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The enzymatic hydrolysis of ribonucleoside 2', 3'-cyclic phospates by a diesterase in nerve tissue Lee, Jack Foo 1967

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THE ENZYMATIC HYDROLYSIS OF RIBONUCLEOSIDE 2',3'-CYCLIC PHOSPHATES BY A DIESTERASE IN NERVE TISSUE by JACK FOO LEE B.S.P., University of B r i t i s h Columbia, 1965 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Pharmacology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1967 In 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 the r e q u i r e m e n t s f o r an advanced deg ree a t t he 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 ag r ee t h a t the 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 ag r ee 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 the Head o f my Depar tment o r by h.i)s r e p r e s e n t a t i v e s . It 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 no t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . JACK P. LEE Depar tment o f Pharmacology  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 Vancouve r 8, Canada Date September 2 7 , 1967 Chairman: Professor George I. Drummond ABSTRACT During the past 50 years, a number of nucleophosphodiesterases have been studied. Of recent i n t e r e s t , i s a br a i n phosphodiesterase which converts ribonucleoside 2',3'-cyclic phosphates to the corresponding ribonucleoside 2'-phosphates. The p h y s i o l o g i c a l r o l e of t h i s enzyme i s e n t i r e l y unknown. The present studies were designed to learn something of i t s 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 , properties and mechanism of a c t i o n . The assay of the enzyme was based on the hydrolysis of adenosine 2', 3'-c y c l i c phosphate by the diesterase followed by the hydrolysis of the reaction product with a l k a l i n e phosphatase and subsequent analysis f o r inorganic phosphate by a modified method of Fiske and SubbaRow. Rabbit brain homogenate was fractionated i n t o nuclear, mitochondrial, microsomal and 100,000 x g supernatant f l u i d f r a c t i o n s by d i f f e r e n t i a l c e n t r i f u g a t i o n . 2',3'-Cyclic phosphodiesterase a c t i v i t y was found i n a l l the f r a c t i o n s with the greatest a c t i v i t y i n the mitochondrial f r a c t i o n . Since the mitochondrial f r a c t i o n was a heterogenous mixture as revealed by ele c t r o n microscopy, i t s components were separated by sucrose density gradient c e n t r i f u g a t i o n by established methods. Five subtractions were obtained (A, B, C, D, E). The l i g h t e s t subfraction (A) contained most of the diesterase a c t i v i t y and electron microscopy revealed that t h i s s u b fraction consisted of fragments of myelin of various s i z e s . The microsomal and nuclear f r a c t i o n s were also subjected to sucrose density gradient c e n t r i f u g a t i o n . Again, the l e a s t dense, myelin-containing sub-f r a c t i o n , contained most of the diesterase a c t i v i t y . I t was also found that r a b b i t b r a i n white matter contained greater diesterase a c t i v i t y than grey matter. The data provide strong evidence that the diesterase i s i i i associated with myelin. I t was found that acetone extraction of the myelin f r a c t i o n from a homogenate of white matter of beef b r a i n resulted i n a doubling of diesterase a c t i v i t y with a f i v e - f o l d p u r i f i c a t i o n . E f f o r t s directed toward s o l u b i l i z a t i o n of the enzyme were unsuccessful. Some properties of the enzyme were also determined. The diesterase opened the c y c l i c d i e s t e r bond of cyclic-ended oligonucleotides, at l e a s t as large as (Ag) and did so, without cleavage of internucleotide bonds. I t was in a c t i v a t e d by the s u l f h y d r y l reagent, para-hydroxymercuribenzoate, and was unaffected by the presence of a wide v a r i e t y of purine and pyrimidine compounds or derivatives and various nucleoside mono-, d i - and -3 triphosphates. The of the diesterase was determined to be 1.9 x 10 M. i v TABLE OF CONTENTS Page INTRODUCTION 1 EXPERIMENTAL PROCEDURE 9 Materials 9 Chromatographic Solvent Systems 10 Enzyme Assays 10 I. The Diesterase Standard Assay 10 I I . The Ribonuclease Standard Assay 11 Determination of Relative Rates of Hydrolysis 14 I. 2',3'-Cyclic AMP 14 I I . Ap(Ap) 2A c y c l i c - P 16 Br a i n - C e l l Homogenization 16 Preparation of the Primary Subcellular Fractions 16 Subfractionation of the Primary Subcellular Fractions 20 Electron Microscopy 22 Homogenization of Beef Brain White Matter and Rabbit Brain White and Grey Matter 22 Preparation of Beef Brain Myelin 22 Acetone Extraction of Beef Brain Myelin 22 Nitrogen Determination 23 I. Preparation of Digestion Mix 23 I I . Preparation of Modified Nessler's Solution 23 Protein Determination 23 TABLE OF CONTENTS (Continued)... Page RESULTS 26 Preliminary 26 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 26 Electron Microscopic Examination of the Primary Fractions 31 Density Gradient Sedimentation of the Primary Fractions 35 I. Subfractionation of the Mitochondrial F r a c t i o n 35 I I . Subfractionation df the Microsomal F r a c t i o n 39 I I I . Subfractionation of the Nuclear Fra c t i o n 43 Diesterase A c t i v i t y i n White and Grey Matter of Rabbit Cerebrum 49 Preparation of Diesterase from Brain Tissue 54 Properties of the Diesterase 56 I. Enzyme Concentration and Time Course 56 I I . S p e c i f i c i t y Studies 56 (a) Action of the enzyme on ApA c y c l i c - P 59 (b) Action of the enzyme on Ap(Ap) 2A c y c l i c - P 62 (c) Action of the enzyme on Ap(Ap)^A c y c l i c - P 65 I I I . Relative Rates of Hydrolysis 69 IV. Possible Activators or Inhi b i t o r s 72 V. E f f e c t of Protein Reagents on the Enzyme 72 VI. E f f e c t of para-Hydroxymercuribenzoate on the the Time Course of the Diesterase A c t i v i t y 74 VII. K i n e t i c s 77 VIII. E f f e c t of pH on K and V 77 m max D i s t r i b u t i o n of Ribonuclease 82 TABLE OF CONTENTS (Continued). DISCUSSION ADDENDUM BIBLIOGRAPHY v i i LIST OF TABLES No T i t l e Page I. Diesterase a c t i v i t y i n the primary f r a c t i o n s 30 I I . Diesterase a c t i v i t y i n the mitochondrial subfractions 36 I I I . Diesterase a c t i v i t y i n the microsomal subfractions 45 IV. Diesterase a c t i v i t y i n the nuclear subfractions 51 V. Diesterase a c t i v i t y i n white and grey matter of of r a b b i t cerebrum 53 VI. Diesterase a c t i v i t y i n the acetone extract of myelin f r a c t i o n from beef b r a i n white matter 55 VII. Hydrolysis of 2',3'-cyclic AMP versus Ap(Ap)„A Cyc l i c - P 71 VIII. Possible a c t i v a t o r s or i n h i b i t o r s 73 IX. E f f e c t of pH on K and V 81 m max X. RN'ase i n the primary f r a c t i o n s 83 XI. RN'ase i n white and grey matter 84 v i i i LIST OF FIGURES No. -Title Page 1. Standard phosphate curve 12 2. Time course of P i color development 13 3. Standard RN'ase curve 15 4. Standard curve of 2',3'-cyclic AMP 17 5. Standard curve of Ap(Ap>2A c y c l i c - P 18 6. Flow-sheet showing a summary of the preparation of the primary f r a c t i o n s 19 7. Sketch of a tube a f t e r c e n t r i f u g a t i o n of the mitochondrial f r a c t i o n 21 8. Sketch of a tube a f t e r c e n t r i f u g a t i o n of the microsomal f r a c t i o n 21 9. Sketch of a tube a f t e r c e n t r i f u g a t i o n of the nuclear f r a c t i o n 21 10. Determination of nitrogen 24 11. E f f e c t of a l k a l i n e phosphatase on the hydrolysis of 2'-AMP produced from the hydrolysis of 2',3'-c y c l i c AMP by the diesterase 27 12. E f f e c t of enzyme concentration on the rate of hydrolysis of 2',3'-cyclic AMP 28 13. Time course f o r the hydrolysis of 2',3'-cyclic AMP by the diesterase 29 14. Electronmicrograph of the nuclear f r a c t i o n 32 15. Electronmicrograph of the mitochondrial f r a c t i o n 33 16. Electronmicrograph of the microsomal f r a c t i o n 34 17. Electronmicrograph of subtraction A of the mitochondrial f r a c t i o n 37 18. Electronmicrograph of subfraction B of the mitochondrial f r a c t i o n 38 LIST OF FIGURES (Continued)... No. T i t l e Page 19. Electronmicrograph. of subfraction C of the mitochondrial f r a c t i o n 40 20. Electronmicrograph of subfraction D of the mitochondrial f r a c t i o n 41 21. Electronmicrograph of subfraction E of the mitochondrial f r a c t i o n 42 22. Electronmicrograph of subtraction a of the microsomal f r a c t i o n 44 23. Electronmicrograph of subfraction b_ of the microsomal f r a c t i o n 46 24. Electronmicrograph of subfraction jc of the microsomal f r a c t i o n 47 25. Electronmicrograph of subfraction d. of the microsomal f r a c t i o n 48 26. Electronmicrograph of subfraction a of the nuclear f r a c t i o n 50 27. Electronmicrograph of subfraction y of the nuclear f r a c t i o n 52 28. E f f e c t of enzyme concentration on the rate of hydrolysis of 2',3'-cyclic AMP 57 29. Time course f o r the hydrolysis of 2',3'-cyclic AMP by the diesterase 58 30. Action of the enzyme on ApA c y c l i c - P (chromatographed i n Solvent A) 60 31. Action of the enzyme on ApA c y c l i c - P (chromatographed i n Solvent B) 61 32. Action of the enzyme on Ap(Ap) 2A c y c l i c - P (chromatographed i n Solvent A) 63 33. Development of eluted spot (as indicated i n Figure 32) i n Solvent B 64 34. Action of the enzyme on Ap(Ap) 2A c y c l i c - P (chromatographed i n Solvent B) 66 X LIST OF FIGURES (Continued)... No. T i t l e Page 35. Action of the enzyme on Ap(Ap)^A c y c l i c - P ( i n a c t i v a t i o n of diesterase by chromatographic solvent) 68 36. Action of the enzyme on Ap(Ap)^ c y c l i c - P ( i n a c t i v a t i o n of diesterase by heat) 70 37. Time course of the preincubation mixture containing p-HMB and diesterase 75 38. E f f e c t of p-HMB on the time course of diesterase a c t i v i t y at various 2',3'-cyclic AMP concentrations 76 39. Time course of reaction at various substrate ( 2 ' , 3 ' - c y c l i c AMP) concentrations 78 40. Plot of i n i t i a l rates of reac t i o n versus substrate (2', 3 ' - c y c l i c AMP) concentrations 79 41. Lineweaver-Burk p l o t of the r e c i p r o c a l of i n i t i a l v e l o c i t y versus that of substrate concentration 80 x i ABBREVIATIONS The following abbreviations are used: 2',3'-cyclic AMP, adenosine 2',3'-cyclic phosphate; RNA, r i b o n u c l e i c a c i d ; RN'ase, ribonuclease; Py, pyrimidine base; ApA c y c l i c - P , 2 1,3'-cyclic-ended adenylyl (3'-5') adenylic a c i d ; Ap(Ap)^A c y c l i c - P , 2',3'-cyclic-ended tetranucleotide containing the base adenine; Ap(Ap)^A c y c l i c - P , 2',3'-cyclic-ended octanucleotide containing the base adenine; DFP, diisopropylfluorophosphate; IAA, iodoacetamide; p-HMB, para-hydroxymercuribenzoate. x i i ACKNOWLEDGEMENTS I wish to extend my sincere appreciation to Dr. George I. Drummond for the time and h e l p f u l advice he contributed throughout the course of thi s p r o j e c t . 1 would also l i k e to thank Mrs. Betty G i l l f o r the able technical assistance i n providing the ele c t r o n micrographs. This p r o j e c t was supported, i n part, by a Medical Research Council Grant award to Dr. G. I. Drummond. 1 INTRODUCTION During the past 50 years, a number of nucleophosphodiesterases have been studied. These enzymes are of two main classes: those that attack internucleotide bonds i n the n u c l e i c acids, and those that are s p e c i f i c f o r c e r t a i n d e r i v a t i v e s of mononucleotides. Included i n the f i r s t c l a s s , are ribonucleases, deoxyribonucleases, and those enzymes that attack i n t e r -nucleotide bonds of both r i b o - and deoxyribonucleic acids. Of these, pancreatic ribonuclease i s the most i n t e n s i v e l y studied. Jones i n 1920 (1) described the presence i n pancreas of a heat stable enzyme capable of digesting yeast n u c l e i c a c i d . He found that the digestion did not l i b e r a t e any inorganic phosphorus. In 1938, Dubos and Thompson (2), who introduced the name "ribonuclease", confirmed Jones' observations; they went on to state that the action of the enzyme on n u c l e i c acid gave r i s e to decomposition products which were soluble i n mineral acids. The enzyme did not degrade any of the other soluble substrates tested, p a r t i c u l a r l y thymus nucle i c a c i d . At that time, there were s t i l l questions concerning the s p e c i f i c i t y of ribonuclease towards RNA and DNA. Two years l a t e r , Kunitz (3) reported that he had c r y s t a l l i z e d ribonuclease from beef pancreas. As a r e s u l t , d e f i n i t e evidence was obtained that t h i s enzyme hydrolyzed only RNA, but not DNA. Furthermore, the proof of absolute s p e c i f i c i t y was obtained when Kunitz (4), i n 1948, c r y s t a l l i z e d deoxyribonuclease. By 1951, much information on ribonuclease had accumulated leading to the general observation that the enzyme attacked pyrimidine s i t e s g i v ing fragments co n s i s t i n g of chains of purine nucleotides terminated by pyrimidine nucleotides, and free pyrimidine nucleotides. Through the combined e f f o r t s of Schmidt et a l (5), Markham and Smith (6,7), M e r r i f i e l d and Woolley (8), 2 Brown, Dekker and Todd (10), Volkin and Cohn (11), and Heppel and W h i t f i e l d (12), the mechanism of a c t i o n of pancreatic ribonuclease was elucidated. I t was found that the enzyme hydrolyzed RNA i n two stages. The f i r s t stage of the reaction involves t r a n s e s t e r i f i c a t i o n forming pyrimidine nucleoside 2',3'-cyclic phosphates with the simultaneous s p l i t t i n g of the internucleotide bond d i s t a l to C,_ M of the l a t t e r : R R 0 \ CH, 0 Py 7 Transes t e r i f i c a t i o n + R*0 0 OH 0 0 I t i s possible to i s o l a t e t h i s c y c l i c intermediate from short-period digestion of RNA (6). The second stage of the reaction involves the hydrolysis of the pyrimidine c y c l i c nucleotides giving pyrimidine nucleoside 3'-phosphates (6,9): 3 Recently, the a c t i v e s i t e of t h i s enzyme has been examined. Studies by C r e s t f i e l d , Stein and Moore, Finn and Hofmann, Richards and many other workers have revealed two important h i s t i d i n e residues necessary for the a c t i v i t y of pancreatic RN'ase — ^ * s i 2 a n < * ^ s 1 1 9 * T h e s e studies have shown that the a c t i v i t y i s dependent on the close proximity of these two h i s t i d i n e residues. Other important amino acid residues were also found, namely Met^.j and G l u ^ . These were not e s s e n t i a l f o r a c t i v i t y , but were e s s e n t i a l for binding. These fin d i n g s , along with t h e i r own, led Findlay et a l (13) to postulate a mechanism of action of pancreatic ribonuclease at the a c t i v e s i t e i n v o l v i n g the two h i s t i d i n e residues. The t r a n s e s t e r i f i c a t i o n r e a c t i o n i s proposed as follows: 4 A h i s t i d i n e i n the acid form (I) transfers a proton to the oxygen atom of the P—0 bond d i s t a l to C ( 3 t ) of the sugar, weakening that P—0 bond and rendering the phosphorus atom more susceptible to attack by n u c l e o p h i l i c reagents. At the same time, a h i s t i d i n e i n the base form (II) accepts a proton from the 2'-oxygen, rendering i t n u c l e o p h i l i c i n character which then f a c i l i t a t e s attack on the phosphorous atom. The r e s u l t i s the formation of a c y c l i c intermediate and the simultaneous s p l i t t i n g of the P—0 bond d i s t a l to C^iy The next stage of the reaction i s the hydrolysis of the pyrimidine c y c l i c nucleotide intermediate: 5 The reaction at t h i s stage i s s i m i l a r to that at the f i r s t stage. The h i s t i d i n e i n the a c i d form (II) transfers a proton to the 2'-oxygen atom, thus weakening the P—0 ( 2 ' ) k° n c* a n c * r e n d e r i n g the phosphorous atom more susceptible to attack by n u c l e o p h i l i c reagents. Simultaneously, i n t e r a c t i o n by the h i s t i d i n e i n the base form (I) with the attacking agent (H^O) renders the oxygen i n the l a t t e r more n u c l e o p h i l i c i n character which then f a c i l i t a t e s attack on the phosphorous atom. The end r e s u l t i s a pyrimidine nucleoside 3'-phosphate. In t h i s representation, (III) i s proposed to represent the s i t e s of a d d i t i o n a l i n t e r a c t i o n with the alcohol, and (IV), the s p e c i f i c i t y s i t e for a pyrimidine. Following the work on pancreatic ribonuclease, other 6 ribonucleases from animal t i s s u e s , plants and microorganisms have been reported, having the same general mode of act i o n but with some differences i n s p e c i f i c i t i e s — spleen (14), l e a f (15,16), rye grass (17), taka diastase (18). Included also i n the f i r s t class of phosphodiesterases which attack internucleotide bonds are those enzymes that can act on both DNA and RNA. There are two types of these phosphodiesterases. The f i r s t type are those which form nucleoside 5'-phosphates of which venom phosphodiesterase i s a good example. The a c t i o n of venom phosphodiesterase on a polynucleotide i s a stepwise hydrolysis beginning from the nucleotide bearing a 3'-hydroxyl end group, r e s u l t i n g i n the successive release of nucleoside 5'-phosphate u n i t s : (etc.) (2) (1) ' ! 1 pNpN \ (pN) 1 pN [pN 3^- pN 's i n ' I t t • Phosphodiesterases having a s i m i l a r mode of action as the venom diesterase have been reported i n i n t e s t i n a l tissues and leukemia c e l l s . The second type are phosphodiesterases which form nucleoside 3'-phosphates. The spleen phosphodiesterase can serve as an example. Again, the hydrolysis i s step-wise, but beginning from the nucleotide bearing a 5'-hydroxyl end group, and r e s u l t i n g i n the successive release of nucleoside 3'-phosphate u n i t s : (1) (2) (etc) i * i Np jNp j (Np) | Np Np 's l I I The second cl a s s of phosphodiesterases i s represented by those enzymes that are s p e c i f i c f o r c e r t a i n d e r i v a t i v e s of mononucleotides. For example, 7 Sutherland and R a i l (19,20) have described a diesterase i n l i v e r , heart, s k e l e t a l muscle, and b r a i n which hydrolyzes nucleoside 3',5'-cyclic phosphates. The product i s e x c l u s i v e l y the corresponding nucleoside 5'-phosphate. Included i n t h i s class are phosphodiesterases hydrolyzing r i b o -nucleoside 2',3'-cyclic phosphates to the corresponding 3'-ribonucleotide (21). Calf spleen and i n t e s t i n a l mucosa contain a diesterase which converts ribonucleoside 2',3'-cyclic phosphates to ribonucleoside 2'-phosphates (22). From rye grass preparations, approximately equal amounts of adenosine 2'-phosphate and adenosine 3'-phosphate are formed from adenosine 2',3'-c y c l i c phosphate (22). In a report from t h i s laboratory, Drummond e_t a l (23,24) described the i s o l a t i o n from beef brain, of a highly a c t i v e diesterase which converted ribonucleoside 2',3'-cyclic phosphates to the corresponding ribonucleoside 2'-phosphates. B r i e f l y , some properties of t h i s enzyme as described by Drummond et^ al^ (24) are: 1. I t attacks a l l ribonucleoside 2',3'-cyclic phosphates tested with c y c l i c phosphates bearing purine bases being hydrolyzed somewhat more r a p i d l y than those bearing pyrimidine bases. 2. I t opens the c y c l i c d i e s t e r linkage of several cyclic-ended dinucleotides, without attacking the internucleotide bond, to form the corresponding 2'-ended dinucleotide. 3. The enzyme i s a c t i v e over a broad range of pH, with an optimum of a c t i v i t y between pH 6 and 7. 4. The hydrolysis of adenosine 2',3'-cyclic phosphate shows no metal requirement. The enzyme i s s l i g h t l y activated (approximately 10%) i n the presence of EDTA, c i t r a t e , and succinate ions. I t i s i n h i b i t e d by cupric s u l f a t e , zinc acetate, and mercuric c h l o r i d e . 8 5. I t i s present i n various t i s s u e s , being most act i v e i n the s p i n a l cord and b r a i n . I t i s also present i n extracts of the vagus and s c i a t i c nerve. In tissues other than nerve, the enzyme i s much l e s s a c t i v e . The p h y s i o l o g i c a l r o l e of t h i s enzyme i s e n t i r e l y unknown and much remains to be learned of i t s c e l l u l a r d i s t r i b u t i o n , properties and mechanism of a c t i o n . The present study was i n i t i a t e d to learn more of the properties of t h i s enzyme i n brain t i s s u e . 9 EXPERIMENTAL PROCEDURE Materials Adenosine 2',3'-cyclic phosphate was prepared by the method of Smith, Moffatt, and Khorana (25). Before use, the barium s a l t was converted to the potassium s a l t by treatment with Dowex-50 (potassium form). The beef b r a i n diesterase preparation used for preliminary assays was prepared by Drummond et a l (24). Ribonuclease was obtained from the N u t r i t i o n a l Biochemicals Corporation. RNA, also from the N u t r i t i o n a l Biochemicals Corporation, was p u r i f i e d by a combined method of Kunitz (3), Bain and Rusch (26) and Woodward (27). A suspension of commercial yeast RNA (2.5 g) i n d i s t i l l e d water (12.5 ml) was cooled i n an ice-water bath to about 4°. 5 N sodium hydroxide was added slowly u n t i l a c l e a r s o l u t i o n was obtained. Care was taken to keep the s o l u t i o n cold and the pH below 6. Next, 5 volumes of g l a c i a l a c e t i c acid were added, and the whole s o l u t i o n was allowed to stand at 25° for 10 minutes. The p r e c i p i t a t e formed was f i l t e r e d with suction on a Buchner funnel, washed four times with an a c e t i c a c i d s o l u t i o n (5 parts g l a c i a l a c e t i c a c i d to 1 part of d i s t i l l e d water), once with ethanol (95%), once with d i e t h y l ether, and then dried i n a vacuum desiccator. A l k a l i n e phosphatase was obtained from the Worthington Biochemicals Corporation and The Sigma Chemical Company. ApA c y c l i c - P , Ap(Ap^A c y c l i c - P , and Ap(Ap)^A c y c l i c - P were obtained from the Miles Laboratories, Inc. 10 Chromatographic Solvent Systems The chromatographic solvent systems used were: (A) n-propanol-ammonium h y d r o x i d e - d i s t i l l e d water (55:10:35 by volume); (B) n-propanol-ammonium h y d r o x i d e - d i s t i l l e d water (6:3:1 by volume); (C) isopropanol-ammonium h y d r o x i d e - d i s t i l l e d water (7:1:2 by volume). Enzyme Assays I. The Diesterase Standard Assay The assay system to be described was used r o u t i n e l y i n the measurement of the diesterase a c t i v i t y . The assay i s i l l u s t r a t e d by the following equation: 2',3'-cyclic 2'-AMP Adenosine inorganic AMP phosphate The assay consisted of measuring the release of inorganic phosphate i n the presence of an excess of a l k a l i n e phosphatase. Thus, one mole of P i would be equivalent to one mole of substrate hydrolyzed. The reaction mixture contained adenosine 2',3'-cyclic phosphate (1.4-1.5 umoles), 0.05 ml of 0.2 M Tris-HCl (pH 7.5) containing 0.25% egg albumin, a l k a l i n e phosphatase (20 y)> and a s u i t a b l e d i l u t i o n of the diesterase sample being tested i n a f i n a l volume of 0.25 ml. This r e a c t i o n mixture was incubated at 30 f o r 20 minutes i n a Dubnoff metabolic shaker. To stop the reaction, cold 3% t r i c h l o r o a c e t i c a c i d (1 ml) was added. The r e s u l t i n g p r e c i p i t a t e was removed by c e n t r i f u g a t i o n , and an a l i q u o t (0.5 ml) of the supernatant s o l u t i o n was analyzed f or inorganic phosphate by the method of Fiske and SubbaRow (28), modified as follows: 0.05 ml of 2.5% ammonium molybdate i n 5 N H 2S0^ was added to a 0.5 ml aliq u o t of the supernatant s o l u t i o n and 0.43 ml of d i s t i l l e d water. Blue color was developed by the a d d i t i o n of 0.02 ml of a reducing s o l u t i o n (described below) and was read at 720 my against a d i s t i l l e d water blank on the Beckman model DU spectrophotometer, with cuvettes of 1 cm l i g h t path. A sample containing substrate but no diesterase served as a c o n t r o l . The reducing s o l u t i o n used consisted of l-amino-2-naphthal-4-sulphonic acid (7.70%), sodium b i s u l f i t e (NaHSO^, 46.15%), and sodium s u l f i t e (Na2S0.j, 46.15%) i n d i s t i l l e d water. The standard curve for inorganic phosphate by the method used i s shown i n F i g . 1. The color development was maximum i n 2 minutes and did not change thereafter up to 20 minutes ( F i g . 2). One unit of enzyme i s defined as that a c t i v i t y c a t a l y z i n g the hydrolysis of 1 umole of 2',3'-cyclic AMP i n 20 minutes under the conditions of the assay. S p e c i f i c a c t i v i t y i s defined as units/mg p r o t e i n . I I . The Ribonuclease Standard Assay Ribonuclease a c t i v i t y was determined by the method of McDonall (29) and Kunitz (30) which i s based on the spectrophotometrie measurement of l i b e r a t e d soluble nucleotides from RNA. The re a c t i o n mixture contained 0.3 ml of p u r i f i e d RNA (0.1%) i n 0.1 M acetate buffer (pH 5), 0.1 ml of a 0.0 0.1 0.2 0.3 Pi (^umoles) Figure 1. Standard phosphate curve. Figure 2. Time course of P i color development by the modified method of Fiske and SubbaRow. 14 test sample and d i s t i l l e d water added to a f i n a l volume of 1 ml. The incubation was carried out at 25° for 10 minutes with shaking and with a proper tissue blank to correct for contaminating nucleotides. A sample containing substrate but no enzyme served as a control. The reaction was stopped by the addition of an equal volume of MacFadyen's reagent (0.25% uranylacetate in 2.5% trichloroacetic acid), and allowed to stand for 30 minutes at 25° for complete precipitation of proteins, nucleic acids, and nucleoproteins. The supernatant solution was collected by centrifugation, suitably diluted with water (1/3 dilution) and the absorbance was read at 260 mu in a Beckman model DU spectrophotometer, with cuvettes of 1 cm light path. The amount of ribonuclease was determined from a standard curve (Fig. 3) using a known amount of ribonuclease reacting with excess RNA under conditions of the standard assay. Determination of Relative Rates of Hydrolysis  I. 2 f,3'-cyclic AMP Each incubation mixture contained 0.01 ml of 0.01 Tris-HCl (pH 7.5), 2',3'-cyclic AMP (0.075 umoles), beef brain myelin acetone extract (0.122 y to 0.912 y protein), and d i s t i l l e d water to a f i n a l volume of 0.06 ml. The mixture was incubated at 30° for 20 minutes with shaking. To stop the reaction, 0.01 ml of glacial acetic acid was added and the tube chilled in ice. The entire volume of reaction solution was spotted on Whatman No. 3MM paper and chromatographed (descending) i n solvent C for 15 hours. Unreacted substrate was cut out, eluted and brought to a volume of 1 ml with d i s t i l l e d water. The absorbance was then determined at 260 mu in a Beckman model DU Spectrophotometer, with cuvettes of 1 cm light path. The amount of unreacted substrate was determined from a standard curve of 0.0 0.4 0.8 1.2 1.6 2.0 R N ' a s e ( j j g ) Figure 3. Standard RN'ase curve under conditions of the standard assay f o r RN'ase. 16 2',3'-cyclic AMP ( F i g . 4), or from the e x t i n c t i o n c o e f f i c i e n t of c y c l i c AMP at 260 mu which i s 14,650 at pH 7.5. I I . Ap(Ap)^A c y c l i c - P Each incubation mixture contained 0.01 ml of 0.01 T r i s - H C l (pH 7.5), Ap(Ap) 2A c y c l i c - P (0.076 umoles), beef b r a i n myelin acetone extract (0.608 y t o 3.65 y p r o t e i n ) , and d i s t i l l e d water to a f i n a l volume of 0.06 ml. Again, the r e a c t i o n mixture was incubated at 30° for 20 minutes with shaking, g l a c i a l a c e t i c a c i d added as above and the tube c h i l l e d i n i c e . 0.025 ml of the r e a c t i o n s o l u t i o n was spotted on Whatman No. 3MM paper and chromatographed (descending) i n solvent A for 15 hours. The spot representing unreacted substrate was cut out, eluted, brought to the desired volume, and absorbance read, as described above. A standard curve of Ap(Ap)^A c y c l i c - P ( F i g . 5) was used to determine the amount of unreacted substrate. B r a i n - C e l l Homogenization The homogenization was done by a modified method of De Robertis et. a l (31). For each experiment one white rabb i t was used. The rabbit's neck was quickly broken by a blow and the r a b b i t bled through the c a r o t i d artery. The cerebrum (approximately 5 g), stripped of i t s surface membranes and blood vessels was cut i n t o small pieces and homogenized i n 0.32 M sucrose i n a glass homogenizer (15-20 passes) with a loosely f i t t e d t e f l o n p e s t l e . The homogenate was then d i l u t e d to 10% (W/V) i n 0.32 M sucrose. Preparation of the Primary Subcellular Fractions The preparation of the primary s u b c e l l u l a r f r a c t i o n s i s i l l u s t r a t e d on the flow-sheet ( F i g . 6). The homogenate, 10% (W/V) i n 0.32 M sucrose, was centrifuged i n the cold for 10 minutes at 755 x g i n a S o r v a l l Superspeed RC-2 Automatic Refrigerated Centrifuge, SS-34 ro t o r . The nuclear p r e c i p i t a t e 1 7 Figure 4. Standard curve of 2',3'-cyclic AMP for determination of the r e l a t i v e rate of hydrolysis by diesterase. Figure 5. Standard curve of Ap(Ap) 2A c y c l i c - P f o r determination of the r e l a t i v e rate of hydrolysis by diesterase. 19 Homogenate (10% i n 0.32 M sucrose) 755 x g 10 min (Nuclear f r a c t i o n ) 11,500 x g washed X2 and r e - 20 min centrifuged. (Mitochondrial f r a c t i o n ) washed X2 and re-centrifuged. Combined a l l S. 100,000 x g 2 hrs. (Microsomal fr a c t i o n ) (100,000 x g supernatant f l u i d ) Figure 6. Flow-sheet showing a summary of the preparation of the primary f r a c t i o n s . P represents p r e c i p i t a t e and S, the supernatant s o l u t i o n . 20 was washed twice i n 0.32 M sucrose by rapid s t i r r i n g on an automatic mixer and centrifuged as above. The combined supernatant and washings were centrifuged i n the cold f o r 20 minutes at 11,500 x g. The r e s u l t i n g mitochondrial p r e c i p i t a t e was washed twice by c e n t r i f u g a t i o n as above. The combined supernatant and washings were centrifuged f o r 2 hours at 100,000 x g i n the Spinco rotor No. 40 of the Beckman model L U l t r a c e n t r i f u g e . The r e s u l t i n g p r e c i p i t a t e was considered as the microsomal .fraction and the resultant supernatant s o l u t i o n , the 100,000 x g supernatant f l u i d . A l l the p r e c i p i t a t e s were redispersed and brought up to a volume of 10 ml with 0.32 M sucrose. Subfractionation of the Primary Subcellular Fractions The three primary s u b c e l l u l a r f r a c t i o n s — nuclear, mitochondrial, and microsomal — were subfractionated i n a discontinuous sucrose density gradient, used by De Robertis et a l (31), of the following steps: 1.4,1.2, 1.0 and 0.8 M (each 5.5 ml). Before use, the gradient was l e f t f o r 2 hours at room temperature and 15 hours i n the cold room. Each redispersed primary s u b c e l l u l a r f r a c t i o n (3 ml) was layered c a r e f u l l y on top of the density gradient and then centrifuged f o r 2 hours at 50,000 x g i n the Spinco SW 25.1 rotor. The bands r e s u l t i n g a f t e r c e n t r i f u g a t i o n of the primary f r a c t i o n s are i l l u s t r a t e d i n F i g s . 7, 8 and 9. The mitochondrial f r a c t i o n resulted i n four layered subfractions (A, B, C, D) and one p e l l e t (E); the microsomal f r a c t i o n , three layered subfractions (a, b_, c_) and one p e l l e t (d) ; the nuclear f r a c t i o n , one layered subfraction (a), one p e l l e t (y)» and a c l e a r supernatant f l u i d (3) i n between. The layered subfractions were c a r e f u l l y separated with a pipet t e and then were d i l u t e d with d i s t i l l e d water and centrifuged at 100,000 x g f o r Figure 7. Sketch of a tube a f t e r c e n t r i f u g a t i o n of the mitochon-d r i a l f r a c t i o n . Four layered f r a c t i o n s A, B, C and D and the p e l l e t were observed. The density gradient of sucrose i n molar concentrations i s indicated at the l e f t . M A _ _ 0 3 2 _ 0.80 WM///, B 1 .00 '///////// C 1 .2 0 W///// D E 1 .40 Figure 8. Sketch of a tube a f t e r c e n t r i f u g a t i o n of the microsomal f r a c t i o n . Three layered f r a c t i o n s a_, b_ and c_ and the p e l l e t ci were observed. Figure 9. Sketch of a tube a f t e r c e n t r i f u g a t i o n of the nuclear f r a c t i o n . One layered f r a c t i o n a and the p e l l e t y were observed. Subfraction g i s the c l e a r super-natant f l u i d inbetween. _M 0.8 0 <LJf US' 9 1.00 1.20 I .40 1 hour. The r e s u l t i n g residues were dispersed by l i g h t homogenization i n a glass homogenizer with a loosely f i t t e d t e f l o n plunger and d i l u t e d to a volume of 5 ml with 0.32 sucrose. The p e l l e t s obtained i n each o r i g i n a l subfractionation were also dispersed and d i l u t e d with 0.32 sucrose. Electron Microscopy A l l p e l l e t s obtained above were fi x e d i n Palades osmium tetroxide (2%); embedded i n Maraglas "Der" Epoxy Resin; cut on Porter-Blum MT-2 u l t r a -microtome; stained with 2% lead hydroxide; and examined i n the Siemens Elmiskop I e l e c t r o n microscope. Homogenization of Beef Brain White Matter and  Rabbit Brain White and Grey Matter A 10% homogenate of these were prepared i n the same manner as described i n " B r a i n - C e l l Homogenization". Preparation of Beef Brain Myelin Beef b r a i n white matter homogenate (10 ml) was subfractionated by sedimentation at 50,000 x g i n a discontinuous sucrose density gradient, used by Whittaker (32), of the following composition: 1.2 M and 0.8 M (each 10 ml). The top layered f r a c t i o n , considered as the myelin f r a c t i o n , was pipetted out, d i l u t e d with d i s t i l l e d water, and centrifuged at 37,000 x g i n the S o r v a l l SS-34 rotor f o r 1 hour. The sediment was resuspended i n a minimum volume of d i s t i l l e d water i n preparation for acetone extraction. Acetone Extraction of Beef Brain Myelin The suspension of myelin was added, with rapid s t i r r i n g , to 15 volumes of acetone at -15°. Throughout the extraction, care was taken to maintain the temperature of the preparation at -15° to decrease rate of denaturation of proteins. The suspension was centrifuged at 12,100 x g, and -15 i n the S o r v a l l centrifuge. The supernatant f l u i d was discarded and the sediment was dispersed i n a small volume of cold acetone (-15°). The dispersed sediment was homogenized i n an Omnimixer for one minute at f u l l speed, d i l u t e d with cold acetone, and recentrifuged as above. The sediment was dispersed, homogenized, and centrifuged two more times and then placed i n t o a cold vacuum dessicator and dried by a s p i r a t i o n . A f t e r 30 minutes the residue was dispersed with a spatula and vacuum evaporation was continued for 3-4 hours u n t i l d r i e d . The f l u f f y dried powder was stored at -18°. Nitrogen Determination Nitrogen was determined by Nesslerimetry (33). The sample was digested i n a pyrex tube i n an oven at 210° with 1 ml of 5 N H 2S0^ (containing 150 mg of copper s e l e n i t e per l i t e r ) f o r 12 hours or more. A f t e r cooling, 5-10 drops of 30% hydrogen peroxide was added and the tube heated over a micro-burner u n t i l the black residue disappeared and white fumes developed. Then, 4 ml of d i s t i l l e d water was added followed by 1.2 ml of 5.5 N KOH and 1.4 ml of modified Nessler's s o l u t i o n . A f t e r 10 minutes the absorbance was measured i n the Klett-Summerson colorimeter with the green (540 mp) f i l t e r . L-glutamic a c i d (9.52% nitrogen) was used as the standard ( F i g . 10). I. Preparation of Digestion Mix 250 mg Se0 2 (selenous acid) and 250 mg CuS0^*5H20 were dissolved i n 1 l i t e r of 5 N E^SO^ II. Preparation of Modified Nessler's Solution Five grams of K l and 7 g of H g l 2 were mixed together i n a mortar with 50 ml of d i s t i l l e d water. The mixture was made up to 500 ml with d i s t i l l e d water and l e f t to stand overnight. Next day, excess Hgl„ was f i l t e r e d o f f . 24 N I T R O G E N (X lOmg) Figure 10. Determination of nitrogen with glutamic acid (9.52% nitrogen) as the standard. 25 To the 500 ml f i l t r a t e was added 160 ml of 1.5% gum indicum. The s o l u t i o n was mixed and d i l u t e d to 1500 ml. Protein Determination Protein was determined by the b i u r e t method modified as follows. A s u i t a b l e volume of the sample was added to 4.5 ml of b i u r e t reagent containing 1% sodium desoxycholate, and d i s t i l l e d water added to a f i n a l volume of 6 ml. A f t e r 30 minutes, the absorbance was measured i n the Klett-Summerson colorimeter with the 540 my f i l t e r . As the standard, egg albumin was used; and i t was found that 1 mg pr o t e i n was equivalent to 25 K l e t t units at 540 mp. 26 RESULTS Preliminary In order to e s t a b l i s h a v a l i d assay for the diesterase coupled with excess a l k a l i n e phosphatase, i t was necessary to determine the amount of the l a t t e r enzyme required. In F i g . 11 i t can be seen, that when quantities of a l k a l i n e phosphatase greater than 15 ug were added, the rate of l i b e r a t i o n of inorganic phosphate did not change, s i g n i f y i n g that t h i s enzyme was i n excess. Therefore i n the phosphodiesterase assay, an excess (20 yg) of a l k a l i n e phosphatase was r o u t i n e l y used. Under conditions of the standard assay f i n a l l y elaborated, the rate of phosphodiesterase a c t i v i t y was proportional to enzyme concentration over a wide range ( F i g . 12). Furthermore the rate of r e a c t i o n was proportional to time up to 40 minutes under the conditions used (Fig. 13). 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 Drummond et a l (24) observed that the diesterase i n nerve ti s s u e was extremely i n s o l u b l e , apparently t i g h t l y bound to tis s u e p a r t i c l e s . Because of t h i s , and because of our i n t e r e s t i n a possible p h y s i o l o g i c a l r o l e f o r the enzyme, i t was of i n t e r e s t to e s t a b l i s h 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 . Rabbit brain homogenate was fractionated as described i n the Experimental Procedure. Table I shows the percentage of diesterase a c t i v i t y i n each of the primary f r a c t i o n s , the a c t i v i t y of the crude homogenate being taken as 100%. From these r e s u l t s , one can see that the diesterase i s present mostly i n the mitochondrial and microsomal f r a c t i o n s . Depending on the c e n t r i f u g a t i o n procedure, one can, by a l t e r i n g the g r a v i t a t i o n a l force, obtain a preparation with the mitochondrial f r a c t i o n having over 50% of the a c t i v i t y . This can be 27 Figure 1 1 . E f f e c t of a l k a l i n e phosphatase on the hydrolysis of 2'-AMP produced from the hydrolysis of 2 ' , 3 ' - c y c l i c AMP by the diesterase whose concentration was constant at 3 . 2 u g . 28 0.5 r 0 4 8 12 PROTEIN (/jg) ( DIESTERASE) Figure 12. E f f e c t of enzyme concentration on the rate of hydrolysis of 2',3'-cyclic AMP. A l k a l i n e phosphatase concentration, constant at 20 ug protein, was used. 29 ~ 0.5 r 0 15 3 0 TIME(min) Figure 13. Time course for the hydrolysis of 2',3'-cyclic AMP by the diesterase. Standard assay conditions were employed with a l k a l i n e phosphatase (2© ug). The beef brain extract (3.02 ug protein) prepared by Drummond et a l (24) was used. 30 TABLE I Diesterase A c t i v i t y i n the Primary Fractions Fractions A c t i v i t y (%) Nitrogen (%) Crude Homogenate (100) (100) Nuclear 5.59 4.63 Mitochondrial 35.5 42.3 Microsomal 24.4 23.0 100,000 x g supernatant 12.9 23.3 To t a l recovery (%) 78.4 93.2 31 done by decreasing the c e n t r i f u g a t i o n speed of the crude homogenate to 450 x g and centrif u g i n g the r e s u l t i n g supernatant f l u i d at 27,000 x g. The values obtained from such a preparation was 52.5% diesterase a c t i v i t y i n the mitochondrial f r a c t i o n , 16.9% i n the microsomal f r a c t i o n , 8.8% i n the 100,000 x g supernatant f l u i d , and 1.1% i n the nuclear f r a c t i o n . Again, from Table I, note that the nitrogen content follows the diesterase a c t i v i t y p r o p o r t i o n a l l y . E l e c t r o n Microscopic Examination of the Primary Fractions Each of the primary f r a c t i o n s were subjected to electron microscopy. The nuclear f r a c t i o n showed e s s e n t i a l l y fragments of myelin sheath. Few i n t a c t n u c l e i were observed, although there were some p a r t i c l e s which might be disrupted n u c l e i . Lovtrup-Rein and McEwen (35) have described the extreme f r a g i l i t y of neuronal n u c l e i during homogenization. Also found i n t h i s f r a c t i o n were a few contaminating mitochondria, nerve endings, and other membranous components (Fig. 14). The mitochondrial f r a c t i o n was seen to be a very heterogenous mixture of myelin sheath fragments, nerve endings, mitochondria, and other membranous materials ( F i g . 15). The microsomal f r a c t i o n showed membranous components along with a few contaminating mitochondria, nerve endings, and smaller fragments of myelin sheath (Fig. 16). Electron micrographs of the 100,000 x g supernatant f l u i d were not a v a i l a b l e ; the gross appearance of t h i s f r a c t i o n was seen to be a yellowish, translucent f l u i d . F i g u r e 14. E l e c t r o n m i c r o g r a p h o f t h e n u c l e a r f r a c t i o n showing e s s e n t i a l l y m y e l i n f r a g m e n t s o f d i f f e r e n t s i z e . (X 5,625) Figure 15. Electronmicrograph of the mitochondrial f r a c t i o n Showing a heterogenous mixture of c e l l components. (X 5,625) Figure 16. Electronmicrograph of the microsomal f r a c t i o n showing membranous components along with other c e l l u l a r materials. (X 22,500) 35 Density Gradient Sedimentation of the Primary Fractions  I. Subfractionation of the Mitochondrial F r a c t i o n Whittaker i n 1959 (32) had separated a crude mitochondrial preparation from brain t i s s u e i n t o three subfractions by density gradient c e n t r i f u g a t i o n . In t h i s study he used a three-layer sucrose gradient. By using a f i v e - l a y e r sucrose gradient, De Robertis et a l (31) could obtain a better r e s o l u t i o n . Since the mitochondrial f r a c t i o n contained 36% of the t o t a l diesterase a c t i v i t y , i t seemed of i n t e r e s t to determine whether the enzyme was associated with mitochondria or with nerve endings which sediment i n t h i s f r a c t i o n . Thus mitochondrial suspensions were subjected to the f i v e - l a y e r sucrose density gradient c e n t r i f u g a t i o n by the procedure of De Robertis et a l (31). Five subfractions were obtained ( F i g . 6) and diesterase a c t i v i t y was determined i n each. In addition, each subtraction was subjected to electron microscopy. The d i s t r i b u t i o n of the enzyme i n these preparations i s shown i n Table I I . The bulk (49%)appeared i n subfraction A at the top of the gradient of density. This was a white narrow layer with w e l l defined boundaries. Electron microscopy revealed the presence i n subfraction A of myelin fragments of d i f f e r e n t s i z e ( Fig. 17)^ Subfraction B was translucent grey and l e s s t i g h t l y packed. The boundaries were d i f f u s e , the upper portion beginning a l i t t l e below subfraction A and the lower portion extending to the upper l e v e l of the 1 M sucrose. This subfraction contained only 7% of the t o t a l enzyme a c t i v i t y and electron microscope observation revealed a heterogenous mixture of c e l l components c o n s i s t i n g of fragments of membranous materials, dense bodies of nerve endings and synaptic v e s i c l e s , and some mitochondria (Fig. 18). Subfraction C was a pale yellow layer with d i f f u s e boundaries extending from the lower portion of the 1 M sucrose to the middle of the 1.2 M sucrose l e v e l . This 36 TABLE I I Diesterase A c t i v i t y i n the Mitochondrial Subtractions Subfraction A c t i v i t y (%) Nitrogen (%) A 48.7 39.1 B 7.35 4.13 C 10.5 11.8 D 13.4 24.3 E 9.04 7.74 Total recovery (%) 89.0 87.1 37 Figure 17. Electronmicrograph of subfraction A of the mitochondrial f r a c t i o n showing e s s e n t i a l l y myelin fragments of d i f f e r e n t s i z e . (X 22,500) 38 Figure 18. Electronmicrograph of subfraction B of the mitochondrial f r a c t i o n showing a heterogenous mixture of c e l l components. (X 22,500) 39 subfraction contained 10.5% of the enzymic a c t i v i t y and electron microscope observation revealed mostly p a r t i c l e s of nerve endings, synaptic v e s i c l e s and some mitochondria ( F i g . 19). Subfraction D was a denser layer than subfraction C with i t s d i f f u s e boundaries extending from j u s t below f r a c t i o n C to the middle portion of the 1.4 M sucrose l e v e l . This subfraction contained 13.4% of the enzyme a c t i v i t y and electron microscope observation showed e s s e n t i a l l y larger p a r t i c l e s of nerve endings with synaptic v e s i c l e s and more mitochondria than i n subfraction C (Fig. 20). Subfraction E was the orange-brown p e l l e t found at the bottom of the tube. This subfraction contained 9% of the enzyme a c t i v i t y and electron microscope observation revealed s t i l l l a r g er p a r t i c l e s , l a r g e l y of mitochondria with some nerve endings and other membranous components (Fig. 21). I t can be seen from these studies that the enzyme i s l a r g e l y associated with myelin (subfraction A) rather than with synaptic v e s i c l e s or mitochondria. In f a c t , the smaller amounts of enzyme a c t i v i t y i n the lower subfractions may be due to contamination of each of these with myelin p a r t i c l e s as evident from the electron micrographs. I I . Subfractionation of the Microsomal F r a c t i o n I t w i l l be r e c a l l e d (Table I) that 25% of the t o t a l enzyme resided i n the microsomal f r a c t i o n . We considered i t f e a s i b l e to subject t h i s f r a c t i o n to density gradient c e n t r i f u g a t i o n , because of the f a c t that any myelin should r i s e i n the gradient of density. Thus the microsomal preparation was fractionated i n a f i v e - l a y e r system according to De Robertis e_t a l (31). Four subfractions were obtained (a, b_, c_ and d_). Subtraction a had the same gross appearance as that of subfraction A. I t was also s i t u a t e d at the upper por t i o n of the 0.8 M sucrose l e v e l ; and therefore, i t was not s u r p r i s i n g that e l e c t r o n microscope observation revealed e s s e n t i a l l y small fragments of myelin 40 Figure 19. Electronmicrograph of subfraction C of the mitochondrial f r a c t i o n showing more nerve endings, synaptic v e s i c l e s , and some mitochondria. (X 25,000) 41 Figure 20. Electronmicrograph of subfraction D of the mitochondrial fraction showing larger particles of nerve ending with synaptic vesicles, and mitochondria. (X 25,000) 42 Figure 21. Electronmicrograph of the p e l l e t E of the mitochondrial f r a c t i o n showing s t i l l l arger p a r t i c l e s . More mitochondria could be seen with nerve ending and other membranous components. (X 25,000) 43 sheath and other membranous components ( F i g . 22). As revealed i n Table I I I , t h i s subtraction contained 60% of the diesterase a c t i v i t y of the microsomal preparation. Subfraction b_ was a l i g h t translucent grey, almost transparent layer extending from j u s t below subfraction a to approximately the 3/4 portion of the 0.8 M sucrose l e v e l . Although not w e l l defined, e l e c t r o n microscope observation revealed a heterogenous mixture of small c e l l u l a r components co n s i s t i n g of myelin fragments, nerve endings and synaptic v e s i c l e s , mitochondria, and other membranous fragments ( F i g . 23). This subfraction contained 16% of the enzyme a c t i v i t y . Subfraction c_ was a yellowish trans-lucent grey l a y e r . I t was s l i g h t l y denser than subfraction b_, extending from the upper portion of the 1 M sucrose to two-thirds of the 1.2 M sucrose l e v e l . This subfraction contained 20% of the enzyme a c t i v i t y and electron microscope observation revealed numerous membrane fragments with dense bodies l i k e synaptic v e s i c l e s , although not w e l l defined (Fig. 24). Subfraction d was the grey p e l l e t found at the bottom of the tube. I t contained 13.8% of the enzyme a c t i v i t y and electron microscope observation revealed numerous tiny and dense v e s i c u l a r bodies ( F i g . 25). Again from these r e s u l t s there i s a strong i n d i c a t i o n that the enzyme i s l a r g e l y associated with myelin. I I I . Subfractionation of the Nuclear Fra c t i o n Although the nuclear f r a c t i o n contained only 6% of the t o t a l diesterase a c t i v i t y (Table I ) , i t was subjected to density gradient c e n t r i f u g a t i o n by the procedure of De Robertis et a l (31). Two subfractions (a and y) were obtained. The transparent region between the two bands i s r e f e r r e d to as subfraction 3. Subfraction a had, on gross inspection, the same appearance and was s i m i l a r l y s i t uated on the gradient as that of subfractions A and a.. The only d i f f e r e n c e observed was a smaller amount of the material present. Figure 22. Electronmicrograph of subfraction a of the microsomal f r a c t i o n showing e s s e n t i a l l y small myelin fragments. (X 22,500) 45 TABLE I I I Diesterase A c t i v i t y i n the Microsomal Subtractions Subtraction A c t i v i t y Nitrogen in (%) a 59.5 59.5 b 16.1 14.6 c 20.1 21.1 d 13.8 12.3 T o t a l recovery (%) 109.5 107.5 4 I fcr>* i -5 Figure 23. Electronmicrograph of subfraction b^  of the microsomal f r a c t i o n showing a heterogenous mixture of small c e l l u l a r components. (X 22,500) Figure 24. Electronmicrograph of subfraction c of the microsomal f r a c t i o n showing numerous membrane fragments with dense bodies l i k e synaptic v e s i c l e s . (X 22,500) •TV r "V . • * Figure 25. Electronmicrograph of the p e l l e t d. of the microsomal f r a c t i o n showing numerous tiny and dense v e s i c u l a r bodies. (X 22,500) 49 Once again, electron microscope observation revealed e s s e n t i a l l y myelin 0 fragments of d i f f e r e n t s i z e and some mitochondria present ( F i g . 26), and remarkably i t contained 92% of the t o t a l diesterase a c t i v i t y of the nuclear f r a c t i o n (Table IV). Subfraction y was the l i g h t and flakey sediment at the bottom of the tube. I t contained 10.6% of the enzyme a c t i v i t y and e l e c t r o n microscope observation revealed membrane fragments with dense bodies which could be nuclear fragments and some mitochondria were present (F i g . 27). Subfraction 0, . the transparent portion of the tube between subfractions a and y, contained 10.6% of the enzyme a c t i v i t y . A l l these studies provided a very strong i n d i c a t i o n that the 2',3'-nucleoside phospho-diesterase was associated with myelin. Diesterase A c t i v i t y " i n White and Grey Matter of Rabbit Cerebrum The evidence thus f a r presented, favored the a s s o c i a t i o n of the diesterase with myelin. The p o s s i b i l i t y , however, s t i l l existed that the enzyme was located i n the soluble cytoplasmic material or some c e l l u l a r fragment, such as that of neuronal or g l i a l process, nerve endings or mitochondria. The enzyme could be released from the c e l l u l a r component during homogenization and then adsorbed onto the myelin fragments. I f the diesterase was t r u e l y associated with myelin, one would expect to f i n d most of the a c t i v i t y i n white matter of the br a i n . So, i n the next experiment, white matter was separated from grey matter and assayed for diesterase a c t i v i t y . Table V indicates that white matter has greater diesterase a c t i v i t y than grey matter. 50 Figure 26. Electronmicrograph of subfraction a of the nuclear f r a c t i o n showing e s s e n t i a l l y myelin fragments of d i f f e r e n t s i z e . (X 9,000) TABLE IV Diesterase A c t i v i t y i n the Nuclear Subfractions Subtraction A c t i v i t y Nitrogen (%) a 92.0 41.0 10.6 (undetectable) Y 10.6 20.8 T o t a l recovery (%) 113.2 61.8 52 F i g u r e 27. E l e c t r o n m i c r o g r a p h o f s u b f r a c t i o n y o f t h e n u c l e a r f r a c t i o n showing membrane fragments w i t h dense b o d i e s and some m i t o c h o n d r i a . (X 22,500) 53 TABLE V Diesterase A c t i v i t y i n White and Grey Matter of Rabbit Cerebrum F r a c t i o n A c t i v i t y / g Nitrogen (mg)/g (wet) tis s u e (wet) tissue White matter 344 16.9 Grey matter 116 16.2 54 Preparation of Diesterase from Brain Tissue Drummond et a l (24) extracted the enzyme from a dried acetone powder of whole beef b r a i n . From the foregoing, i t would seem that the enzyme could be more r e a d i l y obtained by extracting white matter. The next phase of the study was to obtain larger quantities of myelin from which to extract the enzyme. Hence, white matter of beef brain was homogenized and the myelin separated by density gradient by the procedure of Whittaker (see Experimental Procedure). An acetone powder was produced from the myelin preparation as described i n the Experimental. Table VI shows the diesterase a c t i v i t y i n the acetone extract as compared to the crude white matter homogenate. The / ( A A A data i n d i c a t e that the acetone extraction resulted i n a doubling of diesterase a c t i v i t y . This increase i n enzyme a c t i v i t y i s probably due to the substrate being more accessible to the enzyme as a r e s u l t of some l i p i d s extracted by the acetone. The acetone powder extract represented a f i v e - f o l d p u r i f i c a t i o n over the s t a r t i n g white matter homogenate. Thus acetone powders prepared from beef b r a i n myelin proved to be an excellent source of the enzyme. Our next e f f o r t s needed to be directed toward s o l u b i l i z a t i o n and further p u r i f i c a t i o n of the enzyme. Attempts were made to s o l u b i l i z e the diesterase. L i s t e d below are the various reagents employed i n the attempt: 1. S a l t - e x t r a c t i o n of the acetone powder extract a) 0.15 M KC1, pH 7.5 b) 3.0 M NaCl, pH 7.5 c) 3.0 M NaCl, pH 5.5 2. Butanol extraction followed by s a l t extraction (0.15 M KC1, pH 7.5) of  the acetone powder extract. TABLE VI Diesterase A c t i v i t y i n the Acetone Extract of Myelin F r a c t i o n from Beef Brain White Matter White matter Acetone extract T o t a l a c t i v i t y 9,930 21,300 % T o t a l a c t i v i t y (100) 215 To t a l proteins (mg) 423 171 S p e c i f i c a c t i v i t y 23.5 130 56 3. Enzyme dig e s t i o n of the acetone powder extract a) Lipase (Type I) from wheat germ b) Lipase (Type II) from hog pancreas c) Lecithinase-C (Type I) from CI. w e l c h i i d) Lecithinase-D (Type II) from cabbage 4. Detergents a) Sodium desoxycholate b) Tween-20, -60 c) A r l a c e l 80 d) T r i t o n X-35, -45, -100, -305 Because our attempts to s o l u b i l i z e the enzyme f a i l e d and because of l i m i t a t i o n i n time, i t was decided to use the acetone powder preparation for further studies of the properties of the enzyme. Properties of the Diesterase I. Enzyme Concentration and Time Course (T< ^ w>ofe^The acetone extract of beef myelin was used i n a l l the following experiments. The conditions of the assay were chosen so that the rea c t i o n rate was d i r e c t l y proportional to the enzyme concentration ( Fig. 28). The reac t i o n was l i n e a r with respect to time throughout the 20 minutes incubation period ( F i g . 29). I I . S p e c i f i c i t y Studies Since the enzyme opens the c y c l i c d i e s t e r linkage of cyclic-ended dinucleotides i n a manner s i m i l a r to which i t acts on c y c l i c mononucleotides (24), i t was of i n t e r e s t to determine i f the enzyme could attack cyclic-ended oligonucleotides. These materials could be considered as possible p h y s i o l o g i c a l substrates f o r the enzyme. O e 3 0.2 Q QJ CO >-_J o cr o >-X UJ r-< CC I-co CD CO 0.1 0 0.6 1.2 PROTEIN (>ig) Figure 28. E f f e c t of enzyme concentration on the rate of hydrolysis of 2',3'-cyclic AMP. Standard assay conditions were employed. A l k a l i n e phosphatase concentration, constant at 20 ug protein, was used. The acetone extract of beef b r a i n myelin was used. 58 0 15 3 0 TIME(min) Figure 29. Time course for the hydrolysis of 2',3'-cyclic AMP by the diesterase. Standard assay conditions were employed with a l k a l i n e phosphatase (20 ug). The acetone extract of beef brain myelin (0.912 ug protein) was used. 59 (a) Action of the enzyme on ApA c y c l i c - P In order to examine the a b i l i t y of the enzyme to open the di e s t e r bond of ApA c y c l i c - P , t h i s substrate was incubated with the enzyme and the product subjected to paper chromatography i n solvent A ( F i g . 30). When the substrate was incubated with diesterase alone, only one product spot appeared (chromatogram 2, F i g . 30). When a l k a l i n e phosphatase was also present i n the re a c t i o n mixture, a new and d i f f e r e n t product spot appeared (chromatogram 4), with an s l i g h t l y greater than that of ApA c y c l i c - P . Since no AMP was present on the chromatogram, i t was evident that the diesterase d i d not s p l i t the internucleotide bond of t h i s cyclic-ended dinucleotide. Chromatogram 2 i n fac t indicated that the diesterase had s p l i t the d i e s t e r bond with formation of ApAp. In the presence of a l k a l i n e phosphatase, t h i s product would be converted to ApA (chromatogram 4). How-ever, t h i s product migrated only s l i g h t l y f a s t e r than ApA c y c l i c - P and these two materials were c l e a r l y not w e l l separated i n t h i s solvent system (chromatogram 4). I t was therefore necessary to provide further evidence that the product of the complete system (diesterase and a l k a l i n e phosphatase) was ApA. In another experiment (Fig. 31), the concentration of both the diesterase and the a l k a l i n e phosphatase were added i n excess so that no unreacted substrate remained. The reaction mixtures were chromatographed i n solvent B, and again, a si n g l e product res u l t e d from the diesterase attack on ApA c y c l i c - P (chromatogram 2, F i g . 31). In the presence of a l k a l i n e phosphatase, a s i n g l e product spot re s u l t e d , c l e a r l y d i s t i n c t from ApA c y c l i c - P and having an i d e n t i c a l R^ to authentic ApA (chromatogram 3 and 4). Thus, conclusive evidence was obtained showing that the diesterase s p l i t the c y c l i c phosphate bond of ApA c y c l i c - P g i v i n g r i s e to the reac t i o n product, ApAp, without 60 i ) 0 0 0 0 < o " o >> o ro Q. 5 < i to CVI F i g u r e 30. A c t i o n o f t h e enzyme on ApA c y c l i c - P . Tube 1, c o n t r o l -w i t h o u t enzymes; Tube 2, d i e s t e r a s e o n l y (1.08 y p r o t e i n o f a c e t o n e powder e x t r a c t o f b e e f b r a i n m y e l i n ; Tube 3, a l k a l i n e p h o s p h a t a s e o n l y (20 y p r o t e i n ) ; Tube 4, d i e s t e r a s e (1.08 y p r o t e i n ) and a l k a l i n e p h o s p h a t a s e (20 y p r o t e i n ) . 0.304 Vmoles o f ApA c y c l i c - P were added t o each o f the f o u r t e s t t u b e s , d e s c r i b e d above, t h a t c o n t a i n e d 0.20 umoles o f T r i s - H C l , pH 7.5, i n a f i n a l volume o f 0.09 m l . The s o l u t i o n s were i n c u b a t e d a t 30° f o r 20 mi n u t e s w i t h s h a k i n g , and 0.01 ml o f g l a c i a l a c e t i c a c i d was added t o s t o p t h e r e a c t i o n . The e n t i r e volume of each i n c u b a t i o n m i x t u r e was s p o t t e d on Whatman No. 3MM paper and chromato-graphed ( d e s c e n d i n g ) i n S o l v e n t A f o r 15 h o u r s . 61 0 0 0 0 < Q. < F i g u r e 31. A c t i o n o f the enzyme on ApA c y c l i c - P . Tube 1, c o n t r o l - w i t h o u t enzymes; Tube 2, d i e s t e r a s e o n l y (30.4 y p r o t e i n ) ; Tube 3, d i e s t e r a s e (30.4 y p r o t e i n o f a c e t o n e powder e x t r a c t o f b e e f b r a i n m y e l i n ) and a l k a l i n e p h o s p h a t a s e (30 y p r o t e i n ) . 0.304 umoles o f ApA c y c l i c - P were added t o each o f t h e t h r e e t e s t t u b e s , d e s c r i b e d above, t h a t c o n t a i n e d 0.10 umoles o f T r i s - H C l , pH 7.5, i n a f i n a l volume o f 0.07 m l . The s o l u t i o n s were i n c u b a t e d a t 30° f o r 20 m i n u t e s w i t h s h a k i n g , and the n c o o l e d i m m e d i a t e l y i n an i c e - b a t h . A s u i t a b l e volume o f each i n c u b a t i o n m i x t u r e was s p o t t e d on Whatman No. 3MM paper and chromatographed ( d e s c e n d i n g ) i n S o l v e n t B f o r 20 h o u r s . 62 rupture of the internucleotide bond. The a l k a l i n e phosphatase, i n turn, converted ApAp to ApA. As shown i n F i g . 31, i t was possible to separate ApA c y c l i c - P from ApA with solvent B. (b) Action of the enzyme on Ap(Ap)^A c y c l i c - P To examine the a c t i o n of the enzyme on Ap(Ap) 2A c y c l i c - P , t h i s substrate was incubated with the enzyme and the product subjected to paper chromatography as shown i n F i g . 32. The r e s u l t s observed were s i m i l a r to those obtained i n the ApA c y c l i c - P experiment i n solvent A. Only one product spot appeared when the substrate was incubated with diesterase alone (chromatogram 2, F i g . 32). I t seemed l o g i c a l that t h i s product was Ap(Ap) 2Ap. In the presence of both a l k a l i n e phosphatase and diesterase, a spot (chromatogram 4) appeared with an almost i d e n t i c a l to Ap(Ap) 2A c y c l i c - P . This new product was evident by an increase i n s i z e of that spot and was presumed to be a mixture of Ap(Ap) 2A and Ap(Ap) 2A c y c l i c - P . No AMP or any other spot that would i n d i c a t e a dinucleotide was present on the chromatogram i n d i c a t i n g that the diesterase did not s p l i t the i n t e r -nucleotide bonds of t h i s cyclic-ended tetranucleotide. As indicated i n chromatogram 2, the diesterase seemed to have s p l i t the d i e s t e r bond with the formation of Ap(Ap) 2Ap which, i n the presence of a l k a l i n e phosphatase, was converted to Ap(Ap) 2A (chromatogram 4). Since Ap(Ap) 2A and Ap(Ap) 2A c y c l i c - P were c l e a r l y not separated i n solvent A (chromatogram 4), the spot (dotted rectangle, chromatogram 4) was eluted, spotted on paper, and developed i n solvent B which was known from preliminary experiments ( F i g . 33) to separate these two compounds. Indeed, two spots appeared from the o r i g i n a l spot (chromatogram 2, F i g . 33) i n d i c a t i n g that the eluted material from chromatogram 4, F i g . 32 contained a mixture of two compounds. With the 63 0 0 0 0 0 0 < o o OJ or < i "to "CM a < in Figure 32. Action of the enzyme on Ap(Ap)2A cyclic-P. Tube 1, control -without enzymes; Tube 2, diesterase (1.08 y protein of acetone powder extract of beef brain myelin); Tube 3, alkaline phosphatase (20 y protein); Tube 4, diesterase (1.08 y protein) and alkaline phosphatase (20 y protein). 0.152 umoles of Ap(Ap)2A cyclic-P were added to each of the four test tubes, described above, that contained 0.20 umoles of Tris-HCl, pH 7.5, in a f i n a l volume of 0.09 ml. The solutions were incubated at 30° for 20 minutes with shaking, and 0.01 ml of glacial acetic acid was added to stop the reaction. The entire volume of each incubation mixture was spotted on Whatman No. 3MM paper and chromatographed (descending) in Solvent A for 15 hours. Figure 33. Development of eluted spot (as indicated i n Figure 32) i n Solvent B for 40 hours. Ap(Ap)„A was obtained by incubating excess a l k a l i n e phosphatase with Ap(Ap) 2Ap under standard conditions. 65 a i d of references (chromatograms 1 and 3, F i g . 33), the two spots of chromatogram 2, F i g . 33 were i d e n t i f i e d as Ap(Ap) 2A c y c l i c - P and Ap(Ap) 2A. To add further evidence, another experiment on the cyclic-ended tetranucleotide was performed as described i n F i g . 34. This time, an excess of diesterase was added so that no unreacted substrate remained, and the reaction mixture was chromatographed i n solvent B. With the complete system (diesterase and a l k a l i n e phosphatase), a s i n g l e spot resulted (chromatogram 2, F i g . 34), c l e a r l y d i s t i n c t from Ap(Ap) 2A c y c l i c - P and having an i d e n t i c a l to the product obtained by incubating Ap(Ap) 2Ap with a l k a l i n e phosphatase (chromatogram 3, F i g . 34). Thus, evidence was obtained showing that the diesterase s p l i t the c y c l i c d i e s t e r bond of Ap(Ap) 2A c y c l i c - P giving r i s e to the r e a c t i o n product, Ap(Ap) 2Ap, without rupture of i n t e r n u c l e o t i d e bonds. The a l k a l i n e phosphatase, i n turn, converted Ap(Ap) 2Ap to Ap(Ap) 2A. (c) Action of the enzyme on Ap(Ap)^A c y c l i c - P In determining the hydrolysis of Ap(Ap)^A c y c l i c - P by the diesterase, one major problem was encountered. This was the problem of f i n d i n g a solvent system which could separate the r e a c t i o n products from the substrate. Many solvent systems were t r i e d but none were adequate. Therefore, other methods were sought. I f the oligonucleotide could be broken up a f t e r i t s reaction with the diesterase into mononucleotides, then, the a v a i l a b l e solvent systems could be used. Spleen phosphodiesterase proved to be u s e f u l as t h i s enzyme was shown not to attack the c y c l i c d i e s t e r bond of 2',3'-cyclic AMP. Hence, the basis f o r the following experiments on Ap(Ap),A c y c l i c - P i s as follows: 66 0 D 0 0 < CL < CL < Figure 34. Action of the enzyme on ApCAp^A c y c l i c - P . Tube 1, con t r o l - without enzymes; Tube 2, diesterase (30.4 y protein of acetone powder extract of beef b r a i n myelin) and a l k a l i n e phosphatase (20 y p r o t e i n ) . 0.152 umoles of Ap(Ap)2A c y c l i c - P were added to each of the two test tubes, described above, that contained 0.10 umoles T r i s - H C l , pH 7.5, i n a f i n a l volume of 0.06 ml. The solutions were incubated at 30° for 20 minutes with shaking, and then cooled immediately i n an ice-bath. A s u i t a b l e volume of each incubation mixture was spotted on Whatman No. 3MM paper and chromatographed (descending) i n Solvent B for 40 hours. ApCAp^A was obtained as described i n Figure 33. 67 Step 1. Ap(Ap) 6A c y c l i c - P diesterase > Ap(Ap) 6Ap Step 2. Ap(Ap),Ap spleen ^ 6 phosphodiesterase r As a c o n t r o l : Step 1. Ap(Ap) 6A c y c l i c - P no diesterase ^ Ap(Ap) 6A c y c l i c - P Step 2. Ap(A P) 6A c y c l i c - P p h o s p g d i e s t e r a s e > 7 Ap + A c y c l i c - P The cyclic-ended octanucleotide i s hydrolyzed by the diesterase followed by the treatment of the re a c t i o n product with the spleen phosphodiesterase. The r e s u l t on a chromatogram should therefore have only one spot (AMP) present. In the f i r s t experiment, as described i n F i g . 35, the diesterase, a f t e r reacting with the cyclic-ended octanucleotide, was i n a c t i v a t e d by the chromatographic solvent B. I t was noticed that at t h i s step, only one spot was observed ( s t i l l on the o r i g i n ) , and since Ap(Ap)^A c y c l i c - P , ApA c y c l i c - P , and AMP have higher values than Ap(Ap)^A c y c l i c - P , i t could be concluded that the diesterase did not attack the internucleotide bonds of t h i s c y c l i c -ended octanucleotide. Next, the spot of each incubation mixture was eluted, treated with spleen diesterase as described i n F i g . 35, and again spotted on paper and developed i n solvent B. If the diesterase did not s p l i t the diester bond of Ap(Ap)^A c y c l i c - P , there should be, a f t e r treatment with the spleen diesterase, two spots appearing on the chromatogram having R^ values corresponding to authentic AMP and 2',3'-cyclic AMP. The two spots should also have i d e n t i c a l R^ values as those from the c o n t r o l (without b r a i n th diesterase and with spleen diesterase) having a 2',3'-cyclic AMP spot, 1/8 as dense (under UV-light) as the AMP spot. In the actual experiment, only one spot (AMP) r e s u l t e d . No spot corresponding to 2',3'-cyclic AMP was present (chromatogram 2, F i g . 35). This provided evidence that the diesterase had 0 0 Ol CO Figure 35. Action of the enzyme on Ap(Ap)^A cyclic-P. Tube 1, control - without diesterase; Tube 2, diesterase (30.4 y protein of acetone powder extract of beef brain myelin). (A) 0.038 umoles of Ap(Ap)gA cyclic-P were added to each of the two test tubes, described above, that contained 0.10 umoles of Tris-HCl, pH 7.5, in a f i n a l volume of 0.03 ml. The solutions were incubated at 30° for 1 hour with shaking, and then cooled immediately in an ice-bath. The entire volume of each incubation mixture was spotted on Whatman No. 3MM paper and chromatographed (descending) in Solvent B for 4 hours (to inactivate the diesterase). The spot of each incubation mixture was eluted and added up to 0.1 ml with water. (B) To each of the above solutions, were added 0.10 umoles of Tris-HCl, pH 7.5, and 0.2-0.3 units of spleen phospho-diesterase to a f i n a l volume of 0.14 ml. The solutions were incubated at 30° for 2 hours with shaking, and then cooled immediately i n an ice-bath. The entire volume of each incubation mixture was again spotted on Whatman No. 3MM paper and chromatographed (descending) in Solvent B for 15 hours. 69 s p l i t the c y c l i c d i e s t e r bond of Ap(Ap)^A c y c l i c - P , as with lower c y c l i c -ended oligonucleotides. Further information on the a c t i o n of the enzyme on t h i s substrate was observed i n the next experiment as described i n F i g . 36. This experi-ment d i f f e r e d from the f i r s t only i n the manner i n which the diesterase was i n a c t i v a t e d . While i n the f i r s t experiment, the diesterase was i n a c t i v a t e d by the chromatographic solvent, the enzyme i n t h i s second experiment was destroyed by plunging the tubes containing the r e a c t i o n mixture (substrate plus diesterase) i n t o a b o i l i n g water-bath for two minutes. Then, a f t e r cooling, the r e a c t i o n mixture was treated with the spleen enzyme, spotted on paper and developed i n solvent B. As with the f i r s t experiment, only one spot (AMP) was observed (chromatogram 2, F i g . 36). In t h i s solvent system, 2',3'-cyclic AMP i s e a s i l y separated from 2'(3')-AMP. Hence, these experiments i n d i c a t e that the diesterase was capable of opening the c y c l i c d i e s t e r bond of the cyclic-ended octanucleotide, Ap(Ap)^A c y c l i c - P , without rupturing internucleotide bonds, the product therefore being Ap(Ap),Ap. o I I I . Relative Rate of Hydrolysis Drummond et a l (24) reported that c y c l i c phosphates bearing purine bases were hydrolyzed more r a p i d l y than those bearing pyrimidine bases. Since i t had been shown that cyclic-ended oligonucleotides were hydrolyzed by the enzyme, i t was of i n t e r e s t to determine the rate of hydrolysis of these compounds as compared with 2',3'-cyclic AMP. I t can be seen i n Table VII that Ap(Ap^A c y c l i c - P was hydrolyzed at l e s s than one-half the rate of 2',3'-cyclic AMP. Because of the d i f f i c u l t i e s i n separating the cyclic-ended octanucleotide from the product of enzymatic attack, the rate 0 Q. < 0. o I ro "c\J o o o! 00 Figure 36. Action of the enzyme on Ap(Ap)^A cyclic-P. Tube 1, control - without diesterase; Tube 2, diesterase (30.4 y protein of acetone powder extract of beef brain myelin). (A) 0.038 umoles of Ap(Ap)^A cyclic-P were added to each of the two test tubes, described above, that contained 0.10 umoles of Tris-HCl, pH 7.5, in a f i n a l volume of 0.03 ml. The solutions were incubated at 30° for 1 hour with shaking, and then, plunged immediately into a boiling water bath for 2 minutes (to inactivate the diesterase). (B) To each of the above, cooled solutions, were added 0.10 umoles of Tris-HCl, pH 7.5, and 0.2-3.0 units of spleen phosphodiesterase to a f i n a l volume of 0.06 ml. The solutions were incubated at 30 for 2 hours with shaking, and then cooled immediately in an ice-bath. The entire volume of each incubation mixture was spotted on Whatman No. 3MM paper and chromatographed (descending) in Solvent B for 15 hours. 71 TABLE VII Hydrolysis of 2',3'-cyclic AMP versus Ap(Ap) 2A c y c l i c - P Substrate Substrate hydrolyzed Relative rate (umole/mg protein/min) 2',3'-cyclic AMP 1.48 100 Ap(Ap) 2A c y c l i c - P 0.602 40 72 of hydrolysis of th i s compound was not examined. A much longer incubation period time was necessary f o r the complete hydrolysis of t h i s compound as compared with 2',3'-cyclic AMP and we can i n f e r that i t i s hydrolyzed much more slowly than 2',3'-cyclic AMP. IV. Possible Activators or In h i b i t o r s In t h e i r report, Drummond et a l (24) described the i n h i b i t o r y action of a number of metal ions on the enzymatic hydrolysis of 2',3'-cyclic AMP. We have examined the e f f e c t s of a number of purine and pyrimidine derivatives on diesterase a c t i v i t y . We were p a r t i c u l a r l y i n t e r e s t e d i n examining the p o s s i b i l i t y that the diesterase may be regulated i n vivo by nucleoside or nucleoside phosphate esters. For each of the compounds l i s t e d i n Table VIII, a concentration of 1.2-2.0 mM was added to the standard assay. Since a l k a l i n e phosphatase had been shown to hydrolyze mono-, d i - , and t r i -phosphonucleotides (34), the e f f e c t s of those nucleotides tested were analyzed by chromatography (descending) i n solvent B. The e f f e c t s of the other compounds were analyzed c o l o r i m e t r i c a l l y by the modified Fiske and SubbaRow method. No e f f e c t on diesterase a c t i v i t y was observed by a l l compounds tested. V. E f f e c t of Protein Reagents on the Enzyme In recent years, various chemical agents have been used f o r e l u c i d a t i n g the a c t i v e s i t e of an enzyme. Usually, the reagent w i l l react with one type of f u n c t i o n a l group i n the protein and give a very s i g n i f i c a n t a l t e r a t i o n of a c t i v i t y such as i n h i b i t i o n . In t h i s study, three such compounds were tested f o r i n h i b i t i o n of diesterase a c t i v i t y (iodoacetamide (IAA), d i i s o -propylfluorophosphate (DFP), and sodium para-hydroxymercuribenzoate (p-HMB)). DFP i s known to s p e c i f i c a l l y react with the hydroxyl function of serine TABLE VIII Possible Activators or Inhi b i t o r s 1. Theophylline 2. Decoyinine 3. Tubercidin 4. Psicofuranine (psicofuranosyl adenine) 5. 3'-deoxy-3'-amino-adenosine 6. Adenosine 7. Adenylyl(3'-5')adenosine[ApA] 8. Parnate (Tranylcypromine) s u l f a t e 9. Sodium pentobarbital 0. Procaine'HC1 1. 6-Chloro-purine ribonucleoside (6-C1-PRN) 2. 2-amino-purine n i t r a t e 3. 6-Ghloro-purine ,4. 2'-Deoxy-adenosine 5. Adenine 6. Adenosine (5') diphosphate (ADP) .7. Adenosine (5') triphosphate (ATP) .8. 2'-deoxy-5'-adenosine monophosphate (d-5'-AMP) .9. 5'-inosine monophosphate (5*-IMP) 0. 5'-adenosine monophosphate (5'-AMP) .1. 3'-adenosine monophosphate (3'-AMP) 2. 2'-adenosine monophosphate (2'-AMP) 74 residues; while IAA and p-HMB react mainly with s u l f h y d r y l functions of cysteine residues. In the preliminary experiments, preincubation of each -3 of the reagents ( t h e i r preincubation concentrations: IAA, 5 x 10 M; -3 -3 DFP, 0.1 x 10 M; p-HMB, 5 x 10 M) with the diesterase acetone extract (1.52 mg pro t e i n per ml) was c a r r i e d out for 1 hour i n an ice-water bath. A s u i t a b l e d i l u t i o n of the preincubation mixtures was added to the standard assay. As a c o n t r o l , a diesterase acetone extract (1.52 mg prot e i n per ml) was also preincubated without reagent. The i n h i b i t i o n observed was as follows: IAA, 0%; DFP, 0%; p-HMB, 47.4%. I t was noticed that when the preincubation time with p-HMB was extended to 5 hours i n h i b i t i o n was greater than 47.5%. Hence, a time course of the i n a c t i v a t i o n r e a c t i o n was ca r r i e d out. The r e s u l t as shown i n F i g . 37 indicated that f o r maximum i n h i b i t i o n , the preincubation period must be extended to 3 hours. Furthermore, i t was shown that by decreasing the concentration of the diesterase to 0.760 mg pro t e i n per ml i n the preincubation mixture, the i n h i b i t i o n of diesterase a c t i v i t y reached 77.2%. VI. E f f e c t of para-Hydroxymercuribenzoate on the Time Course of the Diesterase A c t i v i t y F i g . 38 shows the e f f e c t of p-HMB on the time course of diesterase a c t i v i t y at various substrate concentrations. Before assaying, p-HMB _3 (5 x 10 M) was preincubated with diesterase (1.52 mg pro t e i n per ml) for 3 hours i n an ice-water bath with shaking every 15 minutes. The preincubation mixture was s u i t a b l y d i l u t e d with 0.01 M Tris-HCl (pH 7.5) and 0.912 ug protein was used f or the assay. As a c o n t r o l , the diesterase was preincubated without p-HMB, and assayed with varying substrate concentrations. The r e s u l t , as shown i n F i g . 38, indicated that the i n h i b i t e d enzyme i s apparently released of i t s i n h i b i t i o n with time and Figure 37. Time course of the preincubation mixture containing p-HMB and diesterase. 76 0 15 30 TIME (min) Figure 38. Effect of p-HMB on the time course of diesterase activity at various 2',3'-cyclic AMP concentrations. Without inhibitor • • • With inhibitor*- Standard assay conditions were employed. The acetone extract of beef brain myelin (0.912 ug protein) was used in the standard assay. 77 that t h i s release of i n h i b i t i o n also varies with the substrate concentration. VII. K i n e t i c s The development of chemical k i n e t i c s as a t o o l f or studying enzyme reactions has contributed greatly to gaining further i n s i g h t i n t o the nature of enzyme reactions and b i o l o g i c a l processes. F i g . 39 shows the time course of diesterase a c t i v i t y at various concentrations of 2',3'-cyclic AMP. When the i n i t i a l v e l o c i t i e s are p l o t t e d against substrate concentration a hyperbolic curve i s obtained ( F i g . 40). According to the M i c h a e l i s -Menten theory of enzyme-substrate i n t e r a c t i o n , the K^ i s equal to the substrate concentration that corresponds to one-half of the maximum v e l o c i t y ; but, since the curve ( F i g . 40) i s a hyperbola, the maximum v e l o c i t y i s vaguely defined. A better method f o r determining the K i s to use a m Lineweaver-Burk p l o t . This method employs the r e c i p r o c a l of the i n i t i a l v e l o c i t y p l o t t e d against the r e c i p r o c a l of the substrate concentration. From such a p l o t ( F i g . 41), the of the diesterase was determined to be 1.9 x 1 0 - 3 M. VIII. E f f e c t of pH on K and V c m max Table IX shows the K and V values at various pH. At pH 5.6, m max 6.0 and 6.5, the buffer used i n the assay was 0.05 ml of 0.2 M c i t r a t e ; at pH 7, 8, and 9, 0.05 ml of 0.2 M Tris-HCl and EDTA (1.2 x 10~ 3 M) was used. EDTA was added to the incubation mixture containing T r i s buffer because i t was observed that both EDTA and c i t r a t e activated diesterase a c t i v i t y , at pH 7, to the same extent. From the data obtained, Lineweaver-Burk pl o t s at each pH were prepared from which K and V values were r r m max determined — K was determined as described above, and V was determined m max from the Y-intercept which i s equal to 1/V. Unfortunately, because of a 78 0 15 30 TIME (min) Figure 39. Time course of reaction at various substrate (2',3'-cyclic AMP) concentrations. Standard assay concentrations were employed. The acetone extract of beef brain myelin (0.912 ug protein) was used. SUBSTRATE CONCENTRATION X I 0 3 M Figure 40. P l o t of i n i t i a l rates of reaction versus substrate (2',3'-cyclic AMP) concentration. 0.5 -0.4 * 0.3 -0.2 -0.1 -j i i i i_ • • i i_ -i i_ • • i • » i i I i i_ 0:5 0.5 1.0 1.5 i i i i i I 2.0 2.5 I / C 2',3-cyclic AMP • X 10 M Figure 41. Lineweaver-Burk p l o t of the r e c i p r o c a l of i n i t i a l v e l o c i t y versus that of substrate concentration. y _ umole substrate hydrolyzed minute-mg protein oo o TABLE IX E f f e c t of pH on K and V m max pH K V m max 5.6 2.63 x 10~ 3 M 8.0 6.0 2.25 x 10~ 3 M 11.78 6.5 1.33 x 10~ 3 M 10.31 7.0 1.24 x 10" 3 M 10.52 8.0 1.10 x 10" 3 M 6.06 9.0 0.885 x 10~ 3 M 2.18 *The acetone extract of beef b r a i n myelin (0.912 pg protein) was used. 82 limited time i n this study, more pH determinations were not possible for determining ionizations of the functional groups on the enzyme. Distribution of Ribonuclease It was mentioned that the physiological role of this enzyme is entirely unknown. As discussed previously, ribonucleoside 2',3'-cyclic phosphates and 23'-cyclic-ended oligonucleotides are intermediates i n the hydrolysis of RNA by RN'ase. Thus, there i s the possibility that these cyclic-ended intermediates may serve as substrates for the diesterase. If the diesterase and RN'ase worked in concert, one might expect the intracellular localization of the two enzymes to be similar. Hence, the next experiment was to study the distribution of RN'ase i n rabbit brain tissue. The primary fractions were prepared as before, and from Table X, i t i s quite clear that RN'ase was present mostly i n the 100,000 x g supernatant f l u i d . Furthermore, from Table XI, i t is apparent that RN'ase activity was approximately evenly distributed between white and grey matter. Thus, from the above data, i t does not seem that the diesterase and RN'ase have any physiological relationship. 83 TABLE X RN'ase i n the Primary Fractions Fr a c t i o n RN'ase (%) Nitrogen (%) Crude Homogenate (100) (100) Nuclear 1.80 4.63 Mi to chondr ia1 17.0 42.3 Microsomal 13.6 23.0 100,000 x g supernatant 57.4 23.3 Tot a l recovery (%) 89.8 93.2 TABLE XI RN'ase i n White and Grey Matter Fract i o n RN'ase (y) per Nitrogen (mg) per gm (wet) t i s s u e gm (wet) tiss u e White matter 58.9 16.9 Grey matter 62.5 16.2 85 DISCUSSION The diesterase i s characterized by extreme i n s o l u b i l i t y , apparently tightlyjbound to t i s s u e particles. Because of t h i s , and because of our i n t e r e s t i n a possible p h y s i o l o g i c a l r o l e f o r the diesterase, our f i r s t experiments were designed to e s t a b l i s h i t s 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 . Using l i g h t homogenization of brain tissue to produce a minimal breakage of c e l l u l a r components, the diesterase a c t i v i t y was found to be mainly i n the mitochondrial f r a c t i o n . With the a i d of electron microscopy, Whittaker (32) and De Robertis (31) have established that the usual preparation of (rat) brain mitochondria i s a heterogenous mixture of c e l l p a r t i c u l a t e s co n s i s t i n g of myelin sheath fragments, nerve endings, mitochondria, and other membranous materials. From our electron micrographs of the mito-chondrial f r a c t i o n , we also established the heterogeneity of this f r a c t i o n and thus the enzyme could have been associated with any one of these o c e l l u l a r components. Whittaker and De Robertis found that the components of the mitochondrial preparation could be separated from each other by discontinuous density gradient c e n t r i f u g a t i o n . A gradient of density serves to eliminate convective currents which occur whenever a dense portion of a s o l u t i o n i s c l o s e r to the axis of r o t a t i o n than i s a l e s s dense portion; thus, enabling the smooth migration of the p a r t i c l e s to occur. By separating the components i n the mitochondrial f r a c t i o n with a sucrose density gradient, we were able to show that the diesterase a c t i v i t y was mainly associated with the l i g h t e s t f r a c t i o n which was composed of fragments of myelin sheaths of various s i z e s as revealed by e l e c t r o n microscopy. Because the other two primary f r a c t i o n s (nuclear and microsomal) contained some diesterase a c t i v i t y , i t was of i n t e r e s t 86 to see which of the c e l l u l a r components i n them were associated with diesterase a c t i v i t y . When microsomal and nuclear f r a c t i o n s were sub-fr a c t i o n a t e d , again diesterase a c t i v i t y was associated with the l i g h t e s t f r a c t i o n which el e c t r o n microscopy revealed as myelin fragments. Furthermore, since i t was observed that white matter contained greater diesterase a c t i v i t y than grey matter, the evidence favors the a s s o c i a t i o n i ^ , — -of the diesterase with myelin and seems to exclude the p o s s i b i l i t y of the enzyme being contained i n , and released from, the soluble cytoplasmic material of some c e l l u l a r component during homogenization. In addition, these findings are i n agreement with those of Drummond et a l (24) who showed that diesterase a c t i v i t y was highest i n those nerve structures that were highly myelinated. " Myelin, as we know i t today, consists of several p r i n c i p a l l i p i d components (plasmalogens, sphingomyelins, cerebrosides, s u l f a t i d e s , diphos-phoinositides and n o n - e s t e r i f i e d cholesterol) along with some p r o t e o l i p i d s and proteins. I t has been shown that peptidase, protease, adenosine triphosphatase, adenosine-5-phosphatase, a l k a l i n e phosphatase, creatine phosphatase, cytochrome oxidase, s u c c i n i c dehydrogenase and pseudo-cholinesterase are enzyme constituents of the peripheral myelin sheath. Current views concerning myelin are that once i t i s deposited, i t remains i n e r t metabolically. Thus, i t i s d i f f i c u l t to imagine how such enzymes that are necessary f o r metabolic reactions, play a r o l e i n t h i s apparently i n e r t s t r u c t u r e . I t i s c l e a r that more studies must be done on the structure and function of myelin. 87 The diesterase c l e a r l y opens the c y c l i c d i e s t e r bond of c y c l i c -ended oligonucleotides, at l e a s t as large as (Ag) and does so, without cleavage of internucleotide bonds. The problem a r i s e s as to the true substrate for the enzyme. I t i s known that 2',3'-cyclic mononucleotides and 2',3 1-cyclic-ended oligonucleotides are intermediates i n the a c t i o n of ribonuclease on RNA. In f a c t these materials can be r e a d i l y i s o l a t e d by rapid d i a l y s i s of a ribonuclease digest of yeast RNA. Ribonuclease i s capable of hydrolyzing 2',3'-cyclic d i e s t e r linkages but the product i s e x c l u s i v e l y pyrimidine 3'-nucleotide or oligonucleotide. In the case of the present diesterase, the product i s e x c l u s i v e l y 2'-nucleotide, although i t attacks those c y c l i c phosphates bearing purine bases more r a p i d l y than those bearing pyrimidine bases (24). This raises the p o s s i b i l i t y of 2'-nucleotides or 2'-ended oligonucleotides having some unique function i n nerve t i s s u e . Nothing i s known however of a p h y s i o l o g i c a l r o l e f or e i t h e r 2',3'-cyclic nucleotides or 2'-nucleotides. In f a c t , as f a r as i s known, measurable l e v e l s of either of these types of materials are not encountered i n l i v i n g t i s s u e . We considered i t possible that the diesterase may act i n concert with ribonuclease. Ribonuclease could produce c y c l i c -ended mono- or oligonucleotides from RNA. Further ribonuclease action would produce 3'-nucleotides. The diesterase could act on these cyclic-ended intermediates to produce 2'-nucleotides. If t h i s were so, one might expect the 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 of the two enzymes to be s i m i l a r . The data show t h e i r d i s t r i b u t i o n to be quite d i f f e r e n t . Such substrates, however, could s t i l l be transported through the c e l l to the s i t e of the diesterase. Our e f f o r t s to s o l u b i l i z e and subsequently p u r i f y the enzyme were not succ e s s f u l . I t might be mentioned that Dr. Stanford Moore of the Rockefeller I n s t i t u t e has, i n a p r i v a t e communication, reported s o l u b i l i z i n g the enzyme, and has engaged on a complete chemical c h a r a c t e r i z a t i o n . Because of the extremely high a c t i v i t y of the diesterase i n the myelin preparation, we considered i t worthwhile to define some of i t s properties beyond those already known (24). The enzyme i s i n a c t i v a t e d by the s u l f h y d r y l reagent, para-hydroxymercuribenzoate. We cannot, however, make any statement as to the p o s s i b i l i t y of a s u l f h y d r y l group being involved i n c a t a l y s i s . The enzyme was unaffected by the presence of a wide v a r i e t y of purine and pyrimidine compounds or d e r i v a t i v e s and various nucleoside mono-, d i - and triphosphates. One might assume, for the time being, that these important materials play no r o l e i n the i n t r a c e l l u l a r r e gulation of the enzyme. 89 ADDENDUM While t h i s thesis was being typed, i t came to our a t t e n t i o n that a paper on the diesterase was presented at the Seventh International Congress of Biochemistry i n Tokyo (August 1967). Kurihara and Tsukada (36) reported studies s i m i l a r to ours i n which they concluded that the diesterase i s located i n myelin and can be employed as a s e n s i t i v e marker f o r myelin of the c e n t r a l nervous system. 90 BIBLIOGRAPHY 1. Jones, W. Am. J . Ph y s i o l . 52, 203 (1920). 2. Dubos, R.J. and Thompson, R.H.S. J . B i o l . Chem. 124, 501 (1938). 3. Kunitz, M. J . Gen. Phy s i o l . 24, 15 (1940). 4. Kunitz, M. Science 108, 19 (1948). 5. Schmidt, G., Cubiles, R., Zo l l n e r , N., Hecht, L., S t r i c k l e r , N., Seraidarian, K., Seraidarain, M. and Thannhauser, S.J. J . B i o l . Chem. 192, 715 (1951). 6. Markham, R. and Smith, J.D. Biochem. J . 52, 552 (1952). 7. Markham, R. and Smith, J.D. Biochem. J . 52, 558 (1952). 8. M e r r i f i e l d , R.B. and Woolley, D.W. J . B i o l . Chem. 197, 521 (1952). 9. Brown, D.M., Dekker, C.A., and Todd, A.R. J . Chem. Soc. p. 2715 (1952). 10. Brown, D.M. and Todd, A.R. J . Chem. Soc. p. 2040 (1953). 11. Volkin, E. and Cohn, W.E. J . B i o l . Chem. 205, 767 (1953). 12. Heppel, L.A. and W h i t f i e l d , P.R. Biochem. J . 60, 1 (1955). 13. Findlay, D., Herries, D.G., Mathias, A.P., Rabin, B.R. and Ross, C.A. Biochem. J . 85, 152 (1962). 14. Kaplan, H.S. and Heppel, L.A. J . B i o l . Chem. 222, 907 (1956). 15. Holden, M. and P i r i e , N.W. Biochem. J . 60, 39 (1955). 16. Frisch-Niggemeyer, W. and Reddi, K.K. Biochim. Biophys. Acta 26, 40 (1957). 17. Shuster, L. J . B i o l Chem. 229, 289 (1957). 18. Sato, K. and Egami, F. J . Biochem. (Tokyo) 44, 753 (1957). 19. Sutherland, E.W. and R a i l , T.W. J . B i o l . Chem. 232, 1065 (1958). 20. Sutherland, E.W. and R a i l , T.W. J . B i o l . Chem. 232, 1077 (1958). 21. Dekker, C.A. Federation Proc. 13, 197 (1954). 22. W h i t f i e l d , P.R., Heppel, L.A. and Markham, R. Biochem. J . 60, 15 (1955). 23. Drummond, G.I. and Perrott-Yee, S. J . B i o l . Chem. 236, 1126 (1961). 24. Drummond, G.I., Iyer, N.T. and Keith, J . J . B i o l . Chem. 237, 3535 (1962). 91 25. Smith, M., Moffatt, J.G. and Khorana, H.G. J . Am. Chem. Soc. 80, 6204 (1958). 26. Bain, J.A. and Rusch, H.P. J . B i o l . Chem. 153, 659 (1944). 27. Woodward, G.E. J . B i o l . Chem. 156, 143 (1944). 28. Fiske, C H . and SubbaRow, Y. J . B i o l . Chem. 66, 375 (1925). 29. McDonall, M.R. In Methods i n Enzymology I I , eds., Colowick, S.P. and Kaplan, N.O., Academic Press, New York (1955), p. 427. 30. Kunitz, M. J . B i o l . Chem. 164, 563 (1946). 31. De Robertis, E., Pellegrino De I r a l d i , A., Rodriguez De Lores Arnaiz, A. and Salganicoff, L. J . Neurochem. 9_, 23 (1962). 32. Whittaker, V.P. Biochem. J . 72, 694 (1959). 33. Umbreit, W.W., B u r r i s , R.H., and Stauffer, J.F. Manometric Techniques  and Tissue Metabolism, Second E d i t i o n , Burgess Publishing Company, Minn. Minn (1951), p. 191. 34. Heppel, L.A., Harkness, D.R. and Hilmoe, R.J. J . B i o l . Chem. 237, 841 (1962). 35. Lovtrup-Rein, H. and McEwen, B.S. J . C e l l B i o l . 30, 405 (1966). 36. Kurihara, T. and Tsukada, Y. 7 International Congress of Biochemistry, Tokyo, Abstract V, J - l l , August 1967. 

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