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Deoxyribonuclease in the intestinal mucosa of rat Lee, C.Y. 1965

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DEOXYRIBONUCLEASE IN THE INTESTINAL MUCOSA OF RAT by C. Y. LEE B. Sc., University of B r i t i s h Columbia, I 9 6 3 . A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Biochemistry. We accept t h i s thesis as conforming to the required standard f o r the degree of MASTER OF SCIENCE. The University of B r i t i s h Columbia A p r i l , I 9 6 5 . 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 b e ' 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 ft -1 nnhmnl Rt,-y 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 , V a n c o u v e r 8, C a n a d a D a t e 5th May, 1965>  - i -ABSTRACT The properties of the deoxyribonuclease a c t i v i t y i n the i n t e s t i n a l mucosa of rat have been studied. I t was found that the a c t i v i t y was extractable from a whole c e l l preparation of the tissue with either Krebs Ringer phosphate buffer (pH 7.8) or p h y s i o l o g i c a l s a l i n e . The former was a better extracting medium, the buffered extract being more stable to storage at -20°C. Two active proteins were pr e c i p i t a t e d on f r a c t i o n a t i o n of the Krebs Ringer phosphate buffer extract with ammonium su l f a t e . One f r a c t i o n p r e c i p i t a t e d at 20% saturation of (NH^JgSO^ was i d e n t i f i e d as DNase I by i t s optimum pH, Ionic requirements and by i t s reaction to known DNase I i n h i b i t o r s such as EDTA, c i t r a t e and arsenate. Ion-exchange chroma-tography on DEAE-cellulose (chloride form) established that the products of reaction ranged from mononucleotides through to oligonucleotides with a degree of polymerization larger than 7. These products were shown to carry a phosphomonoester linkage on the 5*-carbon. This f r a c t i o n was designated as the 20$P enzyme and represented a 20 f o l d p u r i f i c a t i o n of the crude deoxyribonuclease extract. The supernatant f r a c t i o n obtained a f t e r the 20$P enzyme was removed by centrifugatlon was found to s t i l l contain - i i -considerable DNase a c t i v i t y . This residual a c t i v i t y disappeared from the solution when the ammonium sulfate concentration was increased to 30% saturation, but a l l attempts to recover the DNase a c t i v i t y from the protein p r e c i p i t a t e d at t h i s s a l t concentration were unsuccessful. Consequently, studies of t h i s r e s i d u a l DNase a c t i v i t y were c a r r i e d out using the crude extract. This second deoxyrlbonuclease was shown to be q u a l i t a t i v e l y d i f f e r e n t from the 20% enzyme. I t did not require a c t i v a t i o n by Mg + +, and was not i n h i b i t e d by EDTA, c i t r a t e or arsenate. Some of the products formed by the crude enzyme extract were shown to terminate i n 3*-phosphoryl groups. These products were not formed by the 20$P enzyme and therefore must be due to the res i d u a l deoxyrlbonuclease. In a l l l i k e l i h o o d then, t h i s second deoxyribonuclease a c t i v i t y was of the DNase II type. The i n t r a c e l l u l a r d i s t r i b u t i o n of the i n t e s t i n a l mucosal deoxyribonuclease was studied by d i f f e r e n t i a l c e n t r i -fugation of the tissue homogenate. DNase I a c t i v i t y was found to occur i n the mitochondria whereas DNase II was associated with some l i g h t e r subcellular p a r t i c l e s . - i i i -ACKNOWLEDGMENTS The author wishes to express his sincere thanks and deep appreciation to Dr. S.H. Zbarsky for h i s guidance and encouragement during the course of t h i s research. The support of the National Research Council i n the form of a studentship i s also g r a t e f u l l y acknowledged. - i v -TABLE OF CONTENTS Page INTRODUCTION 1 EXPERIMENTAL I. Preparation of Deoxyribonucleic Acids . . . 16 ( i ) Preparation of DNA from small i n t e s t i n a l mucosa of r a t 16 ( i i ) Deproteinization of salmon sperm DNA 18 ( i i i ) Estimation of nitrogen content of deoxyribonucleic acids 19 (iv) Determination of phosphorus content of DNA 19 (v) Determination of £(P) values of DNA preparations 20 I I . Preparation and P u r i f i c a t i o n of Deoxyribonuclease from Rat I n t e s t i n a l Mucosa 21 ( i ) Measurement of enzyme a c t i v i t y . . . 21 ( i i ) Preparation of crude enzyme extract 27 ( i i i ) P u r i f i c a t i o n of i n t e s t i n a l mucosal deoxyribonuclease by ammonium sulfate f r a c t i o n a t i o n 28 I I I . Enzymatic Degradation of Deoxyribonucleic Acids 30 ( i ) Digestion of deoxyribonucleic a c i d by DNase 30 - V -Page ( i i ) Ion-exchange chromatography of DNA hydrolysates 32 ( i i i ) Preparation of snake venom phosphodiesterase 35 (iv) Assay f o r phosphodiesterase a c t i v i t y 38 IV. Characterization of the Products of a DNA Hydrolysate 42 ( i ) Degree of polymerization of the oligonucleotides 42 ( i i ) Secondary degradation of DNA hydrolysates by venom and spleen phosphodiesterases 43 V. E f f e c t of Heat, pH, Magnesium and Inhibitors on the Rate of Digestion of DNA by the Rat I n t e s t i n a l Mucosal Extract. . 49 ( i ) E f f e c t of Heat 49 ( i i ) _ E f f e c t of pH 49 ( i i i ) Magnesium requirements of the i n t e s t i n a l extract 49 (iv) E f f e c t of i n h i b i t o r s 50 VI. I n t r a c e l l u l a r D i s t r i b u t i o n of Deoxyribo-nuclease i n the Rat I n t e s t i n a l Mucosa . . . 52 RESULTS AND DISCUSSION I. Preparation, P u r i f i c a t i o n and Characteri-zation of Substrate Deoxyribonucleic Acids 55 I I . Preparation and P u r i f i c a t i o n of Deoxyribonuclease from Rat I n t e s t i n a l Mucosa 59 - v i -Page (i ) E f f e c t of extracting media 59 ( i i ) Fractionation with ammonium sulfate 63 I I I . Enzymatic Degradation of Deoxyribonucleic Acids and Analysis of the Products . . . . 66 ( i ) K i n e t i c s of reaction , 67 ( i i ) Ion-exchange chromatography and characterization of the products i n DNA hydrolysates 70 ( i i i ) Secondary degradation of DNA hydrolysates by phosphodiesterases . 81 IV. E f f e c t of Heat, pH, Mg + + and some Inhibitors on the Rat I n t e s t i n a l Deoxyribonuclease 86 V. I n t r a c e l l u l a r D i s t r i b u t i o n of Deoxyribonucleases i n the Rat In t e s t i n a l Mucosa 88 VI. General Discussion 91 SUMMARY 92 BIBLIOGRAPHY 95 - v i i -LIST OF FIGURES Page 1. Change i n UV-absorption Spectrum of Salmon Sperm DNA on Digestion with DNase 23 2. Rate of Increase i n UV-absorption of Solution of Salmon Sperm DNA on Digestion with DNase I (Worthington Enzyme) 24 3 . Nomogram fo r Ammonium Sulfate Solution . . . 29 4. Sodium Chloride Gradient i n Ion-exchange Chromatography as Measured by the Refractive Indices of the E f f l u e n t Fractions 34 5. Time Course of the Hydrolysis of p-nitrophenyl-thymidine-5'-phosphate by Venom Phosphodiesterase 40 6. Comparison of Rate of Degradation of DNA by Crude Extracts of Rat I n t e s t i n a l Mucosa - 6 l 7. Time Course of the Degradation of DNA by Crude I n t e s t i n a l DNase Extract as Measured by the Spectrophotometric Method of Kunitz . 68 8 . Time Course of the Degradation of DNA by Crude I n t e s t i n a l DNase Extract as Measured by Viscosimetry 69 9 . Ion-exchange Chromatography of the Oligonucleotides i n Rat I n t e s t i n a l Mucosal DNA Hydrolysate Formed by Exposing the DNA to a Crude I n t e s t i n a l DNase Preparation 71 10. Ion-exchange Chromatography of the Oligonucleotides i n Salmon Sperm DNA Hydrolysate Formed by Exposing the DNA to a Crude I n t e s t i n a l DNAse Preparation . . 72 - v i i i -Page 11. Ion-exchange Chromatography of the Oligonucleotides i n Ca l f Thymus DNA Hydrolysate Formed by Exposing the DNA to a Crude I n t e s t i n a l DNase Preparation 73 12. Ion-exchange Chromatography of the Oligonucleotides i n Rat I n t e s t i n a l Mucosal DNA Hydrolysate Formed by Exposing the DNA to the 20$P Enzyme . . . . 7k 13. Ion-exchange Chromatography of the Oligonucleotides i n Salmon Sperm DNA Hydrolysate Formed by Exposing the DNA to the 20^P Enzyme 75 lk. Ion-exchange Chromatography of the Oligonucleotides i n Calf Thymus DNA Hydrolysate Formed by Exposing the DNA to the 20$P Enzyme 76 15. Rate of Release of Mononucleotides by Snake Venom Phosphodiesterase from Salmon Sperm DNA Hydrolysate 83 16. E f f e c t of pH on Deoxyribonuclease A c t i v i t y of Rat I n t e s t i n a l Mucosal Extracts 8k - i z -LIST OF TABLES Page I. Elementary Composition and Some Other Properties of the DNA Preparations Used 58 I I . S p e c i f i c A c t i v i t y of Deoxyribonuclease i n Crude Extracts of Eat I n t e s t i n a l Mucosa 62 I I I . P u r i f i c a t i o n of I n t e s t i n a l Mucosal Deoxyribonuclease Extract by Fractionation with Ammonium Sulfate . . . . 64 IV. Some A n a l y t i c a l Data of DNA Hydrolysates 78 V. Nucleoside Content of Salmon Sperm DNA Hydrolysate a f t e r Successive Degradation by Deoxyribonuclease, Spleen Phosphodiesterase and E. C o l i Phosphomonoesterase . 85 VI. I n t r a c e l l u l a r D i s t r i b u t i o n of Deoxyribonuclease i n Bat I n t e s t i n a l Mucosa 90 -X-L I S T OF A B B R E V I A T I O N S DNA d e o x y r i b o n u c l e i c a c i d R NA r i b o n u c l e i c a c i d s -RNA s o l u b l e r i b o n u c l e i c a c i d D N a s e d e o x y r i b o n u c l e a s e R N a s e r i b o n u c l e a s e PME p h o s p h o m o n o e s t e r a s e E D T A e t h y l e n e d i a m i n e t e t r a a c e t a t e d-AMP, e t c . t h e 5 ' - p h o s p h a t e o f 2 * - d e o x y r i b o s y l a d e n i n e , e t c . P u p u r i n e P y p y r i m i d i n e p o l y d - U a p o l y m e r o f d e o x y u r i d y l i c a c i d g g r a v i t a t i o n a l f o r c e gm g r a m mg m i l l i g r a m jig m i c r o g r a m M m o l a r ju m o l e m i c r o m o l e mu m i l l i m i c r o n u l m i c r o l i t e r -1-INTRODUGTION Many, i f not a l l , tissues have been shown to possess an enzymatic complement capable of degrading nucleic acids (1). I t i s more than l i k e l y that the function of t h i s enzyme complement i s not a passive one, i . e . they do not represent merely a means of degrading nucleic acids i n dead or lysed c e l l s so that the products formed may be used as substrates i n nucleic a c i d synthesis by l i v i n g members of the c e l l population. The p o s s i b i l i t i e s are that they perform a c t i v e l y i n many aspects of nucleic a c i d metabolism. Genetic recombination has recently been shown to occur by a "breakage and reunion" of the DNA molecule (2). DNases, by analogy with pancreatic RNase (3), may under appropriate conditions act as a transoligonucleotidase t r a n s f e r r i n g oligonucleotide segments from one polynucleotide to another. Studies of the r e p l i c a t i o n of DNA by the polymerase enzyme i s o l a t e d from E. C o l i have demonstrated that DNA synthesis requires the presence of 3'-hydroxyl groups i n the primer DNA (4). The rate of DNA synthesis can be greatly increased i f the primer DNA i s f i r s t subjected to treatment with DNase I to provide the necessary -2-3 1-hydroxyl groups. Although indiscriminate cleavage of dies t e r bonds by DNA endonuclease attack i s i n i m i c a l to b i o l o g i c a l i n t e g r i t y , the s p e c i f i c introduction of 3 * -hydroxyl groups by a c a r e f u l l y controlled endonucleolytic f i s s i o n may f a c i l i t a t e DNA synthesis i n vivo. Recently Setlow (64) proposed that one possible mechanism fo r correcting errors i n the genetic code i s by excision of the e r r a t i c segment by an endonuclease followed by repair of the excised portion. Thus deoxyrlbonucleases might represent a defence mechanism to counteract the e f f e c t of mutation. DNases might also serve as "scavengers" within the c e l l , removing g e n e t i c a l l y superfluous and nonfunctional DNA segments formed, f o r example during r e p l i c a t i o n (5)» Doty et a l . have used the exonuclease-I of _E_. c o l i to remove single-stranded polynucleotide ends from newly-synthesized, double-stranded DNA (5). Despite the great v a r i e t y and sometimes high concen t r a t i o n of n u c l e o l y t i c enzymes present i n c e l l s , the nucleic acids do not undergo any precipitous breakdown except under extreme conditions of c e l l death and l y s i s . Obviously, mechanisms must exis t to keep t h i s powerful n u c l e o l y t i c potency under cont r o l . -3-Studies of p a r t i a l l y p u r i f i e d enzymes have i n many instances given clues as to how they may be Immobilized within the c e l l . For example, endonuclease-I i n E. c o l i , which i s DNA-specific, has been shown to be quantitatively i n h i b i t e d by very low concentrations of RNA (6). This enzyme may thus be immobilized i n vivo, by combination with the c e l l u l a r RNA. One of the two E. c o l i ribonucleases i s found exclusively i n the ribosomal f r a c t i o n of c e l l - f r e e extracts. Elson (7) showed that t h i s ribonuclease a c t i v i t y i s not detectable i n c a r e f u l l y prepared ribosomal suspensions or ribonucleoprotein p a r t i c l e s derived from them, but enzymatic a c t i v i t y appears upon disruption of the ribosomes by t r e a t -ment with urea or t r y p s i n . I t i s suggested that the ribosomal RNase exi s t s only i n a lat e n t form and i s therefore incapable of degrading RNA i n a normally functioning c e l l . I n t r a c e l l u l a r compartmentization i s of course an e f f e c t i v e way of preventing nucleases from acting on t h e i r substrates. But i t i s not always necessary to invoke i n d i v i d u a l p e c u l a r i t i e s of c a t a l y t i c s p e c i f i c i t y or i n t r a -c e l l u l a r l o c a t i o n to explain the i n a c t i v i t y of these enzymes i n vivo. The nucleic acids of the c e l l may be p h y s i c a l l y enveloped by, or otherwise bound to some structures ( f o r -4-example, histone or protamines) and thus are inaccessible to the active s i t e s of the enzymes. Another reason which has prompted the study of various n u c l e o l y t i c enzymes i s the use to which these enzymes might be put as reagents i n studying the structure and nucleotide sequence of nucleic acids, a branch of nuclei c a c i d research which has received great impetus i n the l a s t few years. In the case of enzymes that attack RNA, i t has been shown ( 8 ) that p u r i f i e d J 3 . c o l l poly-nucleotide phosphorylase can rapidly phosphorolyse randomly c o i l e d polyribonucleotides such as p o l y u r i d y l i c acids but ordered, hydrogen-bonded structures such as s-RNA are r e l a t i v e l y r e s i s t a n t to attack by t h i s enzyme. This property of polynucleotide phosphorylase suggests that the enzyme may be used i n studying the secondary structure of poly-ribonucleotides. Recently, Holley et a l (9) were able to determine completely the nucleotide sequence i n alanyl-s-RNA by ex p l o i t i n g the high degree of s p e c i f i c i t y of the two RNA depolymerases, pancreatic RNase and T^-RNase. Ba c t e r i a l phosphatase, by v i r t u e of i t s a b i l i t y to phosphorolyse the terminal phosphomonoester linkage i n oligonucleotides, and i t s lack of diesterase a c t i v i t y has - 5 -proved to "be an excellent a n a l y t i c a l tool f o r the deter-mination of polynucleotide chain length and for the f a c i l i t a t i o n of the synthesis and depolymerization of polynucleotides by enzymes requiring free hydroxyl terminals f o r a c t i v i t y . In the case of enzymes attacking DNA, depolymerases with any high degree of s p e c i f i c i t i e s are sadly lacking. The exonuclease-I of E. c o l i ( 1 0 ) , because of i t s s e l e c t i v i t y f o r single-stranded DNA, may be used for the i d e n t i f i c a t i o n of t h i s conformation i n a DNA sample. The enzyme i s incapable of attacking the phosphodiester bond between the l a s t two nucleotide residues at the 5*-terminus of a polydeoxyribonucleotide chain; t h i s , coupled with i t s lack of endonuclease a c t i v i t y , permits the use of the enzyme as an endgroup reagent. The present trend of nucleic a c i d research i s directed c h i e f l y towards the "cracking" of the genetic code. To t h i s end, a d i r e c t comparison of the nucleotide sequence i n DNA, the genetic material, and the primary structure of the polypeptide chain f o r which i t codes, appears to be the ultimate aim. Such a feat cannot be accomplished u n t i l enzymes attacking DNA are discovered which show a high degree of s e l e c t i v i t y towards the internucleotide bonds -6-which they hydrolyse. None of the DNases examined so f a r has shown any marked s p e c i f i c i t y , and the search for new enzymes capable of degrading DNA at s p e c i f i c i n t e r -nucleotide bonds must remain an imminent concern of the present-day biochemists. The discovery of deoxyribonuclease dated as f a r back as 1903» when Araki observed that i t was possible to l i q u e f y gels of the y8-form of thymus nucleic a c i d with extracts of tissues of l i v e r , spleen and thymus (11). Iwanoff (12) demonstrated i n various molds the presence of enzymes capable of l i q u e f y i n g thymus nucleic a c i d , and he applied the term nucleases to these enzymes. Iwanoff was also able to show experimentally that t h i s n u c l e o l y t i c a c t i v i t y did not rest with the proteases also present i n the extract. This was confirmed by Sachs (13) who further-more, found evidence that t r y p s i n has a destructive action on nuclease. He was also able to demonstrate the presence of nucleases i n fresh pancreas, i n which the p r o t e o l y t i c enzymes are s t i l l i n the zymogen state. Abderhalden and Schittenhelm (14) found that the pancreatic juice of dogs effected the l i q u e f a c t i o n of thymus nucleic acid without l i b e r a t i o n of inorganic phosphate or of nitrogenous bases. Feulgen (15) made the important observation that the - 7 -degradation of deoxyribonucleic a c i d did not progress to the mononucleotide stage, but stopped at the formation of oligonucleotides since the s p l i t products were s t i l l p r e c i p i t a b l e with mineral acids. Schmidt, Pickels and Levene (16) found i n 1939 that the degradation of DNA to oligonucleotides by the pancreatic enzyme was an es s e n t i a l intermediary step f o r the enzymic cleavage of DNA to mononucleotides (and subsequently to nucleosides) by the phosphatase of i n t e s t i n a l mucosa. Highly polymerized deoxyribonucleic a c i d was not attacked by p u r i f i e d i n t e s t i n a l phosphatase (nucleotidase); but i t hydrolysed a l l i n t e r -linkages of the mononucleotide groups i n the oligonucleotide mixture obtained by the action of deoxyribonuclease ( 1 6 ) . The evidence f o r the existence of a s p e c i f i c polynucleotidase, bothfor ribonucleic and deoxyribonucleic acids, though more or l e s s convincing, has been e n t i r e l y circumstantial even as l a t e as 1939. I t was only with the i s o l a t i o n of c r y s t a l l i n e ribonuclease by Kunitz (17) i n 19^0 that the presence of a s p e c i f i c polynucleotidase, at l e a s t f o r RNA, was d e f i n i t e l y established. Since then, attempts to obtain p u r i f i e d preparations of deoxyribonuclease from pancreas preparations were made by Laskowski (18) and -8-McCarty (19). They were able to separate deoxyribonuclease from the very powerful ribonuclease of pancrease and to provide evidence f o r the s t r i c t s p e c i f i c i t y of deoxyribo-nuclease f o r DNA. The c r y s t a l l i z a t i o n of the enzyme was achieved by Kunitz i n 1950 (20). The pH optimum of t h i s pancreatic deoxyribonuclease l i e s close to 7.0, but i t i s somewhat influenced by the nature of the ions i n the medium (20). The enzyme requires divalent cations for a c t i v i t y , magnesium, manganese and cobaltous ions being the most e f f e c t i v e (20, 21). Another DNase which has an optimum pH i n the range 4.5-5*5 --id- i s i n h i b i t e d by the addition of Mg + + was observed i n spleen by Catcheside and Holmes (22) and i n thymus by Maver and Greco (23). These observations were soon confirmed and extended to other t i s s u e s . The name DNase II was suggested f o r t h i s enzyme i n contrast to the pancreatic enzyme, which was termed DNase I. At present, the DNases are subdivided i n t o two classes, the DNase I enzymes and DNase II enzymes. These two classes were introduced by Laskowski (24) to compare and to contrast enzymes d i s t r i b u t e d In d i f f e r e n t tissues with t h e i r prototypes i n respect to pH optimum and a c t i v a t i n g and i n h i b i t i n g agents. Pancreatic DNase I of Kunitz and - 9 -thymus DNase II of Maver and Greco served as prototypes. This terminology i s useful f o r the purpose of characterization of enzymes i n d i f f e r e n t t i s s u e s . In accordance with t h i s , the term " i n t e s t i n a l mucosal DNase I" implies an enzyme is o l a t e d from the i n t e s t i n a l mucosa and resembling pancreatic DNase i n i t s properties. Laskowski (25) proposed four, c r i t e r i a f or the characterization of deoxyribonucleases: the susceptible substrates, the type of attack, the products and the preferen-t i a l linkage. When DNase I i s considered under these headings, i t i s to be seen that DNase I i s s p e c i f i c for DNA. I t i s an endonuclease because i t rapid l y decreases the v i s c o s i t y of a DNA solution and because the products of hydrolysis are predominantly large oligonucleotides. T i t r a t i o n data indicated that approximately 25$ of a l l avai l a b l e internucleotlde linkages could be hydrolysed by DNase I (20). This means that the average size of the product i s that of a t e t r a -nucleotide. Ion-exchange chromatography showed that the products were oligonucleotides of a variable s i z e , ranging from mononucleotides through d i - , t r i - etc., probably to octanucleotides (27). The products were i d e n t i f i e d as terminated i n 5 ,-phosphates (26). The mononucleotides were susceptible to the action of 5 f-nucleotidase and the l a r g e r 10-fragments yielded 5 '-nucleotides exclusively on digestion with venom phosphodiesterase. A systematic i d e n t i f i c a t i o n of products (28) established that a l l four of the common mononucleotides were present, with thymidylic a c i d being the predominant member. Sinsheimer has carried out extensive studies of the d l -nucleotides i n DNase I digests ( 2 9 ) . The dinucleotides with sequences pPy-pPy, pPu-pPu and pPy-pPu were found to be abundant but those with sequences pPu-pPy were very rare. This was interpreted to mean that the pPu-pPy bond was p r e f e r e n t i a l l y hydrolysed. Additional evidence was obtained by i s o l a t i n g a trinucleotide. - ApApTp from a digest of DNA by DNase I I , and then digesting i t with DNase I ( 3 0 ) . The tr i n u c l e o t i d e was s p l i t into d-ApA and pTp by DNase I, as would be expected from the postulated preference. However, t h i s preference cannot be expressed i n quantitative terms. Koerner and Sinsheimer ( 3 D showed that DNase II from spleen was also an endonuclease. Exhaustive reaction resulted i n about 25-30$ of the internucleotide linkages being hydrolysed. The d i s t r i b u t i o n of the fragments as evidenced by the chromatographic pattern was quite d i f f e r e n t from that of a digest by DNase I. DNase II produced more mononucleotides, considerably l e s s dinucleotides and considerably -11-more of the higher oligonucleotides. A l l the reaction products were terminated i n 3 '-phosphates. DNase II d i d not seem to show any preference f o r s p e c i f i c purine and pyrimldine bases adjacent to the sensitive linkages. Both DNase I and DNase II are widely d i s t r i b u t e d ( 1 ) . Pancrease has long been recognized as the best source of DNase I, whereas spleen and thymus are much r i c h e r i n DNase II than DNase I a c t i v i t i e s . Recently, Stewart and Zbarsky (32) reported the loss of DNA from r at i n t e s t i n a l mucosa during incubation i n v i t r o of mucosal scrapings or i n t e s t i n a l s l i c e s and have pointed out the possible existence of a DNase i n the t i s s u e . This l o s s of nucleic acids from the incubated tissue was not i n h i b i t e d by removal of Mg"^ from the buffer, nor by the addition of 0.1 M arsenate. Addition of EDTA however reduced the loss of DNA to only 11$ i n one hour. These r e s u l t s appeared to indicate that both types of DNases were present i n the rat i n t e s t i n a l mucosa and i t seemed that the i s o l a t i o n and characterization of the i n t e s t i n a l enzyme(s) were desirable and might provide some in s i g h t into the nucleic a c i d metabolism of t h i s a c t i v e l y p r o l i f e r a t i n g t i s s u e . A method was developed f o r preparing crude enzyme extracts of the i n t e s t i n a l mucosal tissue using a var i e t y of -12-extracting media. The a c t i v i t i e s of these extracts were tested against deoxyribonucleic acids from several sources. Methods used for the determination of DNase a c t i v i t i e s were based on v i s c o s i t y and o p t i c a l properties of nucleic acids and t h e i r components. Employing the technique of f r a c t i o n a t i o n by means of ammonium sul f a t e , i t was found that two fractio n s of DNase a c t i v i t y were pr e c i p i t a b l e from the mucosal extract. One of these p r e c i p i t a t e d at 20$ saturation of ammonium sulfa t e and the a c t i v i t y was recoverable by red i s s o l v l n g the protein p r e c i p i t a t e i n buffer. The other type p r e c i p i t a t e d at 30-35$ saturation of ammonium sulfate, but a l l attempts to recover the enzymatic a c t i v i t y were unsuccessful. In subsequent experiments performed, both the crude extract and the p a r t i a l l y p u r i f i e d enzyme preparation were therefore used separately so that a q u a l i t a t i v e comparison between the two could be made. The e f f e c t of pH, the i o n i c requirements and the e f f e c t of various i n h i b i t o r s were tested and found to d i f f e r markedly f o r the two enzyme preparations. Thus whereas the p a r t i a l l y p u r i f i e d enzyme extract showed an absolute require-ment f o r Mg + +, removal of magnesium by exhaustive d i a l y s i s of -13-the crude extract did not completely abolish i t s a c t i v i t y . A protein i n h i b i t o r prepared from the pigeon crop gland, while completely i n h i b i t i n g the p u r i f i e d extract, had only l i m i t e d e f f e c t on the crude preparation. These findings would seem to indicate that the crude preparation contained an a d d i t i o n a l DNase d i f f e r e n t from the one preci p i t a t e d at 20% saturation of ammonium s u l f a t e . To further c l a r i f y t h i s point, the digests obtained by exposing DNA to the two fractions were analysed. The el u t i o n p r o f i l e s from a DEAE-cellulose column were found to be d i f f e r e n t . The oligonucleotides obtained with the p a r t i a l l y p u r i f i e d enzyme extract were exclusively those which contained a 5 ,-Phosphomonoester bond, but the products obtained with the crude complete extract contained both 5 ' - and 3'-Phospho-monoester linkages. Moreover, the corresponding oligonucleo-t i d e f r a c t i o n s obtained with the two extracts were found to d i f f e r both q u a l i t a t i v e l y and qua n t i t a t i v e l y . Thymidylic a c i d was predominant i n the mononucleotide frac t i o n s ; but whereas the d - c y t i d y l i c a c i d content i n the higher o l i g o -nucleotides was low when the digest was prepared with the p u r i f i e d extract, no such discrimination was observed when the crude enzyme was used. - 1 4 -Th e p o s s i b i l i t y was considered that the deoxyribonuclease a c t i v i t y of the i n t e s t i n a l mucosal extract might be due to contamination of b a c t e r i a l or pancreatic o r i g i n . Extra care has been taken to flush clean the i n t e s t i n a l lumen during the preparation of the enzyme extract; and i t has also been found that a f t e r a whole c e l l preparation of the i n t e s t i n a l mucosa was extracted, further deoxyribo-nuclease was released i n t o the medium when the tissue was homogenized. To further pinpoint the mucosal o r i g i n of the enzyme, i t was f e l t that an i n t r a c e l l u l a r l o c a l i z a t i o n and d i s t r i b u t i o n of the deoxyribonuclease a c t i v i t y must be established. To t h i s end, a mucosal homogenate was subjected to d i f f e r e n t i a l centrifugation and the various subcellular f r a c t i o n s assayed for DNase a c t i v i t y . I t was found that whereas 54$ of the t o t a l DNase a c t i v i t y was located i n the heavy mitochondrial f r a c t i o n , 43$ of the a c t i v i t y was associated with some l i g h t e r p a r t i c u l a t e f r a c t i o n . On the basis of the r e s u l t s obtained i n the experiments to be described, i t has been concluded that the deoxyribonuclease a c t i v i t y extracted from the i n t e s t i n a l mucosa of the rat may contain both DNase I and DNase II types. The experiments have, however, not unequivocally established the existence of DNase I I . A p o s i t i v e i d e n t i f i -cation of t h i s enzyme has to await i t s i s o l a t i o n from the -15-t i s s u e and the c h a r a c t e r i z a t i o n of i t s products of r e a c t i o n . The problem of b a c t e r i a l contamination has a l s o not been s a t i s f a c t o r i l y s o l v e d . The use o f germ-free r a t s would undoubtedly c l a r i f y t h i s p o i n t . -16-EXPERIMENTAL I. Preparation of Deoxyribonucleic Acids ( i ) Preparation of DNA from Small I n t e s t i n a l Mucosa of Bat - Method of Colter et a l . Reagents: (a) 1M NaCl-0.03M EDTA - 0.02M phosphate buffer, PH 7 . 3 . (b) Water-saturated Phenol: t h i s solution contains 75 gm. of r e d i s t i l l e d phenol and 25 ml. of d i s t . H2Q. (c) 0.15M NaCl-0.03M EDTA. The pH of the solution was adjusted to 7.0 with 0.1N NaOH. (d) 10% Na deoxycholate s o l u t i o n . Procedure: Male Wistar rats weighing 180-200 gm. each were starved f o r 2h hours. They were then k i l l e d by a blow on the head and decapitated. The small Intestine was removed and cut into segments approximately 10 cm. i n length. These segments were flushed e n t i r e l y free of contents with c h i l l e d phosphate buffer. They were then s l i t open and applied to a cold glass surface with the mucosal side facing upwards. The mucosal epithelium was then scraped o f f with the edge of a microscope s l i d e and Immediately placed i n l i q u i d nitrogen. -17-Five rats afforded about 7 gms. wet weight of mucosal t i s s u e . The pooled frozen tissue was transferred to the blender cup of a S e r v a l l Omni Mixer and 45 ml. of NaCl-EDTA-Phosphate buffer solution, 2.5 ml. of 10$ sodium deoxycholate solution and 50 ml. of water-saturated phenol solution were added for each 5 gm. of tissue used. The blender cup was set i n an i c e bath and the mixture was homogenized at a low speed, the powerstat setting being at 20v . Following homogenlzatlon, the emulsion was centrifuged f o r 10 min. at 27,000g i n a S e r v a l l r e f r i g e r a t e d centrifuge. The upper aqueous lay e r containing the DNA was c a r e f u l l y removed from the lower phenol l a y e r and denatured protein which c o l l e c t e d at the i n t e r f a c e . The aqueous solution was subjected to two a d d i t i o n a l extractions with equal volumes of water-saturated phenol. These extractions were c a r r i e d out i n the cold room on a mechanical shaker and were of 5 min. duration. A f t e r each extraction, the aqueous and phenol layers were separated by centrifugation at 4 ,000g i n the S e r v a l l centrifuge f o r 10 min. Traces of phenol were then removed from the DNA so l u t i o n by 6 or 7 extractions with ether, and the residual ether removed by bubbling nitrogen through the solu t i o n . The DNA solution was then kept overnight at 4°C; the high molecular weight RNA which prec i p i t a t e d out of solution was - 1 8 -r e m o v e d b y c e n t r i f u g a t i o n a t 4 , 0 0 0 g f o r 10 m i n . T h e DNA w a s p r e c i p i t a t e d b y t h e a d d i t i o n o f a n e q u a l v o l u m e o f e t h a n o l . T h e DNA p r e c i p i t a t e w a s d i s s o l v e d a g a i n i n t h e m i n i m u m a m o u n t o f n e u t r a l s a l i n e - E D T A s o l u t i o n a n d c e n t r i -f u g e d i f n e c e s s a r y t o o b t a i n a c l e a r s o l u t i o n . DNA w a s p r e c i p i t a t e d w i t h e t h a n o l a s p r e v i o u s l y , a n d t h e f i b r o u s m a t e r i a l w a s w a s h e d s e v e r a l t i m e s w i t h ?5$ E t O H a n d t h e n d r i e d i n v a c u o o v e r ?2Q5 a t r o o m t e m p e r a t u r e . T h e d r y p r e p a r a t i o n w a s d i s s o l v e d i n t h e a p p r o p r i a t e b u f f e r s o l u t i o n , u s u a l l y t o a c o n c e n t r a t i o n o f 1 mg. D N A / m l . T h e s o l u t i o n s w e r e s t o r e d a t - 2 0 ° C . ( i i ) D e p r o t e i n i z a t i o n o f S a l m o n S p e r m DNA A s a m p l e o f c r u d e s a l m o n s p e r m DNA, a g i f t f r o m D r . T e n e r w a s f o u n d t o b e h e a v i l y c o n t a m i n a t e d w i t h p r o t e i n . I t w a s f e l t d e s i r a b l e t o r e m o v e t h e p r o t e i n b e f o r e t h e DNA w a s u s e d a s s u b s t r a t e f o r e n z y m a t i c d e g r a d a t i o n . D e p r o t e i n -i z a t i o n w a s c a r r i e d o u t e s s e n t i a l l y a c c o r d i n g t o t h e m e t h o d d e s c r i b e d b y Z u b a y (34). P r o c e d u r e : F i v e h u n d r e d mg. o f t h e c r u d e s a l m o n s p e r m DNA w a s d i s s o l v e d i n a m i n i m u m a m o u n t o f d i s t i l l e d w a t e r . T h e v o l u m e o f t h e s o l u t i o n w a s m e a s u r e d a n d e n o u g h N a C l a d d e d t o g i v e a s o l u t i o n 1M i n s t r e n g t h w i t h r e s p e c t t o N a C l . - 1 9 -To approximately kOO ml. of t h i s DNA solution, 100 ml. of chloroform-amyl alcohol mixture (3:1 v/v) were added. The mixture was emulsified by shaking i t i n the cold f o r 10 min. using a vibratory shaker, and was then centrifuged at 4 ,000g on the S e r v a l l r e f r i g e r a t e d centrifuge for 10 min. The upper aqueous layer was c a r e f u l l y removed with a syringe and separated from the lower organic phase and the la y e r of denatured protein c o l l e c t e d at the i n t e r f a c e . The extraction was repeated u n t i l no more protein was observed at the i n t e r f a c e . The DNA was then p r e c i p i t a t e d by adding an equal volume of ethanol and the preparation treated as described i n I ( i ) . ( i i i ) Estimation of Nitrogen Content of Deoxyribonucleic Acids The nitrogen content of deoxyribonucleic acids was determined by the method of Pregl (35)« The recovery of nitrogen by t h i s procedure was very s a t i s f a c t o r y . When r e c r y s t a l l i z e d urea was used as nitrogen standard, i t was found to contain, on the average of three experiments, k6.2% of nitrogen as compared to the t h e o r e t i c a l value of 46 . 6 ? $ . (iv) Determination of Phosphorus Content of DNA The color reaction of Piske and SubbaRow (36) was used f o r phosphorus determination. One mg. of dry DNA was used routinely f o r such determinations. The colour produced - 2 0 -was found to "be proportional to the concentration of phosphorus up to 40 ug for 10 ml. of the reaction mixture. A c a l i b r a t i o n curve was constructed (using KHgPO^ as standard) from which the phosphorus content of the DNA samples was determined. (v) The Determination of £(P) Values of DNA Preparations (37)> (a) 0.1M ammonium acetate buffer - 6 gm. of g l a c i a l a c e t i c a c i d and 2 .03 gm MgCl 2 6H 20 were dissolved i n approx. 900 ml. of d i s t . H 2 0 . The pH of the solution was adjusted to 7 .0 with cone. NH^OH. The f i n a l volume of the solution was made up to j 1 ,000 ml. Procedure: Accurately weighed DNA samples were dissolved In the acetate buffer so that the f i n a l concentration of DNA was 1 mg./ml. One ml. of t h i s stock solution was made up with the same buffer to 25 ml. The u l t r a v i o l e t absorption spectra of the solutions were then recorded with a Gary spectrophotometer. From the o p t i c a l density value at maximum absorption (usually around 258 mu) the 6(P) value of the sample was calculated according to the equation ( 3 7 ) : --21-where £(P) = P.P. at max. C x d £(P) = the atomic extinc t i o n c o e f f i c i e n t with respect to phosphorus O.D. = o p t i c a l density of the solution C = phosphorus concentration of the solution i n m o l e s / l i t e r d = i n t e r n a l c e l l length i n cm. I I . Preparation and P u r i f i c a t i o n of Deoxyribonuclease from Rat I n t e s t i n a l Mucosa, (i ) Measurement of Enzymatic A c t i v i t y nuclease on DNA i s the increase i n the absorption of u l t r a -v i o l e t l i g h t by the digested a c i d . The rate of increase i n the l i g h t absorption i s gradual and can be r e a d i l y determined. I t was found (20) that the rate of increase of the o p t i c a l density at 260 mu, brought about by slow digestion of DNA i n d i l u t e solution at the appropriate pH, was constant for several minutes and was proportional to the concentration of enzyme i n solution. I t can thus be used as a convenient and r a p i d method f o r measuring deoxyribonuclease a c t i v i t y . Reagents: (a) Substrate DNA solutions: 40 Ug/ml i n 0.1M ammonium (a) Spectrophotometric Method of Kunitz (20) One of the e f f e c t s of the action of deoxyribo-- 2 2 -acetate buffer, 0.01M i n MgCl 2, pH 6 . 5 . The solutio n was stable f o r weeks when stored i n the r e f r i g e r a t o r at about 5°C. (b) DNase I solution - 2 mg. DNase I (Worthington) per 10 ml. acetate buffer. Procedure: Three ml. of the substrate DNA was pipetted into a quartz cuvette (1 cm. l i g h t path). At zero time, 0.1 ml. of enzyme solut i o n was added and the solution was mixed thoroughly. The increase i n o p t i c a l density was measured at 260 ima with a Cary recording spectrophotometer equipped with a thermostatable c e l l compartment maintained at 3 7°C The spectra of the DNA solution before the addition of the enzyme and a f t e r the completion of the reaction were measured. Figure 1 shows the increased U.V. absorption at 260 wji obtained during the course of depolymerization of DNA by DNase I. In Figure 2, i t can be seen that when the increase i n o p t i c a l density was plotted against the incubation time, the i n i t i a l portion of the graph was a straight l i n e . The a c t i v i t y of the enzyme solution i s expressed i n terms of the I n i t i a l slope of the plotted curve. This divided by the concentration of the enzyme i n mg. of protein per ml. of the f i n a l digestion mixture gives the s p e c i f i c a c t i v i t y of the -23-PIGURE 1 Change i n UV-absorption spectrum of Salmon Sperm DNA on Digestion with DNase. -24-PIGURE 2 : Rate of Increase i n UV-absorption of Solution of Salmon Sperm DNA on Digestion with DNase I (Worthington Enzyme). - 2 5 -enzyme i n units of deoxyribonuclease a c t i v i t y per mg. of protein. One unit of a c t i v i t y i s thus defined as the amount of enzyme capable of bringing about an increase i n o p t i c a l density at 260 mu of 1 per minute under the given conditions of concentration of the substrate, pH, temperature and s a l t concentration. (b) Viscosimetric Method (38) Measurement of the progressive f a l l i n v i s c o s i t y of DNA solutions during treatment with DNase has yielded constant and reproducible r e s u l t s and has proved a r e l i a b l e method fo r determining enzymatic a c t i v i t y . Reagents and Apparatus: (a) Substrate DNA solution - 1 mg. DNA/ml. i n 0.1M ammonium acetate buffer, 0.01M i n MgClg, pH 6 . 5 . (b) Commercial DNase I (Worthington) solution (c) Ostwald type c a p i l l a r y viscosimeter Procedure: A thoroughly cleaned and dry viscosimeter was clamped v e r t i c a l l y i n the thermostated bath (37°C) i n such a p o s i t i o n that i t could be viewed e a s i l y , and 5 ml* of DNA s o l u t i o n was added from a pipette. The l i q u i d was drawn up i n t o the enlarged bulb and above the upper mark. The l i q u i d was then permitted to run down through the c a p i l l a r y -26-and the stop watch was started when the meniscus passed the upper mark and stopped when i t passed the lower mark. Two or three check determinations on the flow rate were made. The flow time f o r the solvent (acetate buffer) was s i m i l a r l y determined. thoroughly mixed with the substrate. The flow time was measured at 5 min. i n t e r v a l s over a period of about 30 minutes. The a c t i v i t y of a given enzyme can be determined from the slope of a curve obtained by p l o t t i n g the r e l a t i v e v i s c o s i t y , ^, of the reaction mixture against time. Relative v i s c o s i t y i s defined by the term Tt/To, where Tt i s the flow time at time t . Where an understanding of the k i n e t i c s of the depolymerization reaction i s desired, the v e l o c i t y constant K of the reaction at any given time t can be determined from the equation The viscosimetric technique was used more extensively i n the e a r l i e r experiments during the course of t h i s research. But because of i t s requirement f o r large amounts of substrate DNA, i t was l a t e r abandoned i n favour of the spectrophotometric At zero time, 0.1 ml. of enzyme was added and K - 2 7 -method and was then used only occasionally f o r the q u a l i t a t i v e demonstration of deoxyribonuclease a c t i v i t y , where the products of digestion could be used for other experiments. ( i i ) Preparation of the Crude Enzyme Extract. Procedure: Five male Wistar r a t s , weighing about 180-200 gms. each, were s a c r i f i c e d as usual. The i n t e s t i n a l mucosal tissue was removed as previously described and immediately placed into a 50 ml. graduated centrifuge tube standing i n an i c e bath. Enough of Kreb*s Ringer phosphate buffer ( 3 9 ) , pH 7.8, was then added to give a 1/10 (v/v) suspension of the t i s s u e . The tissue clumps were broken up by drawing the suspension through a size 14 syringe needle. The sus-pension was then centrifuged at 105,000g f o r 60 minutes at 0°C i n the Spinco Model L u l t r a c e n t r i f u g e . The supernatant solution was c o l l e c t e d and tested f o r deoxyribonuclease a c t i v i t i e s as described under I I ( i ) . In some experiments, the p e l l e t s a f t e r centrifugation were washed with water several times and then centrifuged. They were then re-suspended In Krebs Ringer phosphate buffer equal i n volume to that used i n the f i r s t extraction. The suspension was then homogenized i n the S e r v a l l Omni Mixer -28-set at 30v. on the rheostat for 15 minutes at 0°C. The homogenate was centrifuged at 105,000g f o r 60 minutes. The supernatant solution was c o l l e c t e d and examined f o r deoxyribonuclease a c t i v i t y . ( i i i ) P u r i f i c a t i o n of I n t e s t i n a l Mucosal Deoxyribo-nuclease by Ammonium Sulfate Fractionation. Procedure: Ammonium sulfate f r a c t i o n a t i o n was car r i e d out by a modification of the method described by Kunitz ( 2 0 ) . A known volume of the crude enzyme extract was brought to % saturation with ammonium sulfate (40). The p r e c i p i t a t e formed was removed by centrifugation at l 6 , 0 0 0 g i n the S e r v a l l r e f r i g e r a t e d centrifuge f o r 60 minutes. A known volume of the supernatant solution (designated 5$S) was removed and examined f o r nitrogen and protein contents and DNase a c t i v i t y . The p r e c i p i t a t e was redissolved i n a volume of buffer equal to the volume of the crude enzyme extract used and s i m i l a r l y examined. This solution was designated 5$P. The rest of the supernatant solution was further charged with ammonium sulfate to a desired saturation of s a l t and the above procedure repeated. The amount of ammonium sulfate required to bring a solu t i o n to a degree of -29-FTGURE 3 s Nomogram fo r Ammonium Sulfate Solution. A straight l i n e through the i n i t i a l saturation and the desired saturation gives the amount of s o l i d (NHjiJgSCV to be added to 1 l i t e r of t h i s solution. A l i n e from t h i s point passing through the volume of the solution gives the amount required. -30-saturation was determined according to Dixon (40). Figure 3 shows how t h i s can be done with the use of a nomogram. The f r a c t i o n a t i o n was done by increasing steps of ammonium sulfate saturation of $% i n t e r v a l s . The various fracti o n s i s o l a t e d were analysed f o r deoxyribonuclease. The s p e c i f i c a c t i v i t y of the enzyme solution was expressed i n terms of enzyme a c t i v i t y per mg. protein. Both the crude and the p a r t i a l l y p u r i f i e d enzyme extracts were stable f o r several months i f stored at -20°C. I I I . Enzymatic Degradation of Deoxyribonucleic Acids ( i ) Digestion of Deoxyribonucleic Acid by DNase. Reagents: (a) Substrate DNA solutions Three types of DNA's were used: rat i n t e s t i n a l DNA, deproteinized salmon sperm DNA and c a l f thymus DNA (sodium s a l t , Mann Assayed product). They were dissolved separately i n 0.1M acetate buffers, 0.01M i n MgClg, pH 6.0, 6.5 or 7.0. When used as substrates f o r DNase I I , the DNA's were dissolved i n 0.33M sodium formate buffer, pH 4 .5. In each case, the concentration of DNA was 1 mg./ml. (b) Enzyme preparations ( i ) The crude enzyme extract ( i i ) "20$ P" enzyme preparation -31-( i i i ) DNase I (Worthington), 100 ^ug/ml i n acetate buffer, pH 7.0 (iv) DNase II (Worthington), 100 ug/ml. i n 0.3M sodium formate buffer, pH 4.5. Procedure: To 20 ml. of substrate solution i n a clean test-tube was added 1 ml. of the enzyme preparation and the mixture incubated at 37°C. The pH of the reaction was maintained constant by addition of 0.001N NaoH at i n t e r v a l s to neutralize the secondary phosphate group released during depolymerization. The reaction was followed by observing the o p t i c a l density changes of the solution at 271 m^ i, and was found to be e s s e n t i a l l y complete a f t e r 4 hrs. However, to ensure complete degradation, the incubation was c a r r i e d out f o r a further 20 hrs., with a drop of chloroform added as preservative. During the l a t t e r period, no attempt was made to adjust the pH of the s o l u t i o n . The reaction mixture was next heated at 100°C fo r 5 min. to p r e c i p i t a t e the enzyme protein. I t was then cooled to room temperature and centrifuged at l6,000g f o r 60 min. The supernatant solution was c o l l e c t e d and stored at -20°C f o r future use. - 3 2 -( i i ) Ion-exchange Chromatography of DNA H y d r o l y s a t e s (41) Reagents • • (a) 7M urea s o l u t i o n (b) 7M urea s o l u t i o n 1M i n NaCl (c) 0.1M T r i s b u f f e r , pH 7.8 (d) 4M NaCl s o l u t i o n (e) 1M NaCl s o l u t i o n ( f ) 2M ammonium carbonate s o l u t i o n (g) 0.5M (NH^) 2 CO^ s o l u t i o n (h) D E A E - c e l l u l o s e (Brown Co., B e r l i n , Procedure: The D E A E - c e l l u l o s e was suspended and decanted r e p e a t e d l y with d i s t . water t o get r i d of f i n e s . A t h i n s l u r r y was made of the s e t t l e d c e l l u l o s e by adding 1M NaCl s o l u t i o n (or 0.5M ammonium carbonate) and poured i n t o a chromatographic column h a l f - f i l l e d with the same s o l u t i o n . (The packing of the column was done i n the presence of s a l t to ensure a uniform bed). A pressure of 5 l b s . / s q . i n . was used f o r the packing. Excess l i q u i d was d r a i n e d from the column and f r e s h s l u r r i e s were added u n t i l the d e s i r e d bed depth was a t t a i n e d . The columns used had an i n n e r diameter of 1 cm. Those i n the c h l o r i d e form were packed to a depth of 30 cm., and those i n the carbonate form to a depth of 10 cm. - 3 3 -A f t e r packing, the columns were washed w i t h 4M NaCl or 2M (NH^) 2C0^ u n t i l the e f f l u e n t showed no a b s o r p t i o n a t 260 mjx. The s a l t s were then removed by washing w i t h d i s t i l l e d water (calcium hydroxide t e s t f o r CO " and s i l v e r n i t r a t e t e s t f o r c h l o r i d e i o n s ) . F i v e ml. of the DNA h y d r o l y s a t e (approx. 450 ug. P) were added to the DEAE column ( c h l o r i d e form) and washed i n w i t h a small amount of water. The e l u t i o n was begun wi t h a l i n e a r g r adient formed by 1 l i t e r of 7M urea i n the mixing chamber t o which 30 ml. of the t r i s - H C l b u f f e r had been added; the other chamber contained 1 l i t e r of 7M urea s o l u t i o n , 1M i n NaCl, a l s o c o n t a i n i n g 30 ml. of the b u f f e r . The flow r a t e was adjusted t o approximately 1 ml. per min. F i v e - m l . f r a c t i o n s were c o l l e c t e d by means of an automatic f r a c t i o n c o l l e c t o r ( G i l s o n , constant volume operation) equipped w i t h a spectrophotometric attachment f o r measuring the u l t r a v i o l e t a b s o r p t i o n of the e l u a t e . The o p t i c a l d e n s i t y of the i n d i v i d u a l f r a c t i o n s was a l s o measured wi t h a Z e i s s s p e c t r o -photometer at 260 mu. The sodium c h l o r i d e gradient was approximated i n a c o n t r o l experiment i n which no DNA d i g e s t was added to the column. The increase i n NaCl co n c e n t r a t i o n of the e f f l u e n t - 3 4 -100 200 300 400 E ^ l u e n t Volume. (v*e.:> 5oo FIGURE 4 : Sodium Chloride Gradient i n Ion-exchange Chromatography as Measured by the Refractive Indices of the Eff l u e n t Fractions. - 3 5 -was checked by measuring the r e f r a c t i v e indices of the e f f l u e n t fractions with an Abbe 60 refractometer (Bellingham and Stanley Inc.). The curve thus obtained i s shown i n Figure 4 . I s o l a t i o n procedure: Fractions comprising a peak were pooled and d i l u t e d with 5 volumes of d i s t i l l e d water to reduce the s a l t concen-t r a t i o n . The solution, i f necessary, was adjusted to pH 8 . I t was next run onto a DEAE-cellulose column i n the carbonate form (10 x 1 cm.) and the column washed with water u n t i l the ef f l u e n t was free of urea and inorganic ions. The o p t i c a l densities of the washings were checked to ensure no leakage of nucleotides from the column occurred. The oligonucleotide was then eluted with 2M ammonium carbonate and located among the fractions with a Zeiss spectrophotometer. The UV-absorbing fractions were combined and evaporated repeatedly i n a f l a s h evaporator with a bath temperature not exceeding o 30 C u n t i l a l l the ammonium carbonate was removed. The residue was dissolved i n the appropriate solvent f o r character i z a t i o n and other experiments. ( i l l ) Preparation of Snake Venom Phosphodiesterase Materials: Lyophillzed Crotalus adamanteus venom was obtained - 3 6 -from the Ross A l l e n Reptile Inst. Carboxymethyl-cellulose was purchased from Carl Schleicher and Schnell Co., Keene, N.H. under the trade name of Selectacel, No. 7 7 , Type 20, capacity 0.72 meq./gm. Reagents: (a) 0.05M acetate buffer, pH 4 (b) 0.05M acetate buffer, pH 6 (c) 0.2M acetate buffer, pH 6 (d) 0.5M acetate buffer, pH 6 Procedure: (a) The preparation of venom phosphodiesterase was done according to the method described by Koerner and Slnsheimer ( 3 1 ) « One gm. of crude venom was dissolved i n 60 ml. of water and the solution was l e f t standing at room temperature fo r one hour. I t was then centrifuged at 3,000g on the S e r v a l l r e f r i g e r a t e d centrifuge f o r 15 min. The supernatant solution was c o l l e c t e d and 40 ml. of 0.05M ammonium acetate buffer, pH 4, was added to i t . A l l subsequent procedures were ca r r i e d out i n the cold room at about 4°C. The solution was transferred into polyethylene tubes and 74 ml. of acetone at -20°C was added to the buffered venom solut i o n . The contents of the tube were mixed thoroughly and l e f t standing f o r 30 min,. The solution was then c e n t r i -- 3 7 -fuged at 3»000g f o r 30 min. and the supernatant solution transferred to another polyethylene tube. Fourteen ml. of acetone were added next and the solution was stored f o r 12 hours at cold room temperature. I t was then centrifuged again and the supernate was further charged with 12 ml. acetone. The f i n a l concentration of acetone i n the solution was 50$. A f t e r standing f o r 30 min., the mixture was c e n t r i -fuged, and the p r e c i p i t a t e was dissolved i n the minimum amount of d i s t i l l e d water. This solution was next dialysed against 0.05M ammonium acetate buffer at pH 6 and further p u r i f i e d by ion-exchange chromatography. (b) Chromatography on carboxymethyl (CM) c e l l u l o s e was done according to Laskowski et al.(42 ) . A CM-cellulose column, 2 x 18 cm., was eq u i l i b r a t e d with 0.05M acetate buffer, pH 6. The dialysed protein solution was then added to the column. The protein was eluted stepwise using 1) 90 ml. of 0.05M acetate buffer, pH 6; 2) 90 ml. of 0.2M acetate buffer, pH 6 and 3) 90 ml. of 0.5M acetate buffer, pH 6. The flow-rate was adjusted to about 6 ml. per hour. The fracti o n s were assayed for UV-absorbing material at 280 mu with a Zeiss spectrophotometer, and for phosphodiesterase a c t i v i t y . - 38-(iv) Assay f o r Phosphodiesterase A c t i v i t i e s (a) Qualitative assay for venom phosphodiesterase a c t i v i t y i n fract i o n s c o l l e c t e d from the CM-cellulose column. Substrate sol u t i o n : 13.6 mg. of p-nitrophenyl-thymidine-5»-phosphate i n 25 ml. of 0.1M tris-0.002M magnesium acetate buffer. Procedure: The assay method was developed by C. Mezei ( 4 3 ) . 0.1 ml. of the substrate solution was pipetted into a micro test-tube and incubated i n a water bath at 3?°C with constant shaking. One drop of solution from a given f r a c t i o n was added to the test-tube and the appearance of the yellow p-nitrophenylate i n one minute was assumed to indicate the presence of phosphodiesterase a c t i v i t y . Two protein peaks were observed i n the e f f l u e n t from the CM-cellulose column, one being eluted by the 0.05M acetate buffer, the other by the 0.2M acetate buffer. Both peaks were found to contain phosphodiesterase a c t i v i t y . This i s i n accordance with observations by Khorana and Razzell ( 4 4 ) . The peak eluted with 0.2M acetate buffer was subsequently used as the stock enzyme solution, as t h i s was found to be free of 5 *-nucleotidase a c t i v i t y . - 3 9 -(b) Quantitative assay f o r phosphodiesterase a c t i v i t y (44). In t h i s method, 1.5 ml. of substrate s o l u t i o n and 1.4 ml. of the Tris-HCl buffer were pipetted into a 1-cm. cuvette and maintained at 37°C i n the thermostated c e l l chamber of the Cary spectrophotometer. At zero time, 0.1 ml. of the enzyme solution was added and the solution mixed thoroughly. The rate of increase i n o p t i c a l density at 400 mjx due to the l i b e r a t i o n of the yellow p-nitrophenylate was a measure of enzyme a c t i v i t y . In a reaction mixture volume of 3 ml., containing 1.5 u mole of substrate, an increase i n o p t i c a l density of 1.2 units i s effected by the hydrolysis of 0.1 ji mole of the substrate. The pooled fractions from the CM-cellulose column had to be d i l u t e d 100 f o l d with the buffer before the diesterase a c t i v i t y contained i n i t could conveniently be measured. Figure 5 shows that when the o p t i c a l density was plot t e d against time, the change i n absorption varied l i n e a r l y with time for at l e a s t ten minutes. During t h i s period, the d i l u t e d enzyme preparation effected an increase of 0.039 O.D. units per minute corresponding to the hydrolysis of 1.95 mole of substrate per hour. -40-5 to 15 20 25 ?o Time mutes) FIGURE 5 Time Course of the Hydrolysis, of p-nitrophenyl-thymidine-5'-phosphate by Venom Phosphodiesterase. -41-When a standard preparation of venom phospho-diesterase (Worthington, 10 ug./ml.) was used, i t was found to hydrolyse 8.2 p. mole of substrate per hour. One ml. of the p u r i f i e d d i l u t e phosphodiesterase solution therefore corresponds to 2.k jig. of the Worthington enzyme. (c) Qualitative assay for spleen phosphodiesterase Reagents: (a) a spleen phosphodiesterase solu t i o n was given by Dr. W.E. R a z z e l l . (b) Substrate solution: a 0.03 solution of synthetic p-nitrophenyl thymidine-3*-phosphate was also a g i f t from Dr. W.E. R a z z e l l . Procedure: The assay method was s i m i l a r to that described for venom phosphodiesterase. The spleen phosphodiesterase solution had been In storage (-20°C) fo r several years, but was s t i l l found to possess considerable hydrolysing a c t i v i t y towards the substrate. Both spleen and venom phosphodiesterases were used i n the characterization of the products of DNA hydrolysates by the i n t e s t i n a l enzyme(s) as described under IV. - 4 2 -IV. Characterization of the Products of a DNA Hydrolysate (I) Degree of polymerization of the oligonucleotides Reagents: (a) Phosphomonoesterase preparation - the Worthington E . - c o l i phosphomonoesterase suspension i n ammonium sulfate was d i l u t e d 10 times with 0.01M t r i s buffer, 0.01M i n MgCl 2, pH 8.0. (b) 60$ HCIO^ (c) 5$ ammonium molybdate solution (d) Flske-SubbaRow reagent (36) Procedure: (a) Estimation of phosphomonoesterase-sensitive phosphorus. This was performed by a s l i g h t l y modified method used by Snider et a l . (45). Each of the dry oligonucleotide f r a c t i o n s i n i t i a l l y prepared as described under I l l ( i i ) was dissolved i n 1 ml. of 0.01M t r i s buffer, 0.01M i n MgClg, pH 8.0. The o p t i c a l density of the solution was measured. * Duplicate samples containing 1 O.D. unit of UV-1 O.D. unit of oligonucleotide i s defined as the amount of material which gives r i s e to an o p t i c a l density of 1 at 260 mu when present i n 1 ml. of solution and when measured i n a/ cuvette with l i g h t path = 1 cm. - 4 3 -absorbing material from each of the oligonucleotide frac t i o n s were made up to 1 ml. with t r i s buffer, pH 8 . 0 . These were transferred to 10-ml. volumetric f l a s k s . Tenyul.of the phosphomonoesterase preparation was added to each f l a s k and the mixture incubated f o r 16 hours at 3 7°C A drop of chloroform was routinely added to eliminate b a c t e r i a l contam-inations. A f t e r the digestion, the mixture was d i l u t e d to 6 ml. with d i s t i l l e d water and 0 .8 ml. of 60$ p e r c h l o r i c a c i d was added, followed by 1 ml. of molybdate solution and 0 . 4 ml. of Fiske-SubbaRow reducing reagent. The solut i o n was well shaken a f t e r each addition. The f i n a l volume was made up to 10 ml. and the o p t i c a l density of the solution was measured at 830 mu. The phosphate l i b e r a t e d by the a l k a l i n e phosphatase was determined from a previously prepared standard curve. (b) Estimation of t o t a l phosphorus. One ml. of solution containing 1 O.D. unit of ol i g o -nucleotide i n t r i s buffer was digested with 2 ml. of cone, perch l o r i c a c i d to l i b e r a t e a l l the phosphate, which was then determined as described under I ( i v ) . ( i i ) Secondary Degradation of DNA Hydrolysates by Venom and Spleen Phosphodiesterases. Materials and Reagents: (a) Sephadex G-25, purchased from Pharmacia, Upsala, Sweden. -44-(b) 0.1M T r i s buffer, 0.002M i n magnesium acetate, pH 8.9. (c) 0.01M T r i s buffer, 0.01M i n magnesium acetate, pH 8. (d) 0.18M ammonium acetate buffer, 0.06M i n EDTA, pH 5.7. (e) spleen phosphodiesterase (f) venom phosphodiesterase (g) "_E_. c o l i phosphomonoesterase (h) Standard deoxyribomononucleotides and de oxyrib onucleosides The four common deoxyribomononucleotides and the four deoxyribonucleosides i n DNA were used: d-adenylic acid, d - c y t i d y l i c acid, d-guanylic a c i d (sodium s a l t ) , d-thymidylic acid, d-adenosine, d-cytidine, d-guanosine and thymidine. These were purchased from the C a l i f . Foundation f o r Biochem. Research, L.A., C a l i f , and standard solutions were prepared by di s s o l v i n g 2 mg. of each substance i n 1 ml. water. Where necessary, d i l . HCl was added to e f f e c t complete d i s s o l u t i o n . ( i ) Chromatographic system I - isopropanol: cone. NH.OH:H 0 i n the rati© (v/v) 7:ls2. 4 2 (j) Chromatographic system II - 100 ml. water saturated butanol plus 1 ml. cone. NH.OH. Procedure: (a) Desalting of DNA hydrolysate "by gel f i l t r a t i o n (46). The Sephadex-G-25 powder was suspended i n d i s t i l l e d water to swell. I t was then washed several times to remove f i n e s . A s l u r r y (10 volumes of water to 1 v o l . of gel) was prepared. The Sephadex column (10 x 1 cm.) was then packed at atmospheric pressure. The sample to be desalted was then poured onto the column and the material eluted with d i s t i l l e d water. The fractions were co l l e c t e d and assayed f o r u l t r a -v i o l e t absorbing material at 260 mu and for chloride ions. The f r a c t i o n s containing oligonucleotides were pooled, dried i n vacuo and the residue redissolved i n the appropriate buffer. (b) Action of venom phosphodiesterase on DNA hydrolysates. The desalted DNA hydrolysate (containing 5 mg. of oligonucleotides) obtained from IV ( l i a ) was dissolved i n 5 ml. of 0.1M t r i s buffer (pH 8 . 9 ) , 0.002M i n magnesium acetate. One ml. of t h i s solution was incubated with ah excess ( 0 . 5 ml.) of venom phosphodiesterase solution at 37°C. At appropriate i n t e r v a l s of time, 100 pi. of the reaction mixture were withdrawn and mixed with 10 pi. of g l a c i a l a c e tic a c i d to stop the reaction. The mixture was then spotted onto -46-Whatman No. 40 f i l t e r paper. F i f t y ^ig. of each standard deoxyribomononucleotlde were also spotted onto the same paper. The chromatogram was then developed i n the Isopropanol: NH^ : H 20 system f o r 72 hours, employing the descending technique. The nucleotide spots were located under u l t r a v i o l e t l i g h t . These were cut out and eluted with approximately 5 ml. of 0.1N HCl (43). The eluates were evaporated to dryness and the residues taken up i n an exact volume of d i s t i l l e d water appropriate f o r the c e l l s i n which the extinctions were to be measured. The u l t r a v i o l e t l i g h t absorption curves were recorded with a Cary spectrophotometer. Blank spots approximately equal i n area to the nucleotide spots and at equal distances from the o r i g i n were cut out, eluted and read at the same wavelengths as the corresponding spots. The amount of deoxyribonucleotides i n the spots was determined from the o p t i c a l densities of t h e i r solutions at the appropriate wavelengths. Since the solvent system used did not give clean separations of d-AMP, d-CMP and TMP even a f t e r the chromatogram had been developed f o r 72 hours, the three nucleotides were eluted together and the solution analysed spectrophotometrl-c a l l y . - 4 7 -(c) Action of spleen phosphodiesterase on DNA hydrolysates (47) The desalted DNA hydrolysates from I I I ( i ) were dissolved i n 5 ml. 0.18M ammonium acetate buffer, 0.06M i n EDTA, pH 5 . 7 . One ml. of t h i s solution was Incubated with 0.2 ml. of spleen phosphodiesterase preparation f o r 16 hours at 3 7°C One drop of chloroform was added as preservative. A f t e r the digestion, the reaction mixture was boi l e d f o r 5 minutes to inactivate the enzyme, and the protein p r e c i p i t a t e removed by centrifugation at l6,000g fo r 60 min. at 0°C. The supernatant solution was then desalted by gel f i l t r a t i o n (zinc uranyl acetate reagent f o r Na +). The eff l u e n t fractions from the Sephadex column which contained the oligonucleotides were pooled and evaporated to dryness. The residue was redissolved i n 5 ml. 0.001M t r i s buffer, pH 8.0. Ten O.D. units of material were incubated with 10 jjl. of the d i l u t e phosphomono-esterase preparation, one drop of chloroform being added as preservative. The reaction was allowed to proceed f o r 16 hours at 37°C, a f t e r which the enzyme protein was removed by b o i l i n g and centrifugation. The supernatant solution was chromatographed on Whatman No. 40 f i l t e r paper. The system employed was water-saturated butanol-conc. ammonia, and the descending technique - 4 8 -was used. Standard solutions of nucleosides were also spotted onto the same chromatogram as markers. The chromatogram was developed f o r about 30 hours. The nucleoside spots were located with the a i d of u l t r a v i o l e t l i g h t . These were extracted with 0.1N HCl as described and quantitatively determined spectrophotometrically. (d) Base Composition analysis The various oligonucleotide fract i o n s ( I l l i i ) i s o l a t e d from a salmon sperm DNA hydrolysate prepared by exposing the DNA to the p u r i f i e d or the crude i n t e s t i n a l mucosal extracts were analysed for t h e i r base compositions. Each f r a c t i o n was subjected successively to enzymatic degradation by venom phosphodiesterase, spleen phosphodiesterase and E. c o l i phosphomonoesterase by a combination of methods (b) and (c) above. The base composition of each f r a c t i o n was determined by estimating the amount of each nucleoside present and converting these values to the corresponding bases. Where the base composition of native deoxyribonucleic acids was desired, the complete desalted DNA hydrolysate obtained by treatment with commercial DNase I was used as substrates f o r venom phosphodiesterase and E. c o l i phosphatase. -49-V. E f f e c t of Heat, pH, Mg + + and Inhibitors on the Rate of Digestion of DNA by the Rat I n t e s t i n a l Mucosal Extract. ( i ) E f f e c t of heat The i n t e s t i n a l mucosal extract was heated f o r 5 minutes at 100°C i n a b o i l i n g water bath. The solution was then centrifuged at l6,000g i n the S e r v a l l r e f r i g e r a t e d c e n t r i -fuge f o r 60 min. to remove the protein p r e c i p i t a t e . The supernatant solution was tested f o r deoxyribonuclease a c t i v i t y as described i n I I ( i ) . ( i i ) E f f e c t of pH Reagents: 0.1M a c e t i c a c i d solutions, 0.01M i n MgCl 2, were adjusted to the desired pH values by addition of NH^OH. Salmon sperm DNA was dissolved i n these ammonium acetate buffer solutions to a concentration of 200 ug./ml. Procedure: 3 ml. aliquots of the buffered DNA solutions were preincubated at 37°C i n the thermostated c e l l chamber of the Cary spectrophotometer. At zero time, 0.1 ml. of enzyme soluti o n was Introduced. The i n i t i a l increase i n o p t i c a l density at 260 mu with time was used as a measure of enzyme a c t i v i t y . ( i i i ) Magnesium requirements of the i n t e s t i n a l extract Rat i n t e s t i n a l mucosal DNA solution, 40yag./ml. i n - 5 0 -0 . 1 M ammonium acetate buffer, pH 6 . 5 , was used as substrate. The crude and the p u r i f i e d i n t e s t i n a l mucosal extracts were dialysed against the acetate buffer to remove the magnesium ions. The r e s u l t i n g enzyme solutions were assayed f o r deoxyribonuclease a c t i v i t y by the spectrophotometric method of Kunitz as described. (iv) E f f e c t of Inhibitors (a) Preparation of a s p e c i f i c DNase I i n h i b i t o r from the pigeon crop gland. The existence of a s p e c i f i c DNase I i n h i b i t o r i n the pigeon crop gland was f i r s t reported by Laskowski (48). The i n h i b i t o r was i s o l a t e d as follows:-Twelve pigeons were etherized, immediately decapitated and the blood drained from the wound. The crop gland was exposed, opened and the contents washed o f f . The inner l i n i n g of the gland was then peeled o f f and the tissue chopped up into very t i n y pieces. The tissue was then homogenized with 9 times i t s own volume of water i n the S e r v a l l Omni mixer with the rheostat se t t i n g on 30V. The homogenate was then centrifuged f o r 120 minutes at l6,000g i n the Serval r e f r i -gerated centrifuge. The supernatant solution was co l l e c t e d , and since repeated thawing and freezing was found to abolish the a c t i v i t y of the i n h i b i t o r protein (48), the supernate was - 5 1 -st ore d at -20°C i n 2 ml. fractio n s to be used separately. (b) Action of known DNase i n h i b i t o r s on the a c t i v i t y of the i n t e s t i n a l enzyme. Reagents: (a) EDTA solution: 0.1M i n water, s o l u b i l i z e d by the addition of a minimum amount of NaOH. When 0.1 ml. of t h i s solution was added to 3 ml. of substrate DNA solution (40 jxg DNA/ml. i n 0.1M acetate buffer, pH 6 . 5 ) , there was no s i g n i f i c a n t r i s e i n pH of the mixture. (b) 1M trisodium c i t r a t e solution (c) 1M disodium hydrogen arsenate solution (d) pigeon crop extract, as previously prepared (e) 1M MgCl-Procedure: Three ml. of substrate DNA solution was warmed to 37°C i n the c e l l chamber of the Cary spectrophotometer. At zero time, 0.1 ml. of the i n h i b i t o r solution was added,followed immediately by 0.1 ml. of enzyme solution. The change i n o p t i c a l density of the mixture with time was observed. Where i n h i b i t i o n was observed, the system was further charged with aliquots of enzyme preparation or Mg + +. The e f f e c t of preincubatlng the substrate with the i n h i b i t o r s was also studied. In these cases, the i n h i b i t o r -52-(0.1 ml.) was added to the substrate solution and the mixture incubated at 37°C f o r 30 minutes. 0.1 ml. of enzyme was then added and the deoxyribonuclease a c t i v i t y measured as before. VI. I n t r a c e l l u l a r D i s t r i b u t i o n of Deoxyribonuclease i n the Rat I n t e s t i n a l Mucosa. The tissue f r a c t i o n a t i o n procedure was performed according to de Duve et a l (49, 50). Five male Wistar rats weighing 180-200 gm. each were s a c r i f i c e d as usual and the small i n t e s t i n e removed. The lumen was flushed thoroughly with cold 0.25M sucrose and the mucosal scrapings prepared as described under 1(1). The tissu e was dispersed with approximately 3 volumes of c h i l l e d sucrose medium In a homogenizer of the Potter & Elvehjem type, consisting of a smooth-walled glass tube f i t t e d with a t e f l o n p e s t l e . The tube, kept i n cracked i c e , was given an upward run against the rot a t i n g pestle (rheostat setting at 90V) u n t i l a l l the material had been forced above the l a t t e r . The r e s u l t i n g s l u r r y was centrifuged at l,000g for 10 min. at 0°C. The p e l l e t was rehomogenized i n about the same quantity of medium and centrifuged again at l,000g f o r 10 min. This was repeated a second time and the nuclear sediment was redispersed by means of the homogenizer i n 4 v o l s , of Krebs-Ringer phosphate buffer, pH 7.8 to give a 1:4 nuclear f r a c t i o n . -53-The supernatant solutions from the three c e n t r i -fugations were combined and made up with the sucrose medium to form a 1:10 cytoplasmic extract. This was designated the 10,000g-min. supernate i n accordance with the scheme of de Duve (4-9). This cytoplasmic extract was next centrifuged at 3»300g f o r 10 minutes. The p e l l e t was washed and resuspended i n Krebs-Ringer phosphate buffer and the suspension dispersed by homogenization. The supernatant solution from the l a s t centrifugation was again centrifuged at 24 ,000g for 10 min. The sediment was col l e c t e d , washed and redispersed with 2 v o l s , of sucrose medium by homogenization. The suspension was made to 0.3N with respect to formic a c i d and adjusted to pH 7.8 with NaOH f o r the following reason: In the f i r s t f r a c t i o n a t i o n experiment, a l l the subcellular fractions were redispersed with Krebs-Ringer phosphate buffer. When the enzyme a c t i v i t i e s were assayed, i t was found that the 3»300g supernatant solution showed higher a c t i v i t i e s towards DNA at the more a c i d i c pH (4.5). This seemed to indicate the presence of DNase II a c t i v i t y i n the f r a c t i o n . In the second experiment, the p e l l e t obtained a f t e r centrifugation at 24 ,000g was therefore homogenized i n 0.3M sodium formate to create optimum io n i c conditions f o r - 5 4 -DNase I I . The three "buffered homogenates together with the 24 ,000g-supernate were then incubated at 3 7°C f o r three hours to release the enzyme into solution. They were next centrifuged at 105,000g for 60 minutes. The supernatant solutions were c o l l e c t e d and assayed for enzyme a c t i v i t i e s at pH 4 .5 and 6 . 5 . - 5 5 -RESULTS AND DISCUSSION I. Preparation, P u r i f i c a t i o n and Characterization of Substrate Deoxyribonucleic Acids. Integrity of substrate i s e s s e n t i a l f o r the study of an enzymatic reaction. Complex molecules such as deoxyribonucleic acids are es p e c i a l l y susceptible to denaturation and degradation. Even mild mistreatments can lead to separation of the strands of the double h e l i x and other states of degradation such as depurination and deamln-a t i o n of the nitrogenous bases. Therefore, i n choosing the methods of preparation and p u r i f i c a t i o n , i t i s important that while the y i e l d i s to be as quantitative as possible, the degradation and impurities must be reduced to a minimum. Several c r i t e r i a are available f o r defining the state of i n t e g r i t y of deoxyribonucleic acids. The nitrogen and phosphorus contents of a DNA sample are r e f l e c t i o n s of the state of purity of the compound, while the atomic extinction c o e f f i c i e n t with respect to phosphorus, designated (:(P), the v i s c o s i t y and spectral data of the a c i d are valuable guides to i t s state of denaturation and degradation. The detergent method of Colter et a l (33) f o r the i s o l a t i o n of DNA from tissues, when applied to the rat i n t e s t i --56-nal mucosa, yielded a white fibrous product resembling asbestos. Average of three experiments showed a y i e l d of 3^ mg. per 10 gm. wet t i s s u e . L e s l i e ( 5 D reported an average DNA content of rat i n t e s t i n a l mucosa as 129 mg. per 10 gm. fresh t i s s u e . The method of Colter et a l . f o r i s o l a t i o n therefore involves a very considerable l o s s of DNA. I t has been reported that the use of s t a i n l e s s s t e e l disintegrator caused degradation of DNA catalysed by traces of ferrous ions (52). However, the use of the S e r v a l l Omni mixer was necessary to ensure complete homo-genization of the tissue which would minimize the contamination of DNA with cytoplasmic RNA and other materials. Deproteinization of the salmon sperm DNA also afforded a product i n the form of white, tough strands of f i b r e s . The DNA yielded a highly viscous, s l i g h t l y turbid solution when dissolved i n acetate buffer, but the solution could be c l a r i f i e d by centrifugation. I t has been found that owing to the s t r u c t u r a l r e g u l a r i t i e s i n a l l deoxyribonucleic acids, the nitrogen and phosphorus contents are clos e l y s i m i l a r even fo r preparations showing large differences i n base compositions. These values cannot be u t i l i z e d f o r precise d e f i n i t i o n of composition, but they are useful as indications of the degree of purity of the samples. - 5 7 -In studying the action of i n t e s t i n a l deoxyribo-nuclease a c t i v i t y , DNA from three d i f f e r e n t tissues were used as substrates. These had been characterized and the a n a l y t i c a l data are summarized i n Table I. As can be seen, the N and P values and also the atomic N/P r a t i o s f o r the rat i n t e s t i n a l mucosal and f o r the commercial c a l f thymus DNA are i n good agreement with values reported i n l i t e r a t u r e f o r DNA preparations from tissues of various species ( 5 3 ) . The salmon sperm preparation showed too high a value f o r nitrogen and too low a value f o r phosphorus. This i s i n t e r -preted as Indicating protein contamination. The £(P) values for the rat DNA and the c a l f thymus DNA are also within the normal range observed with cautiously i s o l a t e d DNA's from various sources ( 6 , 5 0 0 - 7 , 0 0 0 ) . According to Chargaff ( 5 3 ) , 6(P) values higher than 7 , 2 0 0 are i n d i c a t i v e of denaturation. The £(P) value f o r the deproteinized salmon sperm DNA preparation of 7 t 3 1 0 would be a sign of degradation. However, as pointed out e a r l i e r , the salmon sperm DNA appears to be contaminated with protein, which shows a cert a i n amount of residual absorption at 260 mu and consequently giving r i s e to a higher atomic e x t i n c t i o n . In the l i g h t of these data, a l l the three DNA preparations examined were considered to be free of extensive denaturation and degradation. T A B L E I E l e m e n t a r y C o m p o s i t i o n a n d Some O t h e r P r o p e r t i e s o f t h e DNA P r e p a r a t i o n s U s e d R e l a t i v e v i s c o s i t y B a s e C o m p o s i t i o n w i t h r e s p e c t N i t r o g e n P h o s p h o r u s A t o m i c t o s o l v e n t S o . u r c e % % N/P r a t i o £ ( P ) ( l m g . / m l . ) r a t i n t e s t i n a l 1 4 . 8 9.1 3.64 6 7 7 0 2 . 5 1 2 8 . 6 2 0 . 3 2 3 . 3 2 7 . 8 m u c o s a s a l m o n s p e r m 1 6 . 8 7.4 5.03 7 3 1 0 3 . 0 3 2 7 . 7 2 2 . 3 2 3 . 7 2 6 . 3 c a l f t h y m u s 1 5 . 2 9.2 3.67 6 9 7 0 3.87 2 8 . 6 1 9 . 8 2 1 . 3 3 0 . 3 - 5 9 -The r e l a t i v e v i s c o s i t i e s of the DNA solutions were found to vary from batch to batch. Those reported i n Table I are averages of values that were observed most consistently. The commercial c a l f thymus preparation was designated "highly polymerized" by the manufacturer and, f o r the purpose of comparison, was used as a standard. The r e l a t i v e v i s c o s i t y values f o r the rat i n t e s t i n a l and the salmon sperm preparations were considered s a t i s f a c t o r y . While i t was Impossible to define an absolute standard of i n t e g r i t y f o r any DNA sample, the three preparations examined were considered to be r e l a t i v e l y free of denaturation and degradation and were deemed sa t i s f a c t o r y for use as substrates i n enzymatic studies. I I . Preparation and P u r i f i c a t i o n of Deoxyribonuclease from Rat I n t e s t i n a l Mucosa, ( i ) E f f e c t of Extracting Media It has been found that deoxyribonuclease i s extremely susceptible to destruction by the action of p r o t e o l y t i c enzymes ( 5 * 0 . Thus, a fresh extract of pancreatic deoxyribo-nuclease was found to lose i t s a c t i v i t y rapidly when the p r o t e o l y t i c enzymes became activated. Moreover, a DNase I i n h i b i t o r had been found i n many tissues (e.g. kidney) and DNase a c t i v i t y was only revealed a f t e r the i n h i b i t o r was -60-destroyed (55)• A variety of solvents was tested i n an attempt to f i n d one most suitable f o r extraction of the enzyme. Two of these were found to be promising: Krebs-Ringer phosphate buffer and physiological s a l i n e . Figure 6 compares the deoxy-ribonuclease a c t i v i t i e s i n the two extracts and Table II summarizes t h e i r protein content and the s p e c i f i c a c t i v i t y . Krebs-Ringer phosphate buffer was the f i r s t medium tested. I t yielded an extract which contained considerable deoxyribonuclease a c t i v i t y . This a c t i v i t y was stable f o r several months when stored at -20°C and i t was also not affected a f t e r repeated thawing and freezing. When phys i o l o g i c a l saline was used as the extracting medium, i t was found that the s p e c i f i c a c t i v i t y of t h i s extract was a l i t t l e b i t higher than that of the Krebs-Ringer phosphate buffer, i n d i c a t i n g that the buffered enzyme preparation contained more extrenuous protein. However, the deoxyribonuclease a c t i v i t y i n the physiological saline extract diminished with storage, probably due to denaturation of the enzyme protein i n the unbuffered medium. Physiological saline was therefore deemed unsuitable as an extracting s o l u t i o n . When a 1M NaCl solution was used f o r extraction, i t was found that addition of the enzyme extract brought about the p r e c i p i t a t i o n of a white fibrous material out of solut i o n . -61-FIGURE 6 : Comparison of Bate of Degradation of DNA by Crude Extracts of Rat I n t e s t i n a l Mucosa. - 6 2 -TABLE I I : S p e c i f i c A c t i v i t y of Deoxyribo-nuclease i n Crude Extracts of Rat I n t e s t i n a l Mucosa. Extracting Medium Optical density of protein solution (280 mu) Cone. pr o t e i n a ) (mg./ml.) Sp e c i f i c a c t i v i t y DNase of extract a c t i v i t y Units ("b) AO.D./min. /mg. (260 mu) protein Krebs Ringer 11.55 9 . 6 0.00467 0.00048 phosphate buffer, pH 7 .8 Normal Saline 7.63 6.3 0.0075 0.0012 (a) O.D. (280 mu) of 1.2 = 1 mg. protein per ml. (20) (b) 1 unit of DNase a c t i v i t y i s the amount of enzyme capable of bringing about an o p t i c a l density change of 1.0 at 260 mu i n one minute under the experimental conditions as defined i n the text. - 6 3 -This p r e c i p i t a t e was analysed q u a l i t a t i v e l y and found to contain both phosphorus and deoxyribose (diphenylamine r e a c t i o n ) . The rest of the reaction mixture, when analysed spectrophotometrically, showed no absorption peak i n the u l t r a v i o l e t region except f o r a s l i g h t shoulder at 280 rji. I t has been found by several authors that DNase I i s i n h i b i t e d when the sodium chloride concentration i s increased above 0.05M (20 , 5 6 ) , and 0.5M NaCl i n the medium i n h i b i t e d about 65$. I t was therefore considered that 1M NaCl did not constitute a good extracting solvent for deoxyribonuclease, at l e a s t f o r the i n t e s t i n a l mucosa. In one experiment, the tissue a f t e r a preliminary extraction with Krebs-Ringer phosphate buffer, was homogenized and re-extracted with fresh buffer. This second extract was also found to contain deoxyribonuclease a c t i v i t y which was stable to storage. This experiment showed that at l e a s t a part of the deoxyribonuclease a c t i v i t y i n the rat i n t e s t i n a l mucosa was located i n t r a c e l l u l a r l y . ( i i ) Fractionation with Ammonium Sulfate In Table I I I , the data f o r the p u r i f i c a t i o n of the i n t e s t i n a l mucosal DNase (Krebs-Ringer phosphate buffer extract) are presented. In the Table, the symbol 5$S i s used to designate the -64-T A B L E I I I P u r i f i c a t i o n o f I n t e s t i n a l M u c o s a l D e o x y r i b o n u c l e a s e E x t r a c t b y F r a c t i o n a t i o n w i t h A m m o n i u m S u l f a t e . O p t i c a l S p e c i f i c D e n s i t y o f A c t i v i t y P r o t e i n C o n c e n t r a t i o n A c t i v i t y u n i t s / m g P r o t e i n S o l u t i o n o f p r o t e i n ( a > O.D./ p r o t e i n F r a c t i o n ( 2 8 0 mp.) m g . / m l . m i n . x l O 3 x 1 0 3 T o t a l . E x t r a c t 5 % S i b ) 1 1 . 5 5 9.6 4.67 0 . 4 8 4 . 2 1 3.5 4.56 1.3 1 0 % S 1.72 1.43 4 . 1 0 2.9 1 5 % S 0.9 5 0 . 7 9 4 . 0 8 5.2 2 0 % S 0. 6 5 0 . 5 4 1.63 3.1 2 5 % S 0.27 0.22 1.33 6.0 3 0 % S 0 . 2 0 0.17 0. 5 3 3.1 5 % P ( c > 7 . 4 4 6.2 0 0 1 0 % P 2. 1 5 1.8 0 0 1 5 % P 0 . 8 2 0 . 6 8 0 0 2 0 % P 0 . 3 6 2 0 . 3 0 3.0 10 2 5 % P 0 . 1 1 0 . 0 9 0 .37 2.9 3 0 % P 0 . 0 8 0.07 0.20 0 . 4 8 O p t i c a l d e n s i t y o f 1.2 ( 2 8 0 mp) = 1 mg. p r o t e i n / m l . ( 2 0 ) 5 % S = s u p e r n a t a n t f r a c t i o n a t 5 % s a t u r a t i o n o f a m m o n i u m s u l f a t e . 5 % P = s o l u t i o n o b t a i n e d b y r e d i s s o l v i n g t h e p r o t e i n p r e c i p i t a t e d a t 5 % s a t u r a t i o n o f a m m o n i u m s u l f a t e . -65-supernatant solution obtained a f t e r centrlfuging o f f the pr e c i p i t a t e brought down at an ammonium sulfate saturation of 5$. The p r e c i p i t a t e was redissolved i n phosphate buffer and the r e s u l t i n g solution designated "5$PM. Nitrogen determination of the various protein solu-tions was found to give e r r a t i c r e s u l t s , presumably due to incomplete removal of the ammonium s a l t by d i a l y s i s . The protein concentration i n each solution was therefore approxi-mated spectrophotometrically at 280 mu, the o p t i c a l density being about 1.2 per mg. of protein per ml. (20). I t can be seen from Table III that the deoxyribonucle-ase a c t i v i t y was brought down separately at two concentrations of ammonium sulfate during f r a c t i o n a t i o n . The f i r s t portion was pr e c i p i t a t e d at 20$ saturation, the other at 30$. The 20$P f r a c t i o n was found to contain 64$ of the a c t i v i t y i n the whole extract. Whereas^ the 25$S f r a c t i o n s t i l l retained 28$ of the t o t a l a c t i v i t y , the 30$S contained only about 10$. However, deoxyribonuclease a c t i v i t y was not observed i n the 30$P f r a c t i o n (less than 5$ of the t o t a l a c t i v i t y i n the crude e x t r a c t ) . In a repeat experiment, s i m i l a r findings were observed. The deoxyribonuclease a c t i v i t y brought down at 30$ saturation of ammonium sulfate was l o s t . A l l attempts to recover the enzymatic a c t i v i t y i n t h i s f r a c t i o n , such as rec o n s t i t u t i o n with the supernate and d i a l y s i s to remove excess -66-s a l t s were unsuccessful. The reason for the loss of activity-i s not clear; i t i s possible that the high s a l t concentration might have i r r e v e r s i b l y denatured the enzyme. Later experiments were therefore c a r r i e d out using both the crude and the p a r t i a l l y p u r i f i e d enzyme preparations and comparisons between the two were made. It can be seen from the s p e c i f i c a c t i v i t y of the 20$P f r a c t i o n that only a modest 20 f o l d p u r i f i c a t i o n of the enzyme was achieved by ammonium sulfate f r a c t i o n a t i o n . The observed ammonium sulfate concentration at which deoxyribonuclease a c t i v i t i e s were pr e c i p i t a t e d was d i f f e r e n t from that f o r pancreatic DNase I reported by Kunitz (57). Kunitz observed that the bulk of the DNase I a c t i v i t y from beef pancrease was pre c i p i t a t e d at 30$ saturation. The difference was not surprising since the extraction of the pancreatic enzyme was performed under extremely a c i d i c conditions (0.25N HgSO^) to i n h i b i t p r o t e o l y t i c a c t i v i t i e s . Moreover, the high concentration of divalent ions (which were found to be capable of binding DNA)(58) i n the Krebs-Ringer phosphate buffer might have contributed towards the l e s s e r s o l u b i l i t y of the enzyme i n the ammonium sulfate solution. I I I . Enzymatic Degradation of Deoxyribonucleic Acids and Analysis of the Products. -67-( i ) K i n e t i c s of Reaction The degradation of deoxyribonucleic acids by the rat i n t e s t i n a l enzyme preparations, whether measured spectro-photometrically or by v i s c o s i t y determinations, was character-ized by an i n i t i a l constant reaction rate which gradually decreased a f t e r a period of time. Two t y p i c a l examples were i l l u s t r a t e d i n Figures 7 and 8. I t can be seen that the rate of increase of u l t r a v i o l e t l i g h t absorption or the rate of decrease i n r e l a t i v e v i s c o s i t y of the reaction mixture vary l i n e a r l y with time at the s t a r t of the reaction. During t h i s period, when the substrate concentration was high, the reaction was of zero-order. When the substrate concentration was reduced, as when the reaction had been permitted to proceed f o r a period of time, the reaction approximated one that was f i r s t - o r d e r i n substrate concentration, as can be seen from the gradually decreasing rate of reaction. The rate of change i n v i s c o s i t y of the solution i s also seen to be much more rapid than the rate of change i n the absorption of u l t r a v i o l e t l i g h t . This i s interpreted as i n d i c a t i n g that the i n t e s t i n a l DNase i s of the endonuclease type, chopping the DNA molecules into large fragments s t i l l containing considerable double h e l i c a l structure. Similar observations had been made by Kunitz (20) for DNase I and by Jungner et a l (59). -68-FIGURE 7 : Time Course of the Degradation of DNA by Crude I n t e s t i n a l DNase Extract as Measured by the Spectrophotometrie Method of Kunitz. -69-FIGURE 8 : Time Course of the Degradation of DNA by Crude I n t e s t i n a l DNase Extract as Measured by Viscosimetry. -70-( i i ) Ion-exchange Chromatography and Characterization of the Products i n DNA Hydrolysates. Chromatographic separations were conducted with DEAE-cellulose columns (chloride form). To e f f e c t good re s o l u t i o n of the oligonucleotides, 7M urea was incorporated i n t o the eluant to remove the secondary binding forces which existed between the ion-exchanger and the purine and pyrimid-ine derivatives (41). The r e s u l t s of these studies are shown i n Figures 9 to 14. DEAE-cellulose, being an anion exchanger, separates oligonucleotides according to the number of net negative charges they carry, i e . according to the degree of polymerization, the smaller nucleotides being eluted f i r s t . As can be seen, each e l u t i o n p r o f i l e contains 7 distinguishable peaks. Peaks I, II and III are usually f a i r l y well separated from each other, but the peaks containing the higher oligonucleotides show consider-able overlap. When the digestion was performed with the crude enzyme extract (Figures 9-11) I t was seen that the size of a given peak (corresponding roughly to the amount of oligonucleo-tides present) varies considerably from one DNA to another. Thus peak VII obtained f o r c a l f thymus DNA i s d e f i n i t e l y much smaller than the corresponding peaks f o r the rat i n t e s t i n a l or the salmon sperm DNA. On the other hand, when the p a r t i a l l y I -o 200 3 0 0 Vo(u.w\_ FIGURE 9 Ion-exchange Chromatography of the Oligonucleotides i n Rat I n t e s t i n a l Mucosal DNA Hydrolysate Formed by.Exposing the DNA to a Crude I n t e s t i n a l DNase Preparation. The Chromatography was Done on a DEAE-cellulose Column Developed with a Linear Gradient of NaCl to 1M. I ro i too FIGURE 10 2oo 3 0 0 £fy\uzn£ VOUL»Y\«_ WV£. Ion-exchange Chromatography of the Oligonucleotides i n Salmon Sperm DNA Hydrolysate Formed by Exposing the DNA to a Crude I n t e s t i n a l DNase Preparation. The Chromatography was Done on a DEAE-cellulose Column Developed with a Linear Gradient of NaCl to 1M. 0 - 5 0 ioo 200 300 4oo 500 600 E ^ t u e Y v t - Volume. w\£. FIGURE 11 : Ion-exchange Chromatography of the Oligonucleotides i n C a l f Thymus DNA Hydrolysate Formed by Exposing the DNA to a Crude I n t e s t i n a l DNase Preparation. The Chromatograply was Done on a DEAE-cellulose Column Developed with a Linear Gradient of NaCl to 1M. E-f^lwer>t: V o l u m e v*£. FIGURE 12 : Ion-exchange Chromatography of the Oligonucleotides i n Rat I n t e s t i n a l Mucosal DNA Hydrolysate Formed by Exposing the DNA to the 2C$P Enzyme. The Chromatography was Done on a DEAE-cellulose Column Developed with a Linear Gradient of NaCl to 1M. o-5 0 l o o 20o 3oo 4 0 0 5oo Goo FIGURE 13 : Ion-exchange Chromatography of the" O l i g o n u c l e o t i d e s i n Salmon Sperm DNA Hydrolysate Formed by Exposing the DNA to the 2C$P Enzyme. The Chromatography was Done on a DEAE-cellulose Column Developed w i t h a L i n e a r Gradient of NaCl to 1M. FIGURE lk : Ion-exchange Chromatography of the O l i g o n u c l e o t i d e s i n C a l f Thymus DNA H y d r o l y s a t e Formed by Exposing the DNA to the 20%? Enzyme. The Chromatography was Done on a D E A E - c e l l u l o s e Column Developed w i t h a L i n e a r G r a d i e n t of NaCl to 1M. -77-p u r i f i e d enzyme was used, the pattern of peak size and d i s t r i b u t i o n i s remarkably constant from one species of DNA to another. The significance of these are discussed l a t e r . The peaks were characterized by the two c r i t e r i a : (a) the r a t i o of phosphomonoesterase-sensitive phosphorus to t o t a l phosphorus and (b) the decreasing £(P) r a t i o with increasing s i z e . The base composition of peaks I and VII obtained from the digests of salmon sperm DNA were analysed. The amount of nucleotide present i n the various peaks was also determined from i t s phosphorus content. Table IV summarizes these r e s u l t s . The ^(P) values and the r a t i o s of t o t a l phosphorus to phosphomonoesterase-sensitive phosphorus indicated that the degradation products of deoxyribonucleic acids were indeed eluted according to t h e i r s i z e s . The mononucleotides were eluted f i r s t (peak I) followed by the d i - , t r i - , etc. nucleo-t i d e s . The overlapping and coincidence of the peaks contain-ing the higher oligonucleotides invar i a b l y caused some contamination and the t o t a l P/PME-P r a t i o s obtained of these peaks were considerably removed from i n t e g r a l values. When the percentage of a p a r t i c u l a r oligonucleotide was examined, some i n t e r e s t i n g r e s u l t s were observed. The crude enzyme extract i n v a r i a b l y gave r i s e to more mononucleo-tid e s and large oligonucleotides (degree of polymerizationy- 7).» T A B L E I V Some A n a l y t i c a l D a t a o f DNA H y d r o l y s a t e s DNA E n z y m e P r e p a r a t i o n U s e d P e a k t ( p ) T o t a l P D e g r e e o f P o l y m e r i -z a t i o n % o f o l i g o -n u c l e o t i d e • p h o s p h o r u s B a s e C o m p o s i t i o n P M E P * a ^ A C G T I 1 0 6 3 0 1. 2 1 1.2 1 4 . 2 8. .2 7.9 6 9 . 7 I I 9 0 2 0 2. 1 2 3.5 - - -I I I 8 5 3 0 3. 3 3 9.4 — - — S a l m o n C r u d e I V 8 2 0 0 4. 2 4 1 0 . 2 - - -V 7 8 5 0 5. 2 5 7.6 - - -V I 7 6 1 0 6. 6 6 7.4 - - -V I I 7 4 6 0 7. 4 7 6 1 . 7 2 8 . 2 2 1 . .9 2 3 . 2 2 6 . 7 I 9 8 9 0 1. 1 1 1.04 1 0 . 5 10. .4 4.9 7 4 . 2 S p e r m I I 9 1 3 0 2. 2 2 4.1 - - -I I I 8 6 4 0 3. 1 3 1 0 . 9 - - -2 0 % P I V 8 1 2 0 4. 3 4 7.2 - - -DNA V 7 7 8 0 5. 4 5 1 0 . 8 - - -V I 7 5 1 0 6. 2 6 8.5 - - -V I I 7 3 8 0 7. 2 7 5 7 . 5 2 9 . 8 4. .2 3 5 . 9 3 0 . 1 I 9 9 4 0 1. 2 1 5.8 I I 9 1 8 0 2. 2 2 4.1 R a t I I I 8 5 4 0 3. 2 3 8.3 I n t e s t i n a l I V 7 9 9 0 4. 5 4 7.6 M u c o s a l V 7 5 7 0 5. 0 5 7.0 DNA V I 7 1 6 0 6. 5 6 7.6 V I I 6 7 1 0 7. 5 7 5 9 . 6 T A B L E I V ( C o n t i n u e d ) E n z y m e D e g r e e o f % o f o l i g o -P r e p a r a t i o n P o l y m e r i - n u c l e o t i d e DNA U s e d P e a k £ ( P ) PM -P z a t i o n p h o s p h o r u s I 9 9 5 0 1.0 1 2.4 R a t I I 9 2 3 0 2.1 2 5.2 I n t e s t i n a l 2 0 % P I I I 8 6 3 0 3.2 3 1 1 . 2 M u c o s a l I V 8 1 4 0 4.2 4 8.4 DNA V 7 6 8 0 5.3 5 1 0 . 1 V I 7 2 6 0 6.3 6 1 0 . 0 V I I 6 9 1 0 7.4 7 5 2 . 7 I 1 0 2 3 0 1.1 1 1 0 . 5 5 I I 9 2 4 0 2.4 2 7.0 I I I 8 5 1 0 2.9 3 1 5 . 6 C a l f C r u d e I V 8 0 3 0 4.3 4 1 2 . 5 V 7 7 8 0 5.7 5 1 2 . 0 V I 7 5 1 0 6.3 6 1 1 . 6 V I I 7 0 5 0 7.6 7 3 1 . 7 5 I 9 9 7 0 1.1 1 2.2 I I 9 4 1 0 2.1 2 3.8 I I I 8 9 0 0 3.1 3 9.5 T h y m u s 2 0 % P I V 8 4 3 0 4.3 4 1 5 . 4 V 7 8 2 0 5.2 5 9.6 V I 7 3 3 0 6.4 6 1 0 . 2 V I I 7 1 1 0 7.4 7 4 8 . 3 ( a ) PME-P = P h o s p h o r u s l i b e r a t e d b y p h o s p h o m o n o e s t e r a s e . -80-but fewer dinucleotid.es than the p u r i f i e d extract, regardless of the source of the DNA preparation. This i s Interpreted to mean that while the p u r i f i e d extract contained one DNase, the crude extract might contain more than one enzyme possessing deoxyribonuclease a c t i v i t y . During f r a c t i o n a t i o n studies, the i n t e s t i n a l mucosal enzyme had been observed to p r e c i p i t a t e out at two d i f f e r e n t s a l t concentrations. This together with the f a c t that the el u t i o n p r o f i l e s of crude-enzyme digests of DNA lack any uniformity from one species of DNA to another were early clues already pointing to the existence of more than one deoxyribonuclease i n the mucosal t i s s u e . When these percentage values were analysed i n the l i g h t of the base composition of the various DNA samples (see Table I ) , i t was found that the higher the A-T content i n the DNA, the more mononucleotides were formed. Thus with salmon sperm DNA, which contains 54$ A-T, the mononucleotide f r a c t i o n constituted only about 1$ of the t o t a l oligonucleotide. With the r at i n t e s t i n a l DNA, (A-T content 5 6 . 4 $ ) , the mononucleotide f r a c t i o n amounted to 2.2$ of the t o t a l oligonucleotide when the p u r i f i e d enzyme was used for digestion, and to 5*8$ when the crude enzyme preparation was used. These observations seem to indicate that the i n t e s t i n a l deoxyribonuclease, or at l e a s t the M20$P" enzyme, shows a preference towards a phosphodiester linkage next to a thymine base. -81-To further c l a r i f y t h i s point, the base composition of the mononucleotide peak and also of peak VII was analysed, and i t was found that thymidylic a c i d constituted approximately 70$ of the t o t a l mononucleotides. The high oligonucleotide f r a c t i o n showed no unusual base frequency features when the crude enzyme was used, except perhaps a s l i g h t preponderance of purines. But i n the M 20$P H digest, the concentration of d - c y t i d y l i c a c i d was extremely low. This f r a c t i o n was also found to be r i c h i n purines. One possible explanation f o r these findings i s that the presence of pyrimidines i s required for hydrolysis by the "20$P" enzyme, and long chains of purines are consequently r e s i s t a n t to attack. Schmidt (60) reported that i n DNase I digests of calf-thymus DNA, thymidylic a c i d counted f o r 50-60$ of the t o t a l mononucleotides formed. No base preference has been observed thus f a r f o r DNase I I . In view of the above findings, i t seems l i k e l y that the , ,20$P" enzyme i s DNase I. ( i i i ) Secondary Degradation of DNA Hydrolysates by phosphodiesterases. (1) Venom phosphodiesterase The rate of degradation of salmon sperm DNA hydro-lysates by venom phosphodiesterase was determined by measuring ( i ) the deoxyguanylic a c i d and ( i i ) the composite dAMP-d-CMP-TMP -82-f r a c t i o n formed by venom phosphodiesterase. The deoxyribo-nucleases used were the crude enzyme preparations, the 20$P f r a c t i o n and commercial DNase I. Results of these studies are shown i n Figure 15. I t has been reported (61) that oligonucleotides bearing 5*-phqsphomonoester groups are hydrolysed by venom phosphodiesterase much more rapidl y than the corresponding members lacking such groups. The rate of release of mononucleo-tides by venom phosphodiesterase from a DNA hydrolysate can therefore serve to indicate the r e l a t i v e abundance of 5 1 -phosphomonoester linkages i n the products. Since DNase I gives r i s e exclusively to oligonucleotides bearing only 5'-¥0^ groups (62), a DNase I digest of DNA was used as a standard of comparison. As can be seen from Figure 16, the rates of release of both d-GMP and (dAMP-dCMP-TMP) from the W20$P M digest were comparable to those from a DNase I digest, while the crude enzyme digest was much more re s i s t a n t to attack by venom phosphodiesterase. These findings are interpreted to mean that the 20$P digest contains much more oligonucleotides terminating i n 5*-phosphate ends than the crude enzyme extract. Moreover, when the p u r i f i e d enzyme digest was used, the presence of undegraded oligonucleotide (presumably bearing 3 ,-P0^ ends) was not detected on the chromatogram a f t e r the secondary digestion -83-20 4o 60 So FIGURE 15 ; Rate of Release of Mononucleotides by Snake Venom Phosphodiesterase from Salmon Sperm DNA Hydrolysate. The Optical Density of the d-GMP Solution was Measured at 2^5 mu, that of (dAMP+d-CMP+TMP) at 26? mu. ' FIGURE 16 : E f f e c t of pH on Deoxyribonuclease A c t i v i t y of Rat I n t e s t i n a l Mucosal Extracts. - 8 5 -had proceeded f o r over 60 minutes. These findings indicated the absence of any detectable amount of oligonucleotides bearing 3'-phosphomonoester groups i n the DNA hydrolysate formed by the 20$P enzyme. This Is i n agreement with the previous proposition that the 20$P enzyme i s DNase I, whereas the crude enzyme extract, giving r i s e to considerable amounts of oligonucleotides lacking a phosphate group at the 5 ' - p o s i t i o n , also contains DNase I I . (2) Spleen phosphodiesterase The amount of nucleosides obtained from the successive secondary degradations of salmon sperm DNA hydrolysate by spleen phosphodiesterase and E. C o l i phosphomonoesterase are presented i n Table V. TABLE V: Nucleoside Content of mixture obtained by successive degradation of salmon sperm DNA with DNase, spleen phosphodiesterase and E. C o l i phosphomonoesterase. DNase Enzyme Used * Nucleoside jx mole adenosine cytidine guanosine thymidine DNase II 28.9 24.3 25.1 29.4 Crude i n t e s t i n a l prep. 9.2 10.5 7.3 8.7 20$P 0 0 0 0.4 Ten O.D. units of the DNase-spleen phospho-diesterase digest used f o r degradation by phosphomonoesterase. - 8 6 -Spleen phosphomonoesterase degrades only those oligonucleotides which carry a 5 '-hydroxyl group (4-7). Prom Table V, i t can be seen that about 30$ of the oligonucleotides present i n the DNA hydrolysate by the crude enzyme preparation did not terminate i n a 5'-phosphomonoester group and was therefore vulnerable to spleen phosphodiesterase. The presence of t h i s type of oligonucleotides can only be explained by assuming that the crude i n t e s t i n a l extract contains DNase I I , which i s known to produce products ending i n 3'-phosphate groups ( 3 1 ) . The 20$P enzyme d i d not y i e l d any detectable amount of t h i s type of oligonucleotides and d i d not therefore contain any DNase a c t i v i t y . That some thymidine was observed when the 20$P enzyme was used for degrading the DNA i s probably due to the presence of some thymidylic a c i d i n the DNase hydrolysate. ++ IV. E f f e c t of Heat, pH, Mg and some Inhibitors on the Rat I n t e s t i n a l Deoxyribonuclease. The i n t e s t i n a l deoxyrlbonuclease a c t i v i t y i n the ra t i n t e s t i n a l mucosa was found to be h e a t - l a b i l e . Heating f o r 5 minutes at 100°C completely abolished the a c t i v i t y of the extract. Both the crude and the 20$P enzyme preparations showed pH optima at about 6 . Figure 16 shows the r e s u l t s obtained. A c t i v i t i e s at various pH are expressed in/terms of - 8 7 -the slopes of the straight l i n e s obtained i n p l o t t i n g increase i n o p t i c a l density versus time. The 20$P enzyme was found to show an absolute require-ment f o r Mg*"*". When the enzyme solution was dialysed against buffer to remove the Mg + + o r i g i n a l l y present i n the medium, i t ++ l o s t i t s a c t i v i t y . But a c t i v i t y was restored by adding Mg to the reaction mixture. Anions such as c i t r a t e , arsenate and EDTA which remove magnesium were a l l found to i n h i b i t DNase a c t i v i t y . EDTA was p a r t i c u l a r l y e f f e c t i v e . A concentration of I . 6 7 x 10"^ M i n h i b i t e d 70$ when the Mg4"4" concentration was - 2 1 x 10 M. When EDTA was preincubated with the substrate DNA solutio n (containing Mg4"4"), the i n h i b i t o r y action was even more -4 pronounced, 1.67 x 10 M EDTA completely i n h i b i t i n g the enzyme. Doubling the Mg resulted i n a s l i g h t recovery of enzyme a c t i v i t y to about 10$ of that of the o r i g i n a l extract. The i n h i b i t o r y power of c i t r a t e and arsenate was not as pronounced as that of EDTA, but s t i l l considerable. In eit h e r case, preincubation with DNA substrate did not enhance the i n h i b i t o r y power. The e f f e c t of these two i n h i b i t o r s was also decreased by increasing the concentration of Mg + +. The pigeon crop extract was found to be an extremely potent In h i b i t o r of the 20$P enzyme. Its e f f e c t was not reversed by increasing Mg4"4". -88-When the crude enzyme extract was s i m i l a r l y tested, i t was found that d i a l y s i s to remove Mg + + decreased the deoxyribonuclease a c t i v i t y to about 2 0 $ . This residual a c t i v i t y was not diminished by further d i a l y s i s , i n d i c a t i n g that i t did not require Mg + + f o r a c t i v i t y . Arsenate, c i t r a t e and EDTA also d i d not a f f e c t t h i s a c t i v i t y , i n accordance with the findings of Stewart and Zbarsky ( 3 2 ) . These studies showed that the DNase a c t i v i t y i n the 20$P f r a c t i o n resembles the pancreatic DNase I i n i t s pH optimum and i o n i c requirements (38, 5 7 ) . They also served to demonstrate that the crude enzyme extract contains i n addition a d i f f e r e n t DNase which i s independent of Mg + + and unaffected by c i t r a t e , arsenate etc. and resembles DNase II i n these respects. V. I n t r a c e l l u l a r D i s t r i b u t i o n of Deoxyribonucleases i n the Rat I n t e s t i n a l Mucosa. E a r l i e r evidence obtained i n the course of t h i s research favoured the idea that the rat i n t e s t i n a l mucosa enzyme was DNase I i n nature. Since DNase I i s abundant i n pancreatic j u i c e , i t was feared that the enzyme a c t i v i t y i s o l a t e d might be due to contamination, both from adsorbed pancreatic enzyme and from i n t e s t i n a l b a cteria. I t was therefore deemed desirable to esta b l i s h the i n t r a c e l l u l a r l o c a t i o n and d i s t r i b u t i o n of the enzyme a c t i v i t i e s i n the subc e l l u l a r f r a c t i o n s . -89-Th e i n t r a c e l l u l a r l o c a t i o n of the enzyme was indicated by the fact that a f t e r a f i r s t extraction by buffer of a whole c e l l preparation was made, a second extraction done on the homogenized tissue yielded a preparation only s l i g h t l y l e s s active than the whole c e l l extract. When tissue f r a c t i o n a t i o n was next performed on the i n t e s t i n a l mucosa, 3 f r a c t i o n s were obtained: a nuclear f r a c t i o n (10,000g-min. p r e c i p i t a t e , i n accordance with the terminology of de Duve et al.)(49), the 33,OOOg-min. p e l l e t and the 33»OOOg supernatant sol u t i o n . I t was found that 48$ of the t o t a l DNase a c t i v i t y was located i n the 33>000g-min. p e l l e t (mitochondrial); 38.7$ i n the supernate and 3.3$ i n the nuclear f r a c t i o n . Moreover, the enzyme present i n the 33»OOOg-min. supernatant solution was found to be more active at pH 4.5 than at pH 6.0. The f r a c t i o n a t i o n was repeated and the 33»OOOg-min. supernatant solution was further separated into a f r a c t i o n termed " l i g h t mitochondrial" f r a c t i o n and a f i n a l supernatant solut i o n by centrifugation at 24,000g for 10 minutes. The enzyme a c t i v i t i e s of these fractions are summarized i n Table VI. The recovery of a c t i v i t y during the f r a c t i o n a t i o n was very s a t i s f a c t o r y (94.6$). As i n the previous experiment, the " l i g h t mitochondrial f r a c t i o n " was found to be more active - 9 0 - A TABLE VI: I n t r a c e l l u l a r d i s t r i b u t i o n of deoxyribonuclease i n rat i n t e s t i n a l mucosa. Fraction % A c t i v i t y Total Extract 100 Nuclear 4.3 Heavy mitochondrial 48.1 Light mitochondrial 38.4 Cytoplasmic 3*8 at pH 4.5 than 6 . 0 . This seems to indicate DNase I I . However, the products of DNA digestion by t h i s enzyme f r a c t i o n had not been characterized. 0th et a l . (63) reported that i n the normal l i v e r c e l l , both DNase I and DNase II were associated mainly with mito-chondria. De Duve et a l . (49) found that DNase I occurred i n l i v e r c e l l mitochondria, but DNase II was associated with the lysosomes. Lysosomes are p a r t i c l e s intermediate i n size between mitochondria and microsomes and would be expected to be c e n t r i -fuged down at 24,000g f o r 10 minutes. In the l i g h t of t h i s , i t seems very l i k e l y that the deoxyribonuclease a c t i v i t y i n the " l i g h t mitochondrial" f r a c t i o n of the rat i n t e s t i n a l mucosa i s of the DNase II type. -91-VI. General Discussion In summarizing the studies c a r r i e d out for the rat i n t e s t i n a l mucosa deoxyribonuclease, i t may be said that two types of enzyme a c t i v i t i e s are present. One protein could be salted out at 20$ ammonium sulfate and i t s a c t i v i t y recovered by r e - d i s s o l u t i o n i n buffer. I t showed a pH optimum of 6.0, required Mg"1^ for a c t i v i t i e s and was i n h i b i t e d by EDTA, c i t r a t e , arsenate and a protein DNase I i n h i b i t o r i s o l a t e d from the pigeon crop. The products of reaction terminated i n 5'-phosphomonoesters. I t therefore f e l l i n t o the category of enzymes termed DNase I. The other protein was pr e c i p i t a b l e at 30$ saturation ammonium su l f a t e . But since i t s a c t i v i t y was not recovered by re d i s s o l u t i o n i n buffer, i t could only be studied i n the crude extract which also contained the f i r s t kind of protein. However, t h i s 'protein could be shown to d i f f e r from the f i r s t i n i o n i c requirements and nature of products formed, and to resemble DNase I I . Other circumstantial evidences such as the i n t r a c e l l u l a r d i s t r i b u t i o n of deoxyribonuclease a c t i v i t y and oligonucleotide frequencies i n DNA hydrolysates by the crude and 20$P enzymes also pointed to the DNase II nature of the second protein. -92-SUMMARY The deoxyribonuclease a c t i v i t y i n the i n t e s t i n a l mucosa of the rat was studied. This a c t i v i t y could be extracted from a whole c e l l preparation of the mucosa with eit h e r Krebs-Ringer phos-phate buffer or physiological s a l i n e . The extract obtained using the l a t t e r solvent as extracting medium was however found to be the l e s s stable of the two. The deoxyribonuclease a c t i v i t y was found to p r e c i p i t a t e from solution at two d i f f e r e n t s a l t concentrations when the enzyme proteins were fractionated with ammonium su l f a t e . One active protein was pr e c i p i t a t e d at 20$ saturation of (NH^) 2S0^ and i t s a c t i v i t y could be recovered by r e d i s s o l v i n g i t i n Krebs-Ringer phosphate buffer. This material was designated as the 20$P f r a c t i o n , and represented a 20 f o l d p u r i f i c a t i o n of the crude extract. The supernatant f r a c t i o n obtained a f t e r t h i s protein was removed by centrifugation s t i l l contained DNase a c t i v i t y . This r e s i d u a l a c t i v i t y p r e c i p i t a t e d from solution when the concentration of ammonium sulfate was increased to 30$ saturation. However, a l l attempts to recover DNase a c t i v i t y from the pre c i p i t a t e d protein were unsuccessful, studies of the residual deoxyribonuclease a c t i v i t y were - 9 3 -c a r r i e d out i n d i r e c t l y i n the crude extract. 4 . The 20$P enzyme was found to be thermolabile. I t showed an optimum pH of 6, and was found to require magnesium ions f o r a c t i v i t y . I t was strongly i n h i b i t e d by EDTA, c i t r a t e , arsenate and a DNase I - s p e c i f i c protein i n h i b i t o r i s o l a t e d from the pigeon crop gland. Ion-exchange chroma-tography on DEAE-cellulose (chloride form) columns established that the products of reaction vary from mononucleotides through to oligonucleotides with a degree of polymerization l a r g e r than 7. These products were shown to terminate i n 5*-phosphates. The base frequencies d i f f e r e d i n d i f f e r e n t oligonucleotides. The mononucleotide f r a c t i o n contained about 70$ thymidylic a c i d and the amount of deoxycytidylic a c i d i n the higher oligonucleotides was shown to be very low. This was interpreted to mean that phosphodiester linkages next to a pyrimidine base were p r e f e r e n t i a l l y attacked by the enzyme. 5 . In the crude extract, the residual deoxyribonuclease a c t i v i t y was shown to be q u a l i t a t i v e l y d i f f e r e n t from the 20$P a c t i v i t y . I t did not require a c t i v a t i o n by Mg + +, and was not i n h i b i t e d by EDTA, c i t r a t e or arsenate. Some of the products formed by the crude enzyme extract were shown to carry a monoesterified phosphoryl group at -94-carbon 3'. These products were not formed by the 20$P enzyme and therefore must be due to the residual deoxyribonuclease. 6. In l i g h t of the above findings, the 20$P enzyme was c l a s s i f i e d as DNase I whereas the residual deoxyribo-nuclease l i k e l y was of the DNase II type. 7. Studies on the i n t r a c e l l u l a r d i s t r i b u t i o n of deoxyribo-nuclease showed that the i n t e s t i n a l DNase I occurred i n mitochondria. DNase II was associated with l i g h t e r s u b c e l l u l a r p a r t i c l e s . - 9 5 -BIBLIOGRAPHY 1. V. A l l f r e y and A.E. Mirsky, J . Gen. Physiol. __5, 227(1952). 2 . M. Meselson and J . J . Weigle, P.N.A.S. 858 ( 1 9 6 l ) . 3 . L.A. Heppel and P.R. Whitfield, B.J. 60, 1, 8 (1955). 4 . C.C. Richardson, C L . Schildkraut, H.V. Aposliran, A. Kornberg, W. Bodmer and J . Lederberg, i n "Informational Macromolecules", (H. Vogel, ed.) p. 13, Acad. Press, New York, 1963. 5 . C.L. Schildkraut, J . Marmur and P. Doty, J . Mol. B i o l . , 2 595 (1961). 6. I.R. Lehman, G.G. Roussos and E.A. Pratt, J.B.C 237, 819 ( 1962) . 7 . D. 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