n fluorescent patterns indicating the presence of DNA fragments of high and low molecular weights. Base spe c i f i c i t y of cleavage of DNA by DNase II Thiery et a l . (1973) have investigated the base sp e c i f i c i t y of the cleavage of DNA by DNase II from hog spleen i n the middle and terminal phases of the reaction, that i s , with oligonucleotide products of average length from 15 to kO nucleotides. They demonstrated that DNase II cleaved specific sets of short nucleotide sequences at least four nucleo-tides long and presented evidence that indicated that DNase II might be more specific, or at least have a different base sp e c i f i c i t y i n the i n i t i a l phase of the reaction which had not been studied. Accordingly the base speci f i c i t y of the cleavage of DNA by intes-t i n a l DNase II was investigated i n the early stages and throughout the reaction i n order to determine whether the base sp e c i f i c i t y of cleavage changed as the reaction progressed, and whether DNase II cleaved DNA more spe c i f i c a l l y i n the i n i t i a l stages of the reaction. 182 The base sp e c i f i c i t y at the 5' terminals Although radioactive labelling using polynucleotide kinase has been a useful technique for determination of the base sequence of cleavage of DNA by restriction enzymes (Smith et a l . , 1974), the method has not been used for DNase I I . Fig. 47 presents a scheme outlining the procedure used to deter-mine the 5*-terminal base sp e c i f i c i t y of cleavage of DNa by DNase II. DNA was digested with DNase II and the products of the reaction were labelled with ,3?p a t the 5»-hydroxyl groups i n a reaction with poly-nucleotide kinase andl/fr - The labelled oligonucleotides were separated from unreacted [V- ^pjATP-.by chromatography on Sephadex 050 and then degraded to 5*-phosphate terminated nucleotides with DNase I and phosphodiesterase I. The nucleotides were separated by thin layer chromatography and the amount of radioactivity incorporated into each terminal nucleotide was determined. The DNase II and polynucleotide kinase reactions DNase II and polynucleotide kinase reactions were carried out as described previously for determination of the mode of cleavage of DNA by DNase II. After 32 minutes of incubation with polynucleotide kinase, 20 u l of 0.2 M EDTA was added, the reaction mixture was heated at 100° C for 5 minutes and then cooled to 0°C. 183 DNA HoJ 32, 32, DNase II 3' polynucleotide kinase + ft- 32P]ATP 5' 3' J V* unreacted [r- 32p]ATP Sephadex G50 chromatography 3' (l) DNase I ' (g) phosphodiesterase I L 3' OH +(p >fOH)n + p^P Fig. k7 Scheme for determination of the base sp e c i f i c i t y at the 5' -terminals of products of cleavage of DNA by DNase H „ The oligonucleotide products form the DNase II reaction were labelled at the 5'-terminals with 32p usingpoly-nucleotide kinase and then were enzyraatically>degraded to 5«-32p,nucleotides. The 5'-32p nucleotides were separated by thin:: layer chromatography and the amount of radioactivity i n each nucleotide was determined by measuring the Cerenkov ton emitted. I81f Separation of the 5»-32p oligonucleotide products of the poly-nucleotide kinase reaction from unreacted [If- 32p] A TP The reaction mixture was chromatographed on Sephadex 050. The column. 0.9 x 50 cm, was eluted at k°C with 50 mM triethyiammonium bicarbonate buffer, pH 7.5. Fractions of 0.5 ml were collected i n k ml glass v i a l s , and after the addition of 3*5 ml of water to each, these v i a l s were placed i n larger s c i n t i l l a t i o n v i a l s and the radioactivity counted by Cerenkov radiation. F i g . 48 shows the elution pro f i l e from the column. The f i r s t small peak of radioactivity eluted from the column was due to the 5'- P-oligonucleotides, and the second large peak of radioactivity was due to unreacted ATP. The fractions containing the 5*-32p-oligonucleotides were pooled and the solution was lyophilized i n order to remove the triethyiammonium bicarbonate. Degradation of the 5*-^P-oligonucleotides to S ' - ^ p nucleotides This was done by a method similar to that used by Richardson (1966). The reaction mixture contained 80 u l of a solution containing the 5'-32p oligonucleotides dissolved i n water, 10 u l of 0.1 M Tris HCl pH 7.2, 10 u l of 0.1 M MgClg, and 5 ugm of pancreatic DBase I. The reaction was incubated at 37° C for 30 minutes; 3 u l of 1 B BB4OH were added to change the pH to 9.0; 5.3 ugm phosphodiesterase I was added, and the reaction was allowed to continue at 37° C for a further 30 minutes. .185 F r a c t i o n n u m b e r ( 0 . 5 m l . / f r a c t i o n ) Pig. 1*8 Separation of S'-^P oligonucleotides from unreacted [1T-^PJATP by chromatography of the polynucleotide kinase reaction mix-_ .f;rrvfeui».oo*. Sephadex G5P«' The polynucleotide kinase reaction mixture which contained radioactlvely phosphorylated products of the digestion of DNA. with DNase II was placed on a column of Sephadex 050, 0.9 x 50 cm. The column was eluted with 50 mM triethylammonium bicarbonate buffer. pH 7.5; each fraction was made up to k ml with water and the amount of radioactivity was measured by Cerenkov radiation. 186 Separation of the S'-^p nucleotides Twenty-five microlitres of the reaction mixture was spotted on a polyethyleneimine (PEl) cellulose plate 1 u l at a time. The plate had been previously spotted i n the same way with 5 u l of a mixture of 1 mg each of 5*aAMP, 5» dCMP, 5» TMP, 5* dGMP i n 1 ml of water. The thin layer chromatograph was run upwards for 5.0 cm i n 1 N acetic acid, transferred while s t i l l wet, and run a further 10-11 cm in the same direction i n 0.3 M lithium chloride (Randerath and Randerath, 1967)* The plate was dried i n a stream of warm a i r , and the nucleotides except for GMP, which fluoresced, appeared as dark spots under ult r a -violet irradiation at 25k nm. The Rfs f o r dGMP, TMP, dAMP, and dCMP were 0.16, 0.3k, 0.k6 amd O.69, respectively. The areas of the chroma-togram containing the nucleotides were cut out and counted by Cerenkov radiation as described i n the Methods section. Table XIV shows the radioactivity incorporated into each 5'-terndnal nucleotide and the method of calculation of the ^ p incorporation. The percent of 32p incorporation into a particular nucleotide was taken as indicative of the prevalence of the corresponding base at the 5'-teimiaals of products formed after DNase II cleavage of DNA. For each nucleotide the 32p incorporation i n the 5' terminal nucleotide was taken to be equal to the 32p incorporated into the nucleotide for the native or denatured 32 product of the DNase II reaction minus the P incorporated into the native or denatured DNA, respectively. The incorporation of radioactivity into each nucleotide was expressed as a percent of the t o t a l amount of 187 TABLE XIV CALCULATION OF THE 3 2 P INCORPORATED INTO EACH NUCLEOTIDE AT 5 , : TERMINAL AS A PERCENT OF THE TOTAL 3 2 P INCORPORATION 32 P Incorporated M a t e r i a l Analysed CPM Percent of T o t a l Inco r p o r a t i o n (1) Native c a l f . . . 25 thymus DNA GMP , , ... 154 , 33 TMP ... 82 , 18 AMP ... 94 20 CMP , ... 131 28 (2) Denatured c a l f . . , 44 thymus DNA GMP , , ... 324 38 TMP ... 132 15 AMP 146 17 CMP , , , ... 260 30 (3) Native products of ... 108 the 5 minute r e a c t i o n of GMP . ., 572 DNase I I with n a t i v e DNA TMP . . . 230 AMP .. . 356 CMP .. . 396 (4) Denatured products ... 105 of the 5 minute r e a c t i o n GMP , . . . 765 of DNase I I with n a t i v e TMP . . 258 DNA. AMP . . . 368 CMP , , , . . , 629 (5) 5' terminal nucleo- GMP ... 418 38 t i d e s produced by double TMP , , strand cleavage of UNA AMP , , ... 262 by DNase I I (3) - (1) CMP , ... 265 24 (6) 5* terminal nucleo- GMP ... 441 t i d e s produced by both TMP , , ... 126 ........ double-strand and s i n g l e AMP strand cleavage of DNA CMP ... 369 by DNase I I (4) - (2) 188 radioactivity Incorporated into a l l four nucleotides. The low amount of radioactivity observed at the origins of the thin layer ehromatograms indicated that the degradation of 5*-32P oligonucleotides 32 to 5'- P nucleotides had been essentially complete. F i g . U$ illustrates the base sp e c i f i c i t y at the 5* -terminals for cleavage of native calf thymus DNA by DBase I I . For the native digestion products the base that was present i n the highest percentage at the 5*-terminals of the cleavage sites was guanine, which ranged from 30-40$ of the t o t a l . The base which was present i n the lowest percentage was thymine, at about 11-17$. Adenine and cytosine has inter-mediate values. For the native products the order of the bases did not change during the middle and terminal phases of the reaction, but i n the terminal' phase there was a greater variation i n the percentages of the bases at the 5*-terminus than during the middle phase of the reaction. These results are i n accord with those of Soave et al.(,1973) for digestion of calf thymus DNA with hog spleen DNase I I . In the early stages of the reaction the percent of guanine was elevated and the order of cytosine and adenine was reversed. These changes may reflect the presence of a preferred sequence that i s selectively degraded and exhausted during the i n i t i a l phase of the reaction. The situation for the denatured digestion products i s more complex because the percentage obtained for each base represents the percentage of the base prrsent at the 5 1-terminals of single-strand cleavages as well as double-strand cleavages of DNA by DNase I I . In the early stages of the reaction less than 15$ of the cleavages of ca l f thymus DNA by 18§ 0 2 0 4 0 6 0 8 0 E x t e n t o f d e g r a d a t i o n ( % ) F i g . k9 Base sp e c i f i c i t y at the 5»-terminals for cleavage of native calf thymus DNA by DNase II . The percent of the 5'-terminal base was calculated as i n Table XIII. The extent of degradation was determined from Fig. k2, (a.) native products of digestion of DNA by DNase II (b) denatured products of digestion of DNA. by DNase II — • — thymine — © — cytosine ^—4— adenine —A— guanine 190 DNase II were single-strand cleavages (Table XII). These may have had a prevalence of cytosine at their 5*-terminals since the percent of cytosine was higher f o r the denatured digestion products (Fig. 49b) than for the native digestion products (Fig. b$s.). The percentage of cleavages that were single-strand cleavages probably increased as the reaction progressed because, due to denaturation of short DNA. fragments, more single-stranded substrates were l i k e l y present at later stages i n the reaction. In the later stages of the reaction higher percentages of adenine at the 5'-terminals were observed for denatured digestion products (Fig. 49b) than for native digestion products (Fig. 49a). This suggests either that DNase II cleaved small single-stranded DNA fragments with a preference for adenine at the 5*-terminals, or that the small fragments that the enzyme was degrading had already been depleted of the preferred sequences with guanine or cytosine at the 5 '-terminals. Table XV compares the base s p e c i f i c i t i e s at the 5*-terminals for DNase II cleavage of DNA from several different sources. Although the order of the bases was similar for a l l the DNAs with guanine present i n ttie highest percent and thymine, i n the lowest, a different pattern was observed for each DNA. For the eukaryotic DNAs the 5*-terminals of the native digestion products had high percentages for guanine, low percen-tages for thymine and intermediate approximately equal percentages for adenine and cytosine. For DNA from Escherichia c o l i and bacteriophage X the percentages of adenine and cytosine at the 5'-terminals of the native digestion products were not equal. The salmon DNA had a higher percent 191 TABLE XV BASE SPECIFICITY AT THE 5' TERMINALS FOR CLEAVAGE OF DNA FROM DIFFERENT SOURCES BY DNase I I DNA Source Extent of Degradation % 5' Terminal Base Native Products Denatured Products C a l f thymus 13% Salmon 13% Rat E. c o l i 9% 12% B a c t e r i o p h a g e ^ 17% Guanine 38 38 Thymine 14 11 Adenine 24 19 Cytosine 24 32 Guanine 46 39 Thymine 10 13 Adenine 22 22 Cytosine 21 26 Guanine 36 44 Thymine 13 4 Adenine 26 26 Cytosine 25 27 Guanine 37 42 Thymine 9 7 Adenine 22 19 Cytosine 33 32 Guanine 46 53 Thymine 11 10 Ademine 16 9 Cytosine 27 28 192 of guanine at the 5 *-terminals of the native digestion products than did the two mammalian DBAs. The patterns for the bases at the 5»-terminals of the native digestion products for Escherichia c o l l DNa and ADNA also differred from each other as illustrated i n Table XV. The patterns for the bases at the 5*-terminals of denatured digestion products resulted from the combined processes of double-strand and single-strand cleavage reactions and were too complex to be compared for DNA from different species, but were compared with the patterns obtained for native digestion products from DNa of the same species. The increase i n the percent of cytosine for the denatured digestion products from calf thymus and salmon DNA with respect to the native digestion products suggested that for these DNAs DNase II preferred cytosine at the 5*-terminals of single-strand cleavages early i n the reaction. For rat, E. c o l i and WDNA, the percent of guanine was elevated i n the denatured digestion products with respect to the native digestion products, suggesting that guanine was preferred at the 5 1 -terminals of early single-strand cleavages. The base sp e c i f i c i t y at the 3*-terminals The terminal addition of riboadenylic acid to deoxyoligonucleotides by terminal deoxynucleotidyl transferase has been used to specifically label deoxyoligonucleotides at the 3* ends (Kossel and Roychoudhury, 1971; Kossel et a l . , 1974). Terminal transferase catalysed radioactive labelling of the 3'-terminals of products of the reaction of DNA. with spleen DNase II has allowed the analysis of the 3*-terminal nucleotides for cleavage of DNA by spleen DNase II (Bertazzoni et a l . , 1973; i b i d , 193 197*0 • Since both mono- and diaddition of riboadenylic acid to the deoxyoligonocleotides occur, i t i s necessary to remove the second riboadenylic acid either by treatment with base and phosphatase (Kossel et al.> 197*0 > or by separation of the ribonucleotide from the deoxyribonucleotides after degradation to nucleotides (Bertazzoni et a l . , 197*0* Terminal deoxynucleotidyl transferase catalyses the polymerization of deoxynucleotide triphosphates, elongating polydeoxynucleotide chains (Bollum, 1974). Perhaps because a ribonucleotide triphosphate and not the preferred substrate was used, large concentration of enzyme and substrate were required for appreciable labelling of 3'-terminals (Bertazzoni, 1973). Products of the reaction of DM with DNase II were labelled with ^ P using terminal transferase and|°<- 32pj^ TP by the method of Kossel et a l . , (197*0 as outlined i n F i g . 50. DNA. was reacted with DNase II to yield 3'-phosphate oligonucleotides. The 3* phosphate group was removed by incubation with acid phosphatase. ^ P labelled 5*-AMP was added to the resulting 3'-hydroxyl group on the oligonucleotide by using terminal transferase and [W- 32pJATP. Since both mono- and diaddition reactions occurred, the second adenosine was hydrolysed with base and the second 32p labelled phosphate was removed with alkaline phosphatase. 32 The radioactivity labelled oligonucleotide was then separated from P labelled phosphate by chromatography on Sephadex 050 and was digested with spleen DNase II and phosphodiesterase II t o yi e l d 3'-^-? labelled nucleotides from the 3'-terminals of the sites of cleavage of DNA by DNaseH. The labelled nucleotides were then separated by thin layer chromatography on PEI cellulose plates and the percent of radioactivity incorporated 19k DNA HO HO 5' HO 1 j I BNase II 3' K acid phosphatase terminal transferase + P^JATP 3' 5' 3' ..... fpA * HOxJ^ ^pApA + PPi 0OH HO HO • ~> s , J . . . . . . ^ + H04 h alkaline phosphatase 3» J . . . . . . h p A + 32P + Sephadex G50 chromatography HO HO 5' . P + ( H O ^ P ) n 1 pap + A (l) spleen DNase II (5) spleen phosphodiesterase u HO >J>p + A F i g . 50 Scheme for determination of the base sp e c i f i c i t y at the 3'-terminals of products of cleavage of DNA. by DNase I I . Dephosphorylated digestion products were labelled at the 3'-terminals with 32p using terminal transferase and, after removal of excess label, were enzymatically degraded to 3*-32P nucleo-tides. The 3»-32p nucleotides were separated by t h i n layer chromatography and the amount of radioactivity i n each nucleotide was determined from the Cerenhov radiation emitted. 195 into each nucleotide was determined by measuring the Cerenkov radiation emitted. The DNase II reaction As for the determination of the base sp e c i f i c i t y at the 5»-terminal. DNase II was incubated at 37° C with hO ugm DNA i n 1.0 ml of 100 mM sodium acetate buffer containing 10 mM EDTA and the reaction was followed by the hyperchromicity assay. The reaction was stopped and the enzyme and DNA denatured by placing the reaction mixtures, i n rubber stoppered test tubes, into a boiling water bath for 15 minutes and then into an ice-water bath. The acid phosphatase reaction Potato acid phosphatase, 5 ugm, was added to the reaction mixture containing the denatured products of the DNase II reaction with DNA. After a 30 minute incubation at 37° C, the solution was heated to 100° C for 5 minutes i n order to inactivate the enzyme The terminal transferase reaction. The method of Kossel et a l . (1974) was followed with some modifi-cations. To 1.0 ml of the acid phosphatase treated reaction mixture 196 were added 50 u l of 1 M Tris HC1*, pH 7*5, 10 u l of 1 M ZnG^**, 8 u l of 1 M MgClg, and 42 u l of 1 H NaOH to give a f i n a l pH of 7.5. To 100 u l of this solution were added 1 u l of 0.1 M dithiothreitol, 149 pmoles of [o(_ 32PJAJP a t 1 # y 2 x 10^ cpm/pmole and 24 units of terminal deoxyribo-nucleotidyl transferase. The reaction was followed by removing 5 u l aliquots from the reaction mixture before the addition of the terminal transferase and at various times during the reaction and spotting the aliquots on DE8l paper which had previously been prespotted with 50 u l of a solution of 1 mM ATP, 50 mM EDTA. Descending paper chromatography was carried out for two hours with 0.35•'ammonium formate buffer, pH 5.5. The DE8l paper was dried, the origins were cut out, and the radioactivity that was retained at the origin was determined by measuring the Cerenkov radiation emitted. Fig. 51 depicts the terminal deoxynucleotidyl transferase catalysed incorporation of P into products of the five minute reaction of DNase II with DNA from various sources and into the indigested DNA molecules. A substantial amount of 32p v?as incorporated into Escherichia c o l i , bacterio-phage A , and rat intestinal DNAs and the respective reaction products • L i t t l e or no radioactive incorporation was observed with the buffer commonly used for terminal transferase reactions: 40 or 200 mM potassium cacodylate pH 6.8 or 7.2 (Bollum, 1974). Tris buffer was used instead of cacodylate buffer because more incorporation of radioactivity into 3*-hydroxyl oligonucleotides was observed using the former buffer. **The ZnClg was added i n order to complex the EDTA present i n the solution from the DNase II reaction and prevent the inhibition of the enzyme which occurred i n the presence of small quantities of EDTA and was probably due to chelation of the Zn from the active site of the enzyme (Chang and Bollum, 1970). F i g . 51 Terminal deaxynucleotidyl transferase catalysed incorporation of 32p into products of the reaction of BNase II with DNA. from various sources. Incorporation of 32p into products of a five minute DNase II reaction with DNA i s indicated by open symbols, and the incorporation of 32p into undigested DNA, by closed symbols. The sources of the DNA were: , O c a l f thymus, A salmon testes, 0 • bacteriophage A , rat intestine, 0 Escherichia c o l i 198 liberated after digestion with DNase I I , but l i t t l e 32p ^ incorporated into DNA from salmon testes and calf thymus and the respective DNase II reaction products. For most of the samples a biphasic curve was observed as was expected for a diaddltion reaction. However, a large amount of 32p was incorporated into DNA from £. c o l i , rat intestine, bacteriophage A and c a l f thymus. The amount of incorporation of 32p into c a l f thymus DNA was greater than into the respective DNase II reaction products. The 32p compounds may have absorbed non-specifically to DNA which was i t s e l f absorbed at the origin of the DE8l cellulose paper. Separation of the 3'-32p oligonucleotide products of the terminal transferase reaction from ^P-labelled inorganic phosphate The terminal transferase reaction was stopped by the addition of 3 u l of 10 N KOH. Upon incubation at 37° C overnight, the base bydrolysed the terminal ribonucleotide residue resulting from two additions of 32P-AMP (Bock, 1967). The next day UO u l of water, 10 u l of 1.0 M Tris HCl, pH 8.0, and 2 u l of 60% perchloric acid were added at 0° C to the 60 u l of the terminal transferase reaction mixture. The mixture was cooled at 0° C for 10 minutes, and then centrifuged at 12,100 xg for 10 minutes at k° C. Alkaline phosphatase, 5 ugm, was incubated with the supernatant solution at pH 8 and 37° C for 15 minutes; 5 u l of 10 N KOH were then added and the solution was incubated for a further 15 minutes at 37° C in order to inactivate the alkaline phosphatase (Ho and Gilham, 1973; Delaney and Spencer, 1976). The solution was neutralized with k u l of 199 concentrated hydrochloric acid, and was applied to a Sephadex G50 column, 0,9 x 50 cm. The column was eluted attk ° C with 50 mM t r i e t h y l -ammonium bicarbonate, pH 7.5 and 0.5 ml fractions were collected i n k ml glass v i a l s . The radioactivity present i n each fraction was determined by adding 3.5 ml of water to the small v i a l s , placing the small v i a l s i n larger vials,and measuring the Cerenkov radiation emitted. The elution p r o f i l e of radioactivity from the Sephadex G50 column i s i l l u s -trated i n F i g . 52. 32p labelled inorganic phosphate was eluted starting at fraction number 30; the elution profile i s not shown i n Fig . 52, Although judging from Fig, 51, about 7,000 counts per minute of radioactivity were incorporated into products of the digestion of DNA from Escherichia c o l i with DNase I I , very l i t t l e radioactivity was eluted from the Sephadex column i n the position expected for oligo-nucleotides, fractions number 19-25. These fractions were pooled, nevertheless, and the solution was lyophilized to remove the t r i e t h y l -ammonium bicarbonate. Similar elution profiles were obtained for the other DNAs and digestion products. Degradation of the 3>-32p-oligonucleotides to 3'-^l? nucleotides A method similar to that of Bertazzoni et a l . (1973) was used. To the y-^¥ oligonucleotides i n 35 u l of water were added 5 u l of 1 M ammonium acetate, pH 5*6, and k2 ugm of hog spleen DNase II. After one hour of Incubation at 37° C, 10 ugm of spleen phosphodiesterase II was added and the solution was incubated for a further hour at 37° C. Separation of 3'-32p oligonucleotides from 32p_labelled inorganic phosphate by chromatography of the base and alkaline phosphatase treated terminal transferase reaction mixture i n Sephadex G50. Products of a f i v e minute reaction of DNase II with DNa from Escherichia c o l i were radioactively phosphorylated with terminal transferase and - 32p]ATP, and placed on a Sephadex G50 column, 0.9 x 50 cm. The column was eluted with 50 mM triethyiammonium bicarbonate, pH 7.5. Radiactivity i n each fraction was determined by measuring the Cerenkov radiation emitted. A large peak of 32p_labelled inorganic phosphate, which eluted after fraction number 30, i s not shown i n Fig . 52. 1,000 E F r a c t i o n n u m b e r (0.5 m l / f r a c t i o n ) 202 Separation of the 3*-^P nucleotides A PEI cellulose thin layer chromatography plate was spotted andk run i n the same manner as for the separation of the 5'-32p nucleotides except that 3'-P deoxyribonucleotides were used as markers, and inter-fering salts were removed, after spotting but before chromatography, by immersing the TLC sheet f l a t i n 500 ml methanol for 10 minutes, and then drying i t (Randerath and Randerath, 1964). The Rfs of 3'd GMP, 3» TMP, 3'd AMP and 3'd CMP were 0.19, 0.30, 0.U2, O.58, respectively. The areas containing the nucleotides were cut out and the radioactivity incorporated into each nucleotide was measured by the Cerenkov radiation emitted. The amount of radioactivity incorporated into each nucleotide was calculated as a percent of the t o t a l incorporation. Table XVI shows the percent of radioactivity incorporated into each nucleotide for the 3'-terminals of products of the reaction of DNase II with different DNAs. The highest percent incorporation was found for guanine, and the lowest, for cytosine. Thymine and adenine were also labelled at f a i r l y low levels. DNase II had a strong preference for guanine at the 3*-terminals of products of DNase II catalysed hydrolysis of DNA from several different sources. The patterns of incor-poration observed for the different DNAs differed primarily i n the percent incorporation for guanine. This may indicate different frequencies i n the various DNAs of the preferred sequences containing guanine at the 3» -terminals of products released from calf thymus DNA by spleen DNase II (Thiery et a l . , 1973). 203 TABLE XVI BASE SPECIFICITY AT THE 3' TERMINALS FOR CLEAVAGE OF DNA FROM DIFFERENT SOURCES BY DNase I I % 3' Terminal Base Extent of DNA Source Degradation Denatured Products C a l f thymus 13% Guanine 49 Thymine 22 Adenine 23 Cytosine 6 Salmon t e s t e s 13% Guanine 58 Thymine 17 Adenine 15 Cytosine 11 Rat i n t e s t i n e 9% Guanine 72 Thymine 10 Ademine 14 Cytosine 6 E s c h e r i c h i a c o l i 12% Guanine 69 Thymine 11 Adenine 14 Cytosine 6 Bacteriophage 17% Guanine 79 Thymine 7 Adenine 6 Cytosine 7 204 Discussion The Hichaelis constant, Km, far the hydrolysis by DNase II of 3H-DN& from Escherichia c o l i was 2.42 x 10"? M DNA-phosphate. This pro-vides a basis for future experiments to test for the presence of a possible DNase II-specific inhibitor i n crude and purified preparations of DNase II . A decreased DNase II act i v i t y i n a crude preparation at a saturating concentration of DNA would indicate the presence of an inhibitor. It i s unlikely that the highly purified DNase II preparation contains a DNase II-specific inhibitor because the sigmoidal kinetics characteristic of interaction of the inhibitor with DNase II (Lesca, 1976) were not observed. Also, although enzymes from different tissues are being compared, Lesca (1976) found that the beef l i v e r DNase II-specific inhibitor was evident at a DNA concentration of 8.9 x 10~? M DNA-phosphate. That DNase II degrades double-stranded substrates such as native DNA and polydAT hairpin loops at a greater rate than single-stranded substrates such as native DNA, polydA and polydT i s i n accord with results reported for spleen DNase II (Bernardi, 1968). DNase II degraded native DNA primarily by a double-strand cleavage mechanism i n the presence and absence of 10 mM EDTA. Addition of 1 mM sodium sulfate to a DNase II assay solution containing 10 mM EDTA had l i t t l e or no effect on the mode of cleavage of DNa. These results are i n agreement with those of Bernardi and Sadron (1964), Young and Sinsheimer (1965), Bernardi and Bach (1968) and Kopecka et a l . (1973) who found that DNase II degraded DNA primarily by a double-strand 205 cleavage mechanism. When products of the reaction of DNase II with DNA were subjected to electrophoresis on polyacrylamlde gels and visualized by fluorescence after i n t e r r e l a t i o n with ethidium bromide, continuous fluoresecent patterns resulting from oligonucleotides ranging from less than 100 to more than 1,300 base pairs were observed. This indicated that DNase II cleaved a large number of different base sequences i n DNA to yield a wide distribution of fragment sizes. The base sp e c i f i c i t y values together with the electrophoresis results indicated that intestinal DNase II cleaved a large number of different base sequences i n DNA because the percentage values for the bases did not indicate a preference for one or two bases to the exclusion of the others. The most susceptible lnternucleotide linkage for intestinal DNase II was GpCr and the most resistant was CpT. These results are i n accord with those of Vanecko and Laskowski (1962) and Bernardi et a l . (1973) for the cleavage of cal f thymus DNA by spleen DNase I I . In addition the most susceptible linkages for single-strand cleavages were probably GpC and GpG i n the i n i t i a l phase: of the reaction and perhaps GpA with small single-stranded fragments i n the later stages of the reaction. The finding that the base spe c i f i c i t y at the 5'-terminal changed during the course of the reaction, especially i n the i n i t i a l and terminal phases of the reaction, i s i n accord with the results of Vanecko and Laskowski (1962) for the reaction of spleen DNase II with calf thymus DNA. Intestinal DNase II was not much more specific early i n the reaction with DNA than i t was at later stages of the reaction. Different patterns for percentages of terminal bases were observed i n cleavage products of DNA from various species 206 l i k e l y because of the different frequencies with which sequences susceptible to cleavage by DNase II occurred i n the DNAS. These results are i n accord with those of Bernardi et a l . (1973) who found similar patterns for the base sp e c i f i c i t i e s of cleavage of different mammalian DNas by DNase I I , but different patterns when the base sp e c i f i c i t i e s for DNase II cleavage of mammalian, yeast nuclear, and bacterial DNa were compared. 207 SUMMARY 1. DBase II activity was isolated from bovine small intestine by homogenization of the mucosa i n an equal volume of Krebs Ringer /phosphate buffer, pH 7.8, and centrifugation of the mixture f i r s t at l6,300xg, and then at 105,000xg. Buffer solutions containing DBase II were made 10 mM i n ethylenediaminetetraacetate (EDTA) i n order to sta-b i l i z e the enzyme. Since addition of diisopropylflurophosphate, a protease inhibitor, also prevented a loss of DBase II a c t i v i t y , i t i s l i k e l y that the decline i n DBase II ac t i v i t y observed i n the absence of both reagents was due to proteolysis of the enzyme. When the 105,000xg supernatant solution was chromatographed on DEAE cellulose, two peaks of DBase n a c t i v i t y were eluted, a major peak with 20 mM phosphate buffer, pH 7.8, and a minor one with a 0-1M potassium chloride gradient i n the same buffer. Similar results indicating the presence of two DBase II a c t i v i t i e s i n several other tissues have been reported by previous investigators (Cordonnier and Bernardi, 1968; Yamanaka et a l . , 197k; Zollner et a l . , 197*0. The two DBase II a c t i -v i t i e s seemed to have different properties. The major DBase II a c t i -v i t y degraded native DBA more rapidly than denatured DBA, whereas the minor one digested both at the same rate. The activation energies for hydrolysis of native DBA by the two DBase l i s were different, a l -though the activation energies for denaturation oof the two enzyme mo-lecules were similar. The two a c t i v i t i e s also differed i n their res-ponse to increasing ionic strength, pH and sulfate ion concentration. Upon rechromatography on DEAE cellulose of the minor DBase II under 208 conditions i n which the BNase II activity remained stable, most of the enzyme was eluted with 20mM phosphate buffer, pH 7*8, containing lOmM EDTA i n the same position as that i n which the major DNase II activity had been eluted. The major DNase II activity could be bound i n low ionic strength buffer to DNA that was physically absorbed to a cellulose column, and could be eluted from the column along with fragments of DNA by a potassium chloride gradient i n the same buffer. The minor DNase II preparation contained a substantial quantity of endogenous DNA and upon chromatography i n Sephadex G100 s p l i t into several peaks of activity that were associated with species of mole-cular weight greater than or equal to that of the major DNase I I . Thus i t was concluded that the appearance of a minor DNase II a c t i v i -t y was an artifact of the DEAE cellulose chromatography, l i k e l y due to the binding of a small amount of DNase II to DNA which was electro-s t a t i c a l l y abound to the positively charged DEAE groups of the column. Apparent differences i n the properties of the two DNase II a c t i v i t i e s were probably due to the presence i n the minor DNase II preparation of a substantial amount of endogenous DNA which interfered i n the DNase II reactions. 2. Intestinal DNase II was pa r t i a l l y purified by lon exchange chro-matography and gel f i l t r a t i o n . A 105,000xg supernatant prepared from bo-vine intestinal mucosa was applied to DEAE cellulose and the DNase II activity was eluted with 20mM phosphate buffer, pH 7.8, containing lOmM EDTA. The pooled DNase II ac t i v i t y from the DEAE cellulose co-lumn was bound to CM cellulose i n lOmM phosphate buffer, pH 6.0, containing lOmM EDTA and later eluted with a 0-1M potassium chloride 209 gradient i n the same buffer. The enzyme was then subjected to gel f i l t r a t i o n on Sephadex 0100 i n 0.25M TrlsHCl, pH 7.5, containing lOmM EDTA. Electrophoresis of the DNase II preparation from the Sepha-dex column on polyacrylamide gels revealed that only a few other pro-te i n bands were present. The only nuclease contaminants present i n significant amounts were acid and alkaline ENase, which do not degrade DNA, and phosphodiesterase I I , an exonuclease. The amounts of these enzymes relative to DNase II were reduced substantially by a repeti-t i o n of the purification procedure using the p a r t i a l l y purified DNase II preparation from the Sephadex column as the starting material. The enzyme was purified 78 fold with a yield of 13$ compared to the l6,300xg supernatant. 3. The enzyme isolated from intestinal mucosa was a DNase II according to the c r i t e r i a of Maver and Greco (1948) and the interna-t i o n a l recommendations on enzyme nomenclature (1972) because i t hy-drolysed DNA to oligonucleotides with 3'-phosphate and 5'-hydroxyl terminals endonucleolyt i c a l l y at acid pH and showed no requirement for a divalent metal lon. DNase II had a broad pH optimum centred at pH 4.8, and was inhibited by magnesium ions, high ionic strength and sulfate ions. The activation energy for hydrolysis of native calf thymus DNa was 19 Kcal/mole. The activation energy for dena-turation of the DNase II molecule was 43 Kcal/mole. The molecular weight of DNase II was estimated to be 4l,000 by gel f i l t r a t i o n on Sephadex G100. 4. The reaction of DNase II with DNA and other subtrates was studied. 210 The Michaelis constants for the reactions of DNA with sonicated %-DNA from Escherichia c o l i and polydAT-methyl-^H were 2.1+2 x 10"? M DNA-phosphate and 2.63 x 10~?M polydAT-phosphate, respectively. DNase II degraded native calf thymus DBA, 6.1+ times faster than denatured calf thymus DNA. The rate of reaction of DNase II with polydAT was at least 10 times that with polydA or polydT, probably because of the presence of double-stranded "hairpin loops" i n the polydAT (Seheffer et a l . , 1968). DNase II can cleave one or both strands of native DBA. Double-strand cleavage released "external" 51 -hydroxyl groups; single-strand cleavage resulted i n "nicks" i n which access to the "internal" 5'-hy-droxyl groups formed was restricted by the surrounding DBA. Upon de-naturation of the reaction products, both external and internal 5»-hydroxyls became equally accessible. Under specific conditions polynucleotide kinase catalyses the incorporation of 32P from [r-3P] ATP into external 5*-hydroxyl groups only. DBase II was reacted 32 with native DNA. P incorporation into the native DNase II diges-t i o n products was into the external 5*-hydroxyls produced by the double-strand cleavage mechanism, whereas 32P incorporation into the denatured DNase II digestion products was into the external plus the internal 5*-hydroxyl groups resulting from double-strand plus single-strand cleavages. Intestinal DNase II degraded native DBA from various sources, at different stages i n the reaction and under different reac-tio n conditions, primarily by a double-strand cleavage mechanism. 5. Products of the reaction of DBase II with DBA were subjec-ted to electrophoresis on polyacrylamlde gels and visualized by 211 fluorescence after i n t e r r e l a t i o n with ethidium bromide. Early i n the reaction continuous fluorescent patterns resulting from oligo-nucleotides ranging from less than 100 to more than 1,300 base pairs i n length were observed. This indicated that DNase II had cleaved a large number of different base sequences i n the DNa to yie l d a wide distribution of fragment sizes. 6. The base sp e c i f i c i t y of cleavages of DNA by DNase II was investigated using radioactive labelling techniques. The 5*-hydroxyl terminals of DNase II digestion products were labelled with 32p using polynucleotide kinase and [T32pJ ATP. The 5«„32p oligonucleotides were degraded to 5'-32p nucleotides. The a-mount of 32p incorporated into each nucleotide as a percentage of the t o t a l 32p incorporation indicated the percent of the corresponding base at the 5'-terminals. Guanine was present i n the highest percent-age; thymine,in the lowest. Adeline and cytosine had intermediate values. The percentage for each base changed during the reaction, especially i n the i n i t i a l and terminal phases. Although the order of the bases was similar with guanine present i n the highest percent, and thymine i n the lowest, different patterns for percentages of terminal bases were observed for the cleavage by DNase I I of DNA from various species. This indicated that sequences susceptible to cleavage by DNase II were present i n different frequencies i n the various DNas. The 3'-phosphate terminals of DNase II digestion products were dephosphorylated and then labelled with 3 2 p . r i D 0 8 4 e n y i i c acid using terminal deoxynucleotide transferase and ATP. The S^^^P oligonucleotides were degraded to 3*-^P nucleotides. The amount 212 of 32p incorporated into each nucleotide as a percent of the t o t a l 32p incorporated indicated the percent of the corresponding base at the 3'-terminals. Guanine was present at the 3 '-terminals i n the highest percentage; cytosine,in the lowest. Thymine and adenine were also present at low levels. DNase II had a strong preference for guanine at the 3 '-terminals of products of the DNase II catalysed hydrolysis of DNA from several sources. The percent of guanine at the 3'-terminals varied with DNA from different species. This i n d i -cated that the preferred sequences containing guanine at their 3'-ter-minals occurred with different frequencies i n the various DNAs. 213 BIBLIOGRAPHY Albert8, B., and Herrick, G. (1971) Methods Enzymol. 21, 193-217 Aldridge, W. N. (1953) Biochem. J . £3, 62-67 A l l f r e y , V., and Mir sky, A. E. (1952) J . Gen. Physiol. 36, 227-21+1 Altenburger, W., Horz, W., & Zachau, H. G. (1976) Nature 261+, 517-522 Ames, B. H. (1966) Methods Enzymol. 8, 115-119 Beaven, G. H., Holliday, E. R., and Johnson, E. A. (1955) i n The  Mucleic Acids (Chargaff, E., and Davidson, J . H., eds) v o l . 1, pp. 493-553, Academic Press, Hew York. Beckner, K. L., and Polk, W.R. (1977) J . B i o l . Chem. 252, 3176-3184 Bernardi, G. (1965) J . Mol. B i o l . 13, 603-605 Bernardi, G. (1966) i n Procedures i n Nucleic Acid Research (Cantoni, G. L., and Davies, D. R., eds) v o l . 1, pp. 102-121, Harper and Row, New York. Bernardi, G. (1968) Adv. Enzymol. 31, 1-49 Bernardi, G. (1971a) l n The Enzymes (Boyer, P. D., ed.) 3rd Ed., vol. IV, pp. 271-287, Academic Press, New York. Bernardi, G. (1971bl Methods Enzymol. 22, 325-329 Bernardi, G., Appella, E., and Zito, R. (1965) Biochemistry 4, 1725-1729 Bernardi, G., and Bachp**IL.4(l968) J . Mol. B i o l . 3 1 , 87-98 Bernardi, G., and Bernardi, A. (1966) i n Procedures i n Nucleic Acid  Research (Cantoni, G. L. and Davies, D. R. eds) v o l . I, pp. 144-153, Harper and Row, New York and London. Bernardi, G., Bernardi, A., & Chersi, A. (1966) Biochim., Biophys. Acta 129, 1-H. Bernardi, G., Ehrlich, S. D., and Thiery, J . P. (1973) Nature New  Biology 246, 36-40 214 Bernard!, 0., and Griffe, M. (1964) Biochemistry 3, 1419-1426 Bernardi, G., and Sadron, C. (1964) Biochemistry 3> l4ll - l4l8 Bertazzonl, U., Ehrlich, S. D., and Bernardi, G. (1973) Biochim.  Biophys. Acta 312, 192-201 Bertazzoni, U., Ehrlich, S. D., and Bernardi, G. (1974) Methods  Enzymol. 2g, 355-359 Bhattacharya, P., Moskal, J . R., & Basu, S. (1977) Proc. Hat. Acad.  Sc i . U.S.A. j 4 , 842-845 Bock, R. M. (1967) Methods Enzymol. 12A, 224-228 Bollum, F. J . , (1974) i n The Enzymes (Boyer, P. D», ed.) 3rd Ed., v o l . 10, pp.145- 171* Academic Press, Hew York. Brewer, J . M., Price, A. J . , & Ashworth, R. B. (1974) Experimental Techniques i n Biochemistry, Prentice-Hall, Inc., Hew York. Burton, K. (1968) Methods Enzymol. 12B, 163-166 Cassani, E. R., and Bollum, F. J . (I969) Biochemistry 8, 3928-3936 Catcheside, D. G., and Holmes, B. (1947) Symposia Soc. Exptl. B i o l . Chaconas, G., van de Sande, J . H., & Church, R. B. (1975) Anal.  Biochem. 6jj», 312-316 Chang, L. M. S., and Bollum, F. J . (1970) Proc. Hat. Acad. Sc i . U.S.A. 65_, 1041-1048 Char gaff, E. (1955) i n The Hucleic Acids (Chargaff, E. and Davidson, J . H., eds) v o l . 1, pp. 307-371* Academic Press, Hew York. Chiu, J.-F., and Sung, S. C. (1972) Biochim. Biophys. Acta 209» 34-42 Cleaver, J . E., and Boyer, H. W. (1972) Biochim. Biophys. Acta 262 116-124 Colter, J . S., Brown, R. A., and E l l e n , K. A. 0. (1962) Biochim.  Biophys. Acta 55, 31-39 Cordonnier, C , and Bernardi, G. (1968) Can. J . Biochem. 46, 989-995 Cunningham, L., and Lashowski, M. (1953) Biochim. Biophys. Acta 11, 590-591 Davidson, J . H. (1972) The Biochemistry of Hucleic Acids, 7th edn. 215 Academic Press, New York. de Duve, C., Wattiaux, R., and Baudhuin, P. (1962) Adv. Enzymol, 2k, 291-358 Delaney, A,D., and Spencer, J , H, (1976) Biochim. Biophys. Acta 435, 269-281 Dingle, J . T., and F e l l , H. B., eds. Lysozomes i n Biology and Pathology  vol.11 & 2 (1969) v o l . 3 (1973) Dulaney, J . T., and Toaster, 0. (1972) J . B i o l . Chem. 24£, 1424-1432 Enzyme Nomenclature, Recommendations (1972) of the International Union of Pure and Applied Chemistry and the International Union of Biochemistry, Elsevier Scient i f i c Pub. C , Amsterdam, 1973 Eschenbach, C. (1971) C l i n . Wocheschr. Jg, 91*9-958 Fahrney, D. E., and Gold, A. M. (1973) J . Amer. Chem. Soc. 8j>, 997.1000 Felsenfeld, G. (1968) Methods Enzymol. 12B, 247-252 Fischer, L. (1969) i n Laboratory Techniques i n Biochemistry and Molecu- l a r Biology, v o l . 1 (Ed. Work, T. S. and Work, E.) pp. 151-396, John Wiley & Sons, Inc., New York Flanagan, P. R., and Zbarsky, S. H. (1977) Biochim. Biophys. Acta 480, 204-218 Gabriel, 0. (1971a) Methods Enzymol. 22, 565-578 Gabriel, ©. (1971b) Methods Enzymol. 22, 578-604 Glynn, I. M., and Chappel, J . B. (1964) Biochem. J . 9_0, l47-ll»9 Ho, N. W. Y., and Gilham, P. T. (1973) Biochim. Biophys. Acta 308, 53-58 Hodes, M. E., Yip, L. C , and Santos, S. R. (19&7) Enzymologia 32, 241-255 Holtzman, E. (1976) Lysozomes: A Survey, Springer-Verlog, New York Hynie, I., and Zbarfeky, S. H. (1970) Can. J . Biochem. 48, 1141-1150 Jarvis, A. W., and Lawrence, R. C. (1969) Can. J . Biochem. kj_, 673-675 Koener, J . F., and Sinsheimer (1957) J . B i o l . Chem. 228, 1039-10^ 7 Kopecka, H., Chevallier, M. R., Prune11, Bernardi, G. (1973) Biochim. 216 Biophys. Acta 319» 37-47 Kossel, H., Royehoudhury, R., Fischer, D., and Otto, A. (1974) Methods  Enzymol. 29_, 322-341 Kunitz, M. (1947) J . Gen. Physiol. 3J>> 291-310 Kunitz, M. (1950) J . Gen. Physiol. 33> 349-362 Laskowski, M. (1961) The Enzymes, 2nd edn. v o l . 5, pp. 123-147 (Boyer, P. D., Lardy, H., & Myrback, K., eds.) Academic Press, New York. Laskowski, M. (1955) Methods Enzymol. 2, 26-36 Lee, G. Y., and Zbarsky, S. H. C1967) Can. J . Biochem. 45 , 39-51 Lee, C. Y., Lawrence, S., and Zbarsky, S. H. (1972) Can. J . Biochem. 50 697-703 Lehman, I. R. (1967) Ann. Rev. Biochem. 3,6, 645-668 LePecq, J.-B., & Paoletti, C. (1967) J . Mol. B i o l . 2£, 87-106 Lesca, P. (1971) Rev. Europ. Etudes C l i n , et B i o l . 16, 117-123 Lesca, P. (1968) Nature 220, 76-77 Lesca, P. (1976) J . B i o l . Chem. 251, 116-123 Lesca, P. and Paoletti, C. (1969) Proc. Nat. Acad. S c i . U.S.A. 64, 913-919 Lieberman, M. W., Sullivan, R. J . , Shull, K. H., Liang, H., and Farber, E. (1971) Can. J . Biochem. 4g, 38-43 Lillehaug, J . R. and Kleppe, K. (1957a) Biochemistry 14, 1221-1225 Lillehaug, J . R. and Kleppe, K. (1957b) Biochemistry 14, 1225-1229 Lillehaug, J . R., Kleppe, R. K., and Kleppe, K. (1976) Biochemistry 15, I858-I865 Loening, U. E. (1967) Biochem. J . 102, 251-257 Lowry, 0. H., Rosenbrough, N, J . , Farr, A. L., and Randall, R. J . (1951) J . B i o l . Chem. 193» 265-275 MacHattie, L. A., Bernardi, G., & Thomas, C. A., J r . (1963) Science l40 59-60 217 Mahler, H, R., and Cordes, £. H. (1971) Biological Chemistry, 2nd ed., Harper & Row, New York and London Mandel, M., & Marmur, J . (1968) Methods Enzymol. 12B, 195-206 Maniatis, T., Jeffrey, A., and van de Sande, H. (1975) Biochem. 14, 3787-3791* Marmur, J . , Round, R«, and Schildkraut, C. L. (19^ 3) i n Prog, i n Nu- c l e i c Acid Res, v o l . 1, pp. 231-360(Davidson, J . N. & Conn, W. E., eds.) Academic Press, New York and London Martin, B. R., & Voorheis, H. P. (1977) Biochem. J . l6l, 555-559 Maver, M. E., and Greco, A. E. (191*8) Federation Proc. 7, 171 Mezel, C. (1964) Ph.D. Thesis, University of Bri t i s h Columbia. Nathans, D., & Smith, H, 0. (1975) Ann. Rev. Biochem. 44, 273-293 N o l l , M. (1974a) Nature 251, 249-251 N o l l , M. (1974b) Nucl. Acids Res. 1, 1573-1578 Okazaki, R., Hirose, S., Okazaki, T., Ogawa, T., and Karosawa, Y. (1975) Biochem. Biophys. Res. Comm. 64, 1018-1024 Oshima, R. G., and Price, P. A. (1973) J . B i o l . Chem. 248, 7522-7526 Oshima, R. G., and Price, P. A. (1974) J . B i o l . Chem. 249, 4435-4438 Oth, A., Fredericq, and Hacha, R. (1958) Biochim. Biophys. Acta 2£, 287-296 Otsuka, A. S., and Price, P. A. (1974) Annal. Biochem. 62, 18O-I87 Parker, R. P., and E l r i c k , R. H. (1970) i n The Current Status of Liquid S c i n t i l l a t i o n Counting (Bradstone, E. D., J r . , ed.), Grune and Stratton, New York Porath, J . (1955) Nature 175, 148 Price, P. A., Li u , T-Y., Stein, W. H., and Moore, S. (I969) J . B i o l . Chem. 244, 917-923 Prival de Garilhe, M. (I967) Enzymes i n Nucleic Acid Research, Hermann, Paris Randerath, K. and Randerath, E. (1964) J . Chromatog. 16, 111-125 Randerath, K. and Randerath, E. (1967) Methods Enzymol. 12A, 323-347 218 Rao, S. K. (1973) Life S c i . 12, 89-96 Reiland, J . (1971) Methods Enzymol. 22, 287-321 Rej, R. and Richards, A. R. (1974) Annal. Biochem. 62, 240-247 Richardson, G. C. (1966) J . Mol. B i o l . 15., 49-61 Richardson, CQ C. (1971) i n Procedures i n Hucleic Acid Research vol. 2, pp. 815-828 (Canton!, G. L. and Davies, D. R., eds.) Harper and Row, Hew York Riesner, D. and Romer, R. (1973) i n Physico-chemical Propertlessof  Hucleic Acids (Duschesne, J . , ed.) v o l . 2, pp. 237-318, Academic Press, Hew York and London Rosenbluth, R. and Sung, S-C. (1969) Can. J . Biochem. 47, IO8I-IO88 Sanger, F., A i r , G. M., Barren, B. G., Brown, H. L., Coulson, A. R., Fiddes, J . C , Hutchison, C. A. I l l , Slocombe, P. M., and Smith, M. (1977) Nature,265, 687-695 Scheffler, I. E., Els on, E. L., Baldwin, R. L. (1968) J . Mol. B i o l . 36, 291-304 Schendel, P. F. and Wells, R. D. (1973) J . B i o l . Chem. 248, 8319-8321 Segel, I. H. (1975) Enzyme Kinetics, John Wiley & Sons, Toronto Sgaramella, V. and Khorana, H. G. (1972) J . Mol. B i o l . 72, 427-444 Shrivastaw, K. P. and Roa, S. K. (1975) J . Neurochem. 2£, 861-865 Sicard, P. J . , Obrenovitch, A., and Aubel-Sadron, G. (1970) FEBS l e t t . 22, 41-44 Sicard, P. J . , Obrenovitch, A., and Aubel-Sadron, G. (1972) Biochim.  Biophys. Acta $68, 468-479 Slor, H. (1970a) Harefuah 38, (12) 594-596 Slor, H. (1970b) Biochem. Biophys. Res. Comm. 38, 1084-1090 Slor, H. and Lev, T. (1971) Biochem. J . 132, 993-995 Slor, H. (1973) Biochem. J . 136, 83-87 Slor, H. (1974) Enzyme 17, 196-204 Smith, H. 0., Kelly, T. J . , and Roy, P. H. (1974) Methods Enzymol. 2g, 282-294 219 Soave, C , Thiery, J-P., Erhlich, S. D., and Bernardi, G. (1973) Eur. J . Biochem. 38, 423-433 Stevens, C. E., Daost, R., and Leblond, C. P. (1953) J» B i o l . Chem. 202, 177-186 Stewart, B. E. and Zbarsky, S. H. (1963) Can. J . Biochem. and Physiol. Ul, 1183-1186 Sulkowski, E. and Laskowski, M. Sr. (1971) Biochim. Biophys. Acta 240, 443-447 Sung, S. C. (1968) J . Heurochem. 15_, 447-481 Swenson, M. K. and Hodes, M. E. (1969) J . B i o l . Chem. 244, l803-l807 Taper, H. S., Bruchev, J-M., and Fort, L. (1971a) Cancer 28, 482-490 Taper, H. S., Bruchev, J-M., and Fort, L. (1971b) Cancer Res. 31, 913-916 Tomlinson, R. V. and Tener, G. M. (1963) Biochemistry 2, 697-702 Torriani, A. (I966) i n Procedures i n Nucleic Acid Research v o l . 1 (Canton!, G. L. and Bavies, D. R., eds.) pp. 224-235, Harper & Row, New York Townend, R. and Bernardi, G. (1971) Arch. Biochem. Biophys. 147, 728-733 Tsubota, Y., Yamanaka, M., and Takagi, Y. (1974) J . B i o l . Chem. 249, 389O-3894 van de Sande, J . H., Kleppe, K., and Khorana, H. G. (1973) Biochemis- t r y 12, 5050-5055 Vaneko, S. and Laskowski, M. Sr. (1962) Biochim. Biophys. Acta 6 l , 547-552 Weiss, B., Live, T., and Richardson, C. C. (1968) J . B i o l . Chem. 243, 4530-4542 Wilson, T. H. (1962) Intestinal Absorption, W. B. Saunders Co., Phila-delphia and London White, A., Handler, P., and Smith, E. L. (1973) Principles of Biochemis- t r y , 5th edn., McGraw-Hill, Toronto Yamanaka, M., Tsubota, Y., Anal, M., Ishimatsu, K., Okumura, M., Katsuki, S., and Takagi, Y. (1974) J . B i o l . Chem. 249, 220 3884-3889 Yaneva, M, and Dessev, G. (1977) Molec. B i o l . Reports 3, 227-234 Young, E. T. I I , and Sinshelmer, R, L. (1965) J . B i o l . Chem. 240, 1274-1280 Zollner, E. J . , Helm, W., Zahn, R. K., Beck, J . , and Reitz, M. (1974) Huoleic Acids Research 1, IO69-IO78 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0094635/manifest

Comment

Related Items