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Studies on glycogen phosphorylase Gilgan, Michael Wilson 1962

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i STUDIES ON GLYCOGEN PHOSPHORYLASE b y MICHAEL WILSON GILGAN B . S c , 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 , 1959 A THESIS SUBMITTED I N P A R T I A L FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n t h e D e p a r t m e n t o f P h a r m a c o l o g y We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA A u g u s t , 1962 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis f o r scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication of th i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Pharmacology The University of B r i t i s h Columbia, Vancouver 8, Canada. Date ^ Q^tvJr Po ,/5>fc*-i i ABSTRACT PART I GLYCOGEN PHOSPHORYLASE IN BRAIN, ILEUM AND UTERUS In recent years considerable a t t e n t i o n has been devoted to the nature of the glycogen phosphorylase enzyme system i n s k e l e t a l muscle, cardiac muscle and l i v e r . I t has been e s t -a b l i s h e d that those f a c t o r s which a f f e c t the a c t i o n of t h i s enzyme c o n s t i t u t e a major metabolic c o n t r o l mechanism. Con-s i d e r a b l y l e s s a t t e n t i o n has been paid to t h i s enzyme system i n t i s s u e s which are known to c o n t a i n low glycogen, s t o r e s . The present study c o n s t i t u t e s a p r e l i m i n a r y examination of glycogen phosphorylase i n b r a i n , uterus and ileum. I t has been found t h a t b r a i n contains c o n s i d e r a b l y higher phosphorylase a c t i v i t y than l i v e r and i s comparable to heart. Uterus and ileum c o n t a i n enzyme l e v e l s comparable to that of l i v e r . The data suggests that the enzyme e x i s t s i n two forms i n these three t i s s u e s , one a c t i v e i n the presence of adenosine-f?'-monophosphate, the other a c t i v e i n the absence of t h i s n u c l e o t i d e . The enzyme i n each t i s s u e i s thus very s i m i l a r to the s k e l e t a l muscle enzyme, but d i f f e r e n t from that i n l i v e r . P r e l i m i n a r y evidence was also obtained which i n d i c a t e s that phosphorylase a c t i v a t i n g and i n a c t i v a t i n g enzymes were present. Levels of a c t i v e phosphorylase were increased i n b r a i n and uterus i n the presence of epinephrine. The i n t e s t i n a l smooth muscle enzyme f a i l e d to respond to epinephrine. The evidence, although p r e l i m i n a r y , i s c o n s i s t e n t w i t h the idea - i i i that catecholamine-induced muscle c o n t r a c t i o n i s a s s o c i a t e d with phosphorylase a c t i v a t i o n . PART I I SYNTHESIS AND ENZYMATIC DEGRADATION OP SEVERAL DE0XYRIB0NUCLE0STDE-3',51-MONOPHOSPHATES I t i s known, that the phosphorylase a c t i v a t i n g a c t i o n of epinephrine i s mediated through adenosine-3',5'-monophosphate. The metabolism of t h i s important n u c l e o t i d e i s a t t r a c t i n g widespread a t t e n t i o n . In order to study i t s seemingly manifold a c t i o n s , the need has a r i s e n f o r s t r u c t u r a l analogues of the compound. Several ribonucleoside-3 ' ,5 '-monophosphates have already been prepared. In the present work s e v e r a l deoxy-ribonucleoside-3 1 ,5 '-monophosphates were synthesized. These included the nucleoside-35'-monophosphates of deoxyadenosine, deoxyinosine, and deoxyuridine." These compounds were shown to be hydrolysed by a phosphodiesterase from b r a i n which i s s p e c i f i c f o r nucleoside-3' ,5 '-monophosphates. The product of the h y d r o l y s i s , i n each case, was i d e n t i f i e d as the corresponding deoxyribonucleoside-5'-phosphate. i v TABIE OF CONTENTS' PART I Glycogen Phosphorylase i n B r a i n , Ileum and Uterus Page Introduction 1 Experimental Procedure 6 M a t e r i a l s 6 Phosphorylase Assay System and Unit 7 Results 9 Phosphorylase D i s t r i b u t i o n i n Rabbit Tissues 9 E f f e c t of Epinephrine i n vivo on Tissue Phosphorylases 1 1 Phosphorylase D i s t r i b u t i o n i n Mammalian B r a i n l l ; E f f e c t of Epinephrine on Smooth Muscle Phosphorylase 1 7 Phosphorylase P u r i f i c a t i o n Attempts 1 9 D i s c u s s i o n 2 0 PART I I Synthesis and Enzymatic Degradation of Several  Deoxyribonucleoside - 3 ' ,5>' -Monophosphates I n t r o d u c t i o n 22 Experimental Methods and Res u l t s Zh, M a t e r i a l s 2\\ Chromatographic Systems 2 i | E l e c t r o p h o r e s i s 2 i | V TABLE OF CONTENTS PART I I (continued) Page Enzyme Preparations and Incubation Conditions 25> a. B r a i n Phosphodiesterase 25" b. Muscle Adenylate Deaminase 26 c. Snake Venom 27 P r e p a r a t i o n and P u r i f i c a t i o n of C y c l i c - 3 ' , 5 ' -Deoxynucleotides 28 a. Deoxyadenosine-3 ' ,5 '-Monophosphate 28 b. N-Benzoyl-Deoxyadenosine -3 ' ,5> ' -Monophosphate 29 c. Deoxyinosine - 3 5 * 1-Monophosphate 31 d. Deoxyuridine-3',5-'•-Monophosphate 33 Enzymatic I d e n t i f i c a t i o n of the C y c l i c - 3 ' , 5 ' -Deoxynucleotides 33 R e l a t i v e H y d r o l y s i s Rates of the C y c l i c - 3 1 , 5 " ' -Deoxynucleotides. by B r a i n Diesterase 38 D i s c u s s i o n I | ! B i b l i o g r a p h y I4.I4. v i LIST OP TABLES T i t l e Phosphorylase A c t i v i t y of Rabbit T i s s u e s Phosphorylase I n a c t i v a t i o n i n Mouse B r a i n • E x t r a c t s A Comparative Study of Rabbit T i s s u e Phosphorylases a f t e r Pretreatment w i t h Epinephrine D i s t r i b u t i o n of Phosphorylase A c t i v i t y i n D i f f e r e n t Areas of Rabbit B r a i n D i s t r i b u t i o n of Phosphorylase i n D i f f e r e n t Areas of Dog B r a i n The E f f e c t of Epinephrine on U t e r i n e and I n t e s t i n a l Phosphorylase i n v i t r o LIST OP FIGURES A Chromatogram of the (5'-) N u c l e o t i d e Products of B r a i n Phosphodiesterase A c t i o n on the Deoxynucleoside-3',5 1 -Monophosphates A Chromatogram of the Products of Snake Venom A c t i o n Time Rate of B r a i n Phosphodiesterase H y d r o l y s i s of Deoxynucleoside-3',5'-Monophosphates' v i i A BBREVIATIONS The f o l l o w i n g a b b r e v i a t i o n s w e re u s e d t h r o u g h o u t t h e t h e s i s : AMP adenosin.e-5' -monophosphate DOC d l c y c l o h e x y l c a r b o d i i m i d e EDTA e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d IR 120 Amberlike IR 120 TCA t r i c h l o r o a c e t i c a c i d T r i s t r i s ( h y d o x y m e t h y l ) a m i n o m e t h a n e - 1 -PART I GLYCOGEN PHOSPHORYLASE IN BRAIN, ILEUM AND UTERUS INTRODUCTION In recent years glycogen phosphorylase has been e x t e n s i v e l y studied i n l i v e r and i n s k e l e t a l muscle. These i n v e s t i g a t i o n s have e s t a b l i s h e d that the conversion of glycogen to g l u c o s e - 1 -phosphate i s mediated by a complex enzyme system which may be in f l u e n c e d by hormonal and other p h y s i o l o g i c a l f a c t o r s . I t has become c l e a r that the phosphorylase enzyme system f u n c t i o n s as an important metabolic c o n t r o l mechanism r e g u l a t i n g the u t i l i z a t i o n of glycogen during work or s t r e s s . Furthermore, there i s evidence that the phosphorylase system may have an even more d i r e c t r o l e i n muscle c o n t r a c t i o n than that of p r o v i d -ing f u e l f o r the c o n t r a c t i l e process. .In order to b r i n g these f a c t s i n t o focus and i n order to o r i e n t the present problem a b r i e f summary of current knowledge i s necessary. Phosphorylase i n s k e l e t a l muscle (1 , 2) and i n heart (3,. k) e x i s t s i n two forms. One form, designated phosphorylase a has been i d e n t i f i e d as the p h y s i o l o g i c a l l y a c t i v e enzyme and was obtained as a c r y s t a l l i n e p r o t e i n from r a b b i t muscle e x t r a c t s s e v e r a l years ago.by Green and C o r i (5). The second form, designated phosphorylase b d i f f e r s from the a form i n that i t has o n l y h a l f the molecular weight (6) and i s c a t a -l y t i c a l l y i n a c t i v e unless assayed i n the presence of 5'-AMP. I t has a l s o been obtained i n c r y s t a l l i n e form from r a b b i t muscle by F i s c h e r and Krebs ( 2 ) and i s regarded as an e s s e n t i a l l y i n a c t i v e enzyme i n v i v o . Enzymes are present i n muscle which e f f e c t the i n t e r c o n v e r s i o n of these two phosphorylase p r o t e i n s . Thus phosphorylase _a i s converted•to the i n a c t i v e phosphorylase b by a s p e c i f i c phosphatase according to r e a c t i o n 1 . phosphorylase a *- k inorganic P + 2 Phosphorylase b ( 1 ) This enzyme was discovered by C o r i and Green i n 19U3 ( 1 ) and more r e c e n t l y has been thoroughly studied by Graves, F i s c h e r and Krebs ( 7 ) . In t u r n phosphorylase b i s converted to phosphorylase a by a s p e c i f i c enzyme, phosphorylase b kinase i n the presence of ATP and Mg + +. In t h i s r e a c t i o n , phosphate i s t r a n s f e r r e d from the nu c l e o t i d e to phosphorylase and a d i m e r i z a t i o n of the enzyme occurs, as expressed i n r e a c t i o n 2 (8, 9 , 1 0 , 1 1 ) . 2 Phosphorylase b + 1+ A T P — — — P h o s p h o r y l a s e a + k ADP ( 2 ) Krebs est a l . ( 1 1 ) have obtained t h i s enzyme al s o i n an i n a c t i v e form and i t can be converted to the a c t i v e enzyme i n a number of ways. Sutherland and h i s colleagues ( 1 2 , 1 3 , Ik) have s t u d i e d a s i m i l a r phosphorylase system i n mammalian l i v e r . In t h i s t i s s u e , conversion of i n a c t i v e phosphorylase to the a c t i v e form i n v o l v e s only phosphorylation of the p r o t e i n . No dimer-i z a t i o n of the molecule occurs. Consequently conversion of the a c t i v e enzyme to the i n a c t i v e form involves only dephos-p h o r y l a t i o n . In a d d i t i o n the l i v e r enzyme d i f f e r s from the muscle enzyme i n that the i n a c t i v e form i s not a c t i v e when assayed i n the presence of 5'-AMP. I t I s t h u s c l e a r t h a t m e c h a n i s m s a r e a v a i l a b l e f o r a l t e r i n g t h e l e v e l s o f a c t i v e p h o s p h o r y l a s e i n t h e c e l l . P h y s i o l o g i c a l , p h a r m a c o l o g i c a l o r h o r m o n a l f a c t o r s w h i c h c o u l d a f f e c t t h e s e m e c h a n i s m s c o u l d t h u s be e x p e c t e d t o a f f e c t t h e b a l a n c e o f t h e two p r o t e i n s and s h o u l d i n t u r n a l t e r g l y c o g e n o l y s i s . S e v e r a l y e a r s ago C o r i ( l 5 ) made t h e i m p o r t a n t d i s c o v e r y t h a t t h e g l y c o g e n o l y t i c a c t i o n o f e p i n e p h r i n e i n s k e l e t a l m u s c l e was due t o i n c r e a s e d l e v e l s o f p h o s p h o r y l a s e _a. S u b s e q u e n t l y S u t h e r l a n d a n d c o - w o r k e r s showed t h a t e p i n e p h r i n e i n c r e a s e d t h e l e v e l s o f a c t i v e p h o s p h o r y l a s e i n dog l i v e r s l i c e s ( l i | , 16) and i n c e l l - f r e e < l i v e r h o m o g e n a t e s ( 1 7 ) . T h i s e f f e c t c o u l d t h e r e f o r e a c c o u n t f o r t h e h y p e r g l y c e m i c a c t i o n o f t h i s h ormone. The d i s c o v e r y t h a t e p i n e p h r i n e p r o d u c e s g l y c o g e n o l y s i s t h r o u g h a c t i v a t i o n o f p h o s p h o r y l a s e and t h a t a c t i v a t i o n a l s o o c c u r s d u r i n g m u s c u l a r work (18) h a s s t i m u l a t e d i n t e r e s t i n a p o s s i b l e r e l a t i o n s h i p b e t w e e n t h e p h o s p h o r y l a s e s y s t e m and t h e c o n t r a c t i l e p r o c e s s . I n p a r t i c u l a r , i n t e r e s t h a s c e n t e r e d on a p o s s i b l e r e l a t i o n s h i p b e t w e e n t h i s enzyme and m y o c a r d i a l c o n t r a c t i o n . I n t h i s r e g a r d Hess a n d H a u g a a r d ( 1 9 ) and K u k o v e t z e t a l . ( 2 0 ) h a v e r e p o r t e d a c o r r e l a t i o n b e t w e e n t h e i n c r e a s e d c o n t r a c t i l e f o r c e a n d p h o s p h o r y l a s e a c t i v a t i o n i n d u c e d b y s y m p a t h o m i m e t i c a m i n e s i n i s o l a t e d p e r f u s e d r a t h e a r t s . M a y e r and M o r a n ( 2 1 ) h a v e shown t h a t t h e p o s i t i v e i n o t r o p i c e f f e c t o f e p i n e p h r i n e , n o r e p i n e p h r i n e , i s o p r o t e r e n o l , a n d s t i m u l a t i o n o f t h e c a r d i a c s y m p a t h e t i c n e r v e s was a c c o m p a n i e d b y a u g m e n t a t i o n o f c a r d i a c p h o s p h o r y l a s e a c t i v i t y . W i t h t h e d a t a a v a i l a b l e t o d a t e , i t i s n o t p o s s i b l e t o d e c i d e w h i c h i s t h e p r i m a r y e v e n t - k - -. . i n adrenergic amine a c t i o n on the heart, the e f f e c t on phos-phorylase, the e f f e c t on the c o n t r a c t i l e mechanism, or whether both are concomitant phenomena wi t h no immediate causal r e l a t i o n s h i p s . With t h i s intense i n t e r e s t i n g l y c o g e n o l y s i s i n s k e l e t a l muscle and l i v e r , very l i t t l e a t t e n t i o n has been paid to the phosphorylase system i n other t i s s u e s . S k e l e t a l muscle, l i v e r and c a r d i a c t i s s u e a l l store s i g n i f i c a n t amounts of glycogen. Nervous t i s s u e and smooth muscle on the other hand c o n t a i n very low glycogen s t o r e s . I t has long been, considered that glycogen metabolism i n the b r a i n i s of v i r t u a l l y no importance and that nerve t i s s u e obtains energy almost e x c l u s i v e l y from blood glucose. I t has been r e c e n t l y shown (22) that b r a i n contains an a c t i v e glycogen s y n t h e s i z i n g system. Various I n v e s t i g a t o r s have expressed the view that the p o s s i b l e im-portance of glycogen metabolism i n the b r a i n should not be overlooked. The nature of phosphorylase i n smooth muscle i s of i n t e r e s t from the Standpoint of the p o s s i b l e r e l a t i o n s h i p between the enzyme and muscle c o n t r a c t i l i t y . Smooth muscle from various t i s s u e s i s known to respond to adrenergic amines i n d i f f e r e n t ways. Uterine muscle responds to epinephrine by vigorous c o n t r a c t i o n , while r a b b i t ileum responds by r e l a x a t i o n . I f phosphorylase a c t i v a t i o n has anything to do w i t h catecholamine-induced c o n t r a c t i l i t y , u s e f u l i n f o r m a t i o n might be uncovered from a study of these two t i s s u e s which respond o p p o s i t e l y to epinephrine. The f i r s t part of t h i s t h e s i s c o n s t i t u t e s the beginning - 5 -of a program designed to examine the nature of phosphorylase and factors which affect i t s a c t i v i t y i n brain, uterus and i n t e s t i n a l muscle. I t was hoped that such a program would not only provide useful information regarding the r e l a t i v e importance of glycogenolysis i n tissues which store only small amounts of glycogen, but that i t might contribute to our understanding of adrenergic amine-induced muscular con-t r a c t i l i t y . - 6 -EXPERIMENTAL PROCEDURE M a t e r i a l s . Commercial preparations of glucose-l-phosphate were u s u a l l y impure and had r a t h e r high concentrations of inor g a n i c phosphate. Since i n o r g a n i c phosphate i s a competitive i n h i b i t o r of phos-phorylase under the assay c o n d i t i o n s , a l l glucose-l-phosphate samples were p u r i f i e d before use. To accomplish t h i s a concentrated s o l u t i o n of the crude glucose-l-phosphate s a l t was s t i r r e d with N o r i t A at pH 6 and the char c o a l removed by f i l t r a t i o n through a charcoal bed. The f i l t r a t e was made st r o n g l y a l k a l i n e w i t h concentrated •ammonium hydroxide and the inorganic phosphate p r e c i p i t a t e d w i t h a small excess of magnesia mixture. A f t e r standing at i c e temperature f o r at l e a s t two hours the p r e c i p i t a t e was removed by f i l t r a t i o n . The f i l t r a t e was n e u t r a l i z e d and excess magnesium removed with potassium Amberlite IR 1 2 0 (as Indicated by t e s t i n g a drop of the s o l u t i o n w i t h ammoniated phosphate s o l u t i o n ) . C r y s t a l l i z a t i o n was induced by adding 1 . 5 volumes methanol and c h i l l i n g . The c r y s t a l s were removed by f i l t r a t i o n , washed with c o l d methanol and d r i e d i n vacuum. •When d e s i r e d , a more uniform p r e p a r a t i o n of glycogen was produced by r e p r e c i p i t a t i o n . An equal volume of ethanol was added to a aqueous s o l u t i o n of crude glycogen and the sus-pension c h i l l e d . The p r e c i p i t a t e was removed by f i l t r a t i o n ; washed with c o l d , absolute ethanol, followed by ethanol-ether ( 1 : 1 ) and d r i e d i n vacuum. Commercial AMP preparations were used throughout. A l l homogenates f o r comparative studies were prepared using NaP to minimize phosphorylase phosphatase a c t i o n . EDTA blockage of phosphorylase kinase a c t i o n was t e s t e d . However, i n the homogenates, phosphorylase a c t i v a t i o n was not found to be a p p r e c i a b l e , therefore EDTA was not r o u t i n e l y used. Phosphorylase Assay System and Unit Phosphorylase a c t i v i t y was measured u s i n g the method of I l l i n g w o r t h and C o r i i ([(.8). The enzyme preparations were incubated f o r 1 0 minutes at 30° i n 0 . 0 1 6 M potassium g l u c o s e - 1 -phosphate and 1% glycogen at pH 6 . 0 ( f i n a l volume 0 . 8 m l ) . When " t o t a l " phosphorylase a c t i v i t y was measured ( i . e . when i n c l u d i n g the a c t i v i t y of the p h y s i o l o g i c a l l y i n a c t i v e form) mM potassium AMP was included i n the r e a c t i o n mixture. Hence, the a b r e v i a t i o n s -AMP and +AMP, used i n the t a b u l a t i o n of the data, i n d i c a t e phosphorylase a c t i v i t y determinations done i n the absence and presence of 5'-AMP, r e s p e c t i v e l y . The r e a c t i o n was stopped by adding one-quarter volume "12% TGA and c h i l l i n g . P r e c i p i t a t e d p r o t e i n was removed by c e n t r i f u g a t i o n i n the c o l d . A l l samples were held at i c e temperature u n t i l a l i q u o t s were assayed f o r i n o r g a n i c phosphate by the method of L e l o i r ( 2 3 ) . Colour i n t e n s i t y was measured with a Klett-Summerson c o l o r i m e t e r using a 660 mu (red) f i l t e r . C o n t r o l s f o r non-enzymatic h y d r o l y s i s and t i s s u e e x t r a c t i n o r g a n i c phosphate were c a r r i e d . The above i n c u b a t i o n mixture was b u f f e r e d with 1 0 mM sodium beta-glycerophosphate (7.5 mM with c y s t e i n e ) , 25 mM succinate or 5 mM T r i s - H C l . Cysteine was not necessary f o r maximal a c t i v i t y with crude enzyme preparations, except those of - - - 8 - •• - - • s k e l e t a l muscle. - P r e l i m i n a r y i n v e s t i g a t i o n s -indicated that assaying at pH 6 . 0 gave a c t i v i t y values not much l e s s than maximal. Hence a l l assay b u f f e r s were used at pH 6 . 0 to minimize phosphoglucomutase a c t i v i t y . A modified Sutherland (12) phosphorylase a c t i v i t y u n i t was used throughout. The u n i t was defined as that amount of enzyme which ca t a l y z e d the l i b e r a t i o n of yamole of in o r g a n i c phosphate i n 10 minutes at 30 degrees, when glucose-l-phosphate h y d r o l y s i s was between 12 and 23$. The s p e c i f i c a c t i v i t y was defined as u n i t s per mg p r o t e i n . P r o t e i n was determined by the spectrophotometric method of Layne {21+). - 9 -RESULTS Phosphorylase D i s t r i b u t i o n i n Rabbit Tissues To i n i t i a t e t h i s study i t was important to e s t a b l i s h the r e l a t i v e phosphorylase a c t i v i t i e s i n e x t r a c t s of b r a i n , uterus and i n t e s t i n a l muscle and to compare them wit h a c t i v i t i e s of other w e l l - s t u d i e d t i s s u e s . I t was a l s o hoped that such an examination would i n d i c a t e whether two forms of the enzyme e x i s t i n these t i s s u e s . This could be determined by assaying the enzyme i n the presence and absence of -AMP. Increased a c t i v i t i e s i n the presence of AMP ( i . e . -AMP/+AMP r a t i o s l e s s than 1) would suggest the presence of a and b forms of the enzyme as found In s k e l e t a l and cardiac muscle. The phosphorylase a c t i v i t y of s e v e r a l r a b b i t t i s s u e s i s shown i n Table I . I t can be seen that i n t e s t i n e , uterus and b r a i n a l l c o n t a i n strong a c t i v i t y . S u r p r i s i n g l y , the t o t a l enzyme a c t i v i t y i n b r a i n i s co n s i d e r a b l y greater than that i n l i v e r when expressed e i t h e r on a p r o t e i n b a s i s or on a t i s s u e wet weight b a s i s . Uterus contains t o t a l a c t i v i t y quite com-parable to that of l i v e r . Thus although b r a i n and uterus c o n t a i n very low glycogen stores and g l y c o g e n o l y s i s , i n b r a i n at l e a s t , has not been considered of importance, these t i s s u e s are endowed with phosphorylase a c t i v i t i e s q u i t e comparable to l i v e r and he a r t . There i s p r e l i m i n a r y evidence i n Table I that the enzyme e x i s t s i n a and b forms i n the three t i s s u e s under c o n s i d e r a t i o n . A c t i v i t i e s i n the absence of AMP are con s i d e r a b l y lower than those with AMP so tha t the -AMP/+AMP r a t i o i n each case i s below - 1 0 -TABLE I Phosphorylase A c t i v i t y of Rabbit Tissues aA pentobarbital anesthetized rabbit was.bled; the required tissues removed, rinsed, c h i l l e d and weighed. The brain, heart,, l i v e r , intestine (ileum) and uterus were homogenized by 120, l 8 0 , 60, 60 and 120 passes respectively, with a Potter-E l vehj em homogenizer, i n 0.05 M Tris-HGl, pH 6.5 and 0.1 M with. NaF, using 2 ml per g tissue (w w) . The skele t a l muscle was homogenized as above but using the Waring blender for 3 minutes at maximum v e l o s i t y . The preparations were cent-rifuged at 20,000 x g for 30 minutes. The assay was conducted using beta-glycerophosphate-cysteine buffer as described i n the t e xt. • Phosphorylase A c t i v i t y Tissue Units/mg Protein Ratio Units/g ; Tissue -AMP +AMP -AMP/+AMP -AMP +AMP Intestine •0.06 0.39 0.15 U.53 29 .5 Liver o.l+i 0.1+2 0.98 1+3.1 . l+l+.l Uterus 0.11+ 0.57 0.25 9.33 38.7 Brain 2.1+1 3.80 0.6*4. i l l . 5 65.1+ Heart k.30 8.10 0.53 159 219 Skeletal Muscle 0.50 hl.h 0.01 22.5 2130 . . . . . 11 _ u n i t y . T h i s i s i n c o n t r a s t t o t h e l i v e r enzyme whose a c t i v i t y i s e s s e n t i a l l y u n a f f e c t e d b y AMP. F u r t h e r e v i d e n c e t h a t t h e b r a i n enzyme e x i s t s i n two f o r m s i s s e e n i n T a b l e I I . I n t h i s e x p e r i m e n t mouse b r a i n h o m o g e n a t e s were p r e p a r e d i n 0.1 M KF a n d i n 0.1 M KG1. The e x t r a c t s p r e p a r e d i n 0.1 M KG1 were a s s a y e d b e f o r e and a f t e r one h o u r i n c u b a t i o n a t 30°. I t c a n be s e e n t h a t a f t e r i n -c u b a t i o n a c t i v i t y i n t h e a b s e n c e o f AMP h a d v i r t u a l l y d i s -a p p e a r e d w h i l e t h e t o t a l a c t i v i t y (+AMP) h a d d e c r e a s e d o n l y s l i g h t l y . T h i s i s a c l e a r I n d i c a t i o n t h a t a and b f o r m s o f t h e enzyme e x i s t i n b r a i n . I t a l s o s t r o n g l y i n d i c a t e s t h e p r e s e n c e o f a n i n a c t i v a t i n g enzyme s i n c e t h i s c o n v e r s i o n c o u l d be b r o u g h t a b o u t o n l y i n e x t r a c t s f r e e o f f l u o r i d e i o n . E x t r a c t s p r e p a r e d i n 0.1 M KF c o u l d n o t be i n a c t i v a t e d i n t h i s m anner. T h i s i s c o n s i s t e n t w i t h t h e l o n g e s t a b l i s h e d f a c t t h a t p h o s p h o r y l a s e p h o s p h a t a s e i s c o m p l e t e l y i n h i b i t e d i n t h e p r e s e n c e o f t h i s a n i o n . E f f e c t o f E p i n e p h r i n e i n v i v o o n T i s s u e P h o s p h o r y l a s e s R e f e r e n c e h a s b e e n made t o t h e f a c t t h a t e p i n e p h r i n e i s c a p a b l e o f i n c r e a s i n g t h e l e v e l s o f a c t i v e p h o s p h o r y l a s e i n s k e l e t a l m u s c l e and l i v e r . I t was o f i n t e r e s t t o d e t e r m i n e w h e t h e r t h e b r a i n enzyme w o u l d b e h a v e i n a s i m i l a r manner. Thus a r a b b i t was i n j e c t e d w i t h a l a r g e d o s e o f e p i n e p h r i n e and no e f f o r t was made t o m i n i m i z e e x c i t e m e n t o r m u s c u l a r a c t i v i t y b e f o r e r e m o v a l o f t h e o r g a n s . Homogenates f r o m l i v e r , b r a i n , h e a r t and s k e l e t a l m u s c l e were t h e n a s s a y e d i n t h e p r e s e n c e a n d a b s e n c e o f AMP. The r e s u l t s a r e shown i n T a b l e I I I . I t - 12 -TABLE I I Phosphorylase I n a c t i v a t i o n i n Mouse B r a i n E x t r a c t s Three mice were decap i t a t e d , the b r a i n s r a p i d l y removed and dropped i n t o l i q u i d n i t r o g e n . The brai n s were weighed' together and covered with 3 volumes of 0.1 M KP c o n t a i n i n g 2 mM K.-EDTA. An a d d i t i o n a l 3 b r a i n s s i m i l a r i l y t r e a t e d were dropped i n t o 3 volumes of 0.1 M KOI c o n t a i n i n g 2 mM K-EDTA. The t i s s u e s were homogenized f o r 1 minute (60 passes) i n a Potter-Elvehjem homogenizer at 0°, then f o r 1 minute i n the micro attachment of a S e r v a l l Omni-mixer. The homogenates were c e n t r i f u g e d f o r 30 minutes at 20,000 x g and the c l e a r supernatant s o l u t i o n s were removed and placed i n i c e . Standard assay c o n d i t i o n s were used. Phosphorylase A c t i v i t y Experiment uhits/m £ 5 P r o t e i n U n i t s / t l Tissue -AMP +AMP -AMP +AMP Homogenate i n 0.1 M KF 1.11 1.22 56.2 61.5 Homogenate i n 0.1 M KG 1 0.97 1.0k 56.2 60.5 Homogenate i n 0.1 M KC1 incubated at 30° f o r 1 hour 0.08 0.97 M 56.2 - 13 -TABLE I I I A Comparative Study o f Rabbit T i s s u e Phosphorylases a f t e r Pre treatment with Epinephrine A r a b b i t was p r e t r e a t e d by subcutaneous i n j e c t i o n o f 20 ug of e p i n e p h r i n e , and immediately stunned by a blow on the head and b l e d . The t i s s u e s , a f t e r removal, r i n s i n g , c h i l l i n g and weighlLhlg, were homogenized i n f i v e volumes of 0.05 M:iTcis,, pH"6.5 , 0.02 M with NaP. The b r a i n , heart v e n t r i c l e s and l i v e r t i s s u e s were homogenized f o r 100, 120 and 180 passes r e s p e c t i v e l y , i n a Pot t e r - E l v e h j e m homogenizer, at i c e bath temperatures. S k e l e t a l muscle was homogenized two minutes i n a Waring b l e n d e r at maximum v e l o c i t y . Debris was removed by c e n t r i f u g a t i o n as before and the supernatant f l u i d s decanted through g l a s s wool. The p r e p a r a t i o n s were assayed as i n d i c a t e d i n the g e n e r a l procedure, u s i n g s u c c i n a t e b u f f e r . Phosphorylase A c t i v i t y T i s s u e Units/mg P r o t e i n R a t i o U n i t s / g Tissue -AMP +AMP -AMP/+AMP -AMP +AMP L i v e r 0.61 0.56 1.08 . 62.5 57.7 B r a i n 1+.55 U.96 0.92 113 123 Heart 5.08 7-. 1+9 0.68 16b, 2i|l S k e l e t a l Muscle 11.8 39.9 0.30 3kl 1170 c a n be s e e n t h a t t h e b r a i n enzyme now e x i s t s p r i m a r i l y i n t h e a c t i v e f o r m ; a -AMP/+AMP r a t i o o f 0.92 was o b t a i n e d . T h i s e x p e r i m e n t may be t a k e n a s a p r e l i m i n a r y i n d i c a t i o n a t l e a s t t h a t t h e a c t i o n o f e p i n e p h r i n e o n t h e b r a i n enzyme i s n o t u n l i k e t h a t o n s k e l e t a l o r c a r d i a c m u s c l e . I t s u g g e s t s t h a t a p h o s p h o r y l a s e a c t i v a t i o n s y s t e m e x i s t s i n b r a i n s i m i l a r t o t h a t i n o t h e r t i s s u e s . P h o s p h o r y l a s e D i s t r i b u t i o n i n Ma m m a l i a n B r a i n B e c a u s e o f t h e u n e x p e c t e d l y h i g h p h o s p h o r y l a s e a c t i v i t y i n b r a i n t i s s u e i t was o f i n t e r e s t t o d e t e r m i n e t h e d i s t r i b u t i o n , o f t h e enzyme I n t h e m a j o r a r e a s o f t h i s o r g a n . T a b l e I V shows t h a t p h o s p h o r y l a s e was g e n e r a l l y e v e n l y d i s t r i b u t e d t h r o u g h o u t d i f f e r e n t a r e a s o f t h e r a b b i t b r a i n . M e d u l l a , c e r e b e l l a r c o r t e x a n d c e r e b r a l c o r t e x c o n t a i n e d t h e h i g h e s t t o t a l a c t i v i t y d e t e r m i n e d o n a p r o t e i n b a s i s . I t c a n a l s o be s e e n t h a t t h e -AMP/+AMP r a t i o s were g e n e r a l l y s i m i l a r . T h i s e x p e r i m e n t was r e p e a t e d u s i n g dog b r a i n w h i c h b e c a u s e o f i t s l a r g e r s i z e p e r m i t t e d a b e t t e r d i f f e r e n t i a t i o n o f m a j o r b r a i n a r e a s . To m i n i m i z e enzyme l o s s e s d u r i n g r e m o v a l o f t h e b r a i n , t h e a n i m a l was k e p t u n d e r p e n t o b a r b i t a l a n e s t h e s i a d u r i n g s u r g e r y . F r o m T a b l e V i t i s a p p a r e n t t h a t p h o s p h o r y l a s e d i s t r i b u t i o n i n dog b r a i n i s v e r y s i m i l a r t o t h a t i n r a b b i t b r a i n . The -AMP/+AMP r a t i o s i n t h i s e x p e r i m e n t a r e g e n e r a l l y n e a r u n i t y , as was f o u n d f o r mouse b r a i n ( T a b l e I I ) . - 15 -TABLE IV D i s t r i b u t i o n of Phosphorylase A c t i v i t y i n D i f f e r e n t Areas of Rabbit B r a i n The b r a i n of a p e n t o b a r b i t a l anesthetized r a b b i t was removed, separated i n t o the areas I n d i c a t e d , and c h i l l e d on i c e . The samples were homogenized with a Potter-Elvehjem homogenizer f o r 90 passes (with the exception of the cortex which r e q u i r e d 120 passes) i n 5 volumes of the usual T r i s b u f f e r , 0.02 M with NaP. The homogenates were c e n t r i f u g e d at 22,000 x g f o r 30 minutes. The supernatant f l u i d s were then assayed f o r phosphorylase a c t i v i t y . Succinate b u f f e r was used. Phosphorylase A c t i v i t y T i ssue Units/mg P r o t e i n Ratio U n i t s / g Tissue -AMP +AMP -AMP/+AMP -AMP +AMP Cerebral Cortex 4.62 6.01 0.77 66.5 86.6 C e r e b e l l a r Cortex k'hk 5.25 0.85 91.0 108 Thalamus 3.49 4.10 0.85 74.0 86.9 Hypothalamus 3.53 4.17 0.82 63.6 74-0 Medulla J+-81 5.61 0.86 103 120 Upper S p i n a l Cord 2.16 3.77 0.57 31.5 55.0 Pyramidal Tract 3.39 4.35 0.78 78.0 100 Superior and I n f e r i o r C o l l i c u l i 1.37 2.03 0.68 44.8 66.5 - 16 -TABLE V D i s t r i b u t i o n of Phosphorylase i n D i f f e r e n t Areas of Dog B r a i n The b r a i n of a p e n t o b a r b i t a l anesthetized dog was removed, -d i v i d e d and homogenized as with the r a b b i t b r a i n . A l l s e c t i o n s , except the cerebellum and c e r e b r a l c o r t e x , were homogenized with 120 passes w i t h a Potter-Slvehjem homogenizer. The cere-bellum was homogenized one minute at one-half maximum v e l o c i t y with a Waring blender. C e r e b r a l c o r t e x was homogenized i n a l i k e manner but f o r one minute at one-half maximum and one-half minute at maximum v e l o c i t y . The homogenates were'centrifuged and assayed immediately by the standard procedure. Phosphorylase A c t i v i t y Tissue I Jnits/mg Proitaln R a tio Units/g Tissue -AMP +AMP -AMP/+AMP -AMP +AMP . Cerebral Cortex 2.16 3.23 0.67 44-0 64.0 Cerebellum 3.40 4.65 o.74 78.0 106 Thalamus 2.82 3.25 0.87 50.6 59.7 Hypothalamus 3.14 3.51 0.90 65.1 73.4 Medulla 4.81 5.08 0.95 64.2 67.0 Pons 4.57 5.18 0.88 64.0 73.8 Superior and I n f e r i o r C o l l i c u l i 4.00 4.27 0.94 65.6 72.1 B a s a l Ganglia 3.71 4.20 0.88 60.6 68.9 E f f e c t of Epinephrine on Smooth Muscle Phosphorylase Various i n v e s t i g a t o r s (see the Int r o d u c t i o n ) have found a c o r r e l a t i o n between increased cardiac c o n t r a c t i l e force and phosphorylase a c t i v a t i o n induced by sympathomimetic amines. Experiments were designed to examine the e f f e c t of epinephrine on phosphorylase i n u t e r i n e and i n t e s t i n a l muscle i n v i t r o , under c o n d i t i o n s i n which uterus was caused to con t r a c t and ileum s t r i p s were rela x e d by the amine. Tissue samples were incubated i n aerated Ringer-Locke s o l u t i o n w i t h low concentrations of epinephrine before homogenizing. Homogenates were then assayed f o r phosphorylase and compared with c o n t r o l samples which had not been t r e a t e d with epinephrine. Prom the data obtained the -AMP/+AMP r a t i o s were c a l c u l a t e d . Mean values were c a l c u l a t e d f o r each experimental group and the s i g n i f i c a n c e of the d i f f e r e n c e of the experimental from the c o n t r o l was determined by the Student " t " t e s t . The r e s u l t s are shown i n Table V I. Ileum s t r i p s showed a b s o l u t e l y no phosphorylase a c t i v a t i o n w ith epinephrine treatment, even though the i n i t i a l -AMP/+AMP' r a t i o s were r e l a t i v e l y low (0 .38) . Uterine s t r i p s however showed s i g n i f i c a n t phosphorylase a c t i v a t i o n with these con-c e n t r a t i o n s of epinephrine. The s i g n i f i c a n c e of these f i n d i n g s to the r o l e of phosphorylase i n epinephrine a c t i o n on these t i s s u e s cannot be assessed at the present time. Considerably more experimentation must ensue before conclusions can be made. I t i s c l e a r from t h i s data that a and b forms of the enzyme e x i s t i n smooth muscle. - 1 8 -TABLE V I The E f f e c t o f E p i n e p h r i n e on U t e r i n e and I n t e s t i n a l P h o s p h o r y l a s e i n v i t r o F o r t h e u t e r i n e e x p e r i m e n t s , y o u n g f e m a l e r a b b i t s were i n j e c t e d s u b c u t a n e o u s l y w i t h 0.125 mg s t i l b o e s t r o l d a i l y f o r 4 t o 8 days b e f o r e u s e . U n s e x e d r a b b i t s were u s e d f o r t h e i l e u m e x p e r i m e n t s . A n i m a l s were a n e s t h e t i z e d w i t h p e n t o -b a r b i t a l , t h e n e c k v e s s e l s were s e v e r e d and t h e t i s s u e s r a p i d l y r e m o v e d a n d c h i l l e d o n I c e . A p p r o p r i a t e l y s i z e d s a m p l e s were b l o t t e d , w e i g h e d and r e c h i l l e d . C o n t r o l a n d e x p e r i m e n t a l s a m p l e s were o b t a i n e d f r o m t h e same a n i m a l a n d were a d j a c e n t i n t h e t i s s u e . A l l s a m p l e s were p r e i n c u b a t e d f o r 5 m i n u t e s i n 30 m l o f a e r a t e d R i n g e r - L o c k e s o l u t i o n a t 37°. E p i n e p h r i n e (5 u g / m l ) was t h e n a d d e d t o t h e e x p e r i m e n t a l s a m p l e s and I n c u b a t i o n ' c'ont I n u e d f o r 3 m i n u t e s . The s a m p l e s were t h e n r a p i d l y r emoved a n d d r o p p e d i n t o l i q u i d n i t r o g e n . The f r o z e n s a m p l e s were h o m o g e n i z e d i n a P o t t e r - E l v e h j e m h o m o g e n i z e r w i t h 6 v o l u m e s o f 0.05 M T r i s - H C l , pH 6.5, 0.1 M w i t h NaF. A f t e r c e n t r i f u g a t i o n a t 20,000 x g f o r 15' m i n u t e s t h e s u p e r n a t a n t s o l u t i o n s were a s s a y e d i m m e d i a t e l y . S t a n d a r d a s s a y c o n d i t i o n s were u s e d . T i s s u e No. o f Sample s Mean A c t i v i t y R a t i o s (-AMP/+AMP) S t a n d a r d D e v i a t i o n s P I l e u m C o n t r o l W i t h E p i n e p h r i n e 8 12 0.376 0.380 0.11+7 0 . 1 2 9 »o.3 U t e r u s C o n t r o l W i t h E p i n e p h r i n e 13 13 0.1+8 0.68 0.051 0.079 «0.01 - 19 -Phosphorylase P u r i f i c a t i o n Attempts The foregoing experiments have shown that u t e r u s , smooth muscle and p a r t i c u l a r i l y b r a i n c o n t a i n s u b s t a n t i a l phosphorylase a c t i v i t y . Moreover they i n d i c a t e that the enzyme i n these t i s s u e s i s not u n l i k e that i n s k e l e t a l muscle and h e a r t . Pre-l i m i n a r y evidence i n d i c a t e s that enzymic mechanisms are a v a i l -able to c a t a l y z e the a c t i v a t i o n and i n a c t i v a t i o n of phosphorylase i n these t i s s u e s and tha t probably i n b r a i n and ut e r u s , the enzyme responds to epinephrine treatment. In order to more c l e a r l y define the nature of t h i s enzyme system I t would be necessary to p u r i f y the enzymes i n v o l v e d and to study them sep a r a t e l y . Attempts were therefore made to p u r i f y phosphorylase from these t i s s u e s . Although considerable time and e f f o r t were expended i n t h i s d i r e c t i o n , a l l e f f o r t s were without success. Numerous c l a s s i c a l procedures f o r enzyme p u r i f i c a t i o n were employed such as i s o e l e c t r i c p r e c i p i t a t i o n , heat de-n a t u r a t i o n , solvent f r a c t i o n a t i o n s , ammonium sulphate f r a c t i o n -a t i o n s , alumina Cy and calcium phosphate g e l adsorption, e t c . A l l p reparations of the enzyme seemed unstable during f r a c t i o n -a t i o n and the enzyme was not s t a b l e upon storage. Because of the d i f f i c u l t i e s encountered and the continued l a c k of success, t h i s approach was abandoned. In order to conserve space, a d e t a i l e d d e s c r i p t i o n of the experiments conducted are not i n -cluded i n t h i s r e p o r t . - 20 -DISCUSSION B r a i n e x t r a c t s have been found to contain higher phosphoryla a c t i v i t y than l i v e r . The s p e c i f i c a c t i v i t y of the enzyme i n b r a i n i s comparable to that i n heart. This h i g h enzyme a c t i v i t y i n b r a i n i s i n t e r e s t i n g but no d e f i n i t e s i g n i f i c a n c e can be attached t o i t at the moment. B r a i n c l e a r l y contains very low glycogen stores (25). Glycogen that i s present, however, may be present i n high l o c a l concentrations and i t probably may have a r a p i d turnover. I n f a c t s e v e r a l i n v e s t i g a t o r s (26, 27) have i n d i c a t e d that high l o c a l i z e d glycogen concentrations may e x i s t . > Gentschev (26) has reported glycogen granules near and on the surface of P e r k i n j e c e l l s , apparently w i t h i n synaptic s t r u c t u r e s , i n the b r a i n of r a t s , mice and guine)a p i g s . He a l s o found that these glycogen granules disappeared during e l e c t r o - s h o c k , or when the animal was subjected to a' blow on the head, or to I n s u l i n coma. Glycogen content i n -creased when the animals were anesthetized w i t h ether or t r e a t e d w i t h chlorpromazine. E l l i s (28) considers that production of hexose phosphates, w i t h r e s u l t a n t h y p e r p o l a r i z a t i o n , i s the important a c t i o n of phosphorylase i n many t i s s u e s . Epinephrine appears to cause increased l e v e l s of a c t i v e phosphorylase i n c e r e b r a l t i s s u e and t h i s might have i n t e r e s t i n g i m p l i c a t i o n s regarding the a c t i o n of t h i s and other catechol amines i n the c e n t r a l nervous system. The present data at l e a s t i n d i c a t e s that the b r a i n phosphorylase system i s c l e a r l y worthy of c l o s e study and t h a t glycogen metabolism i n nerve t i s s u e should no - 21 -longer be ignored. E l l i s has reported (29) that epinephrine causes incr e a s e d g l y c o g e n o l y s i s , and increased phosphorylase a c t i v i t y i n u t e r i n e muscle, i n agreement with the data reported here. The above author (30) has expressed the view that the epinephrine e f f e c t on u t e r i n e c o n t r a c t i o n s i s independent of energy metabolism, which might point to some other r o l e f o r phosphorylase i n c o n t r a c t i l i t y . Leonard ( 3 1 ) , however, found t h a t u t e r i n e phosphorylase a c t i v i t y one hour a f t e r epinephrine i n j e c t i o n i n t o r a b b i t s , was-lower than c o n t r o l values. The r e s u l t s are questionable since the e f f e c t s of epinephrine are r a p i d i n onset and of short d u r a t i o n . The absence of any epinephrine e f f e c t on i n t e s t i n a l smooth muscle phosphorylase i s i n agree-ment with the f i n d i n g s of E l l i s ( 2 9 , 3 2 ) . Axelsson et a l . (33) on the other hand have reported that guinea p i g taenia c o l i responds to epinephrine w i t h increased a c t i v e phosphorylase l e v e l s but o n l y during h y p e r - p o l a r i z a t i o n . I t may be that phosphorylase a c t i v a t i o n occurs i n i n t e s t i n a l muscle under s p e c i a l c o n d i t i o n s and i n c e r t a i n areas. One might speculate, from the data here and from that of E l l i s , t h a t epinephrine causes phosphorylase a c t i v a t i o n only i n those t i s s u e s that respond to the amine by c o n t r a c t i o n , and that no enzyme a c t -i v a t i o n occurs i n those c o n t r a c t i l e t i s s u e s i n which the amine causes r e l a x a t i o n . This must remain p u r e l y s p e c u l a t i v e u n t i l much more work i s done and i t would be d i f f i c u l t to r e c o n c i l e w i t h what i s a l r e a d y known about the enzymatic mechanism i n -volved. Further i n v e s t i g a t i o n i n t h i s area i s c l e a r l y necessary. - 22 -PART I I SYNTHESIS AND ENZYMATIC DEGRADATION OP SEVERAL DEOXYRIBONUCLEOSlDE-3' ,5'-MONOPHOSPHATES INTRODUOTION Sutherland and h i s colleagues (ll+, 16, 17) c l e a r l y e s t a b l i s h e d that epinephrine caused increased hepatic g l y -cogenolysis by i n c r e a s i n g the l e v e l s of a c t i v e phosphorylase i n the l i v e r . This epinephrine induced phorsphorylase a c t i v i t y increase was shown to be mediated by a heat s t a b l e f a c t o r (31+) which was i s o l a t e d , c h a r a c t e r i z e d (35) and shown to be adenosine - 3 ' , 5 '-monophosphate. The presence of t h i s new nucleotide was not confined to l i v e r , but was also found i n dog h e a r t , s k e l e t a l muscle, and b r a i n (36, 3 7 ) . In recent months our knowledge of the r o l e of adenosine - 3 ' , 5 '-mono-phosphate has g r e a t l y increased and the l i t e r a t u r e Is too extensive to review here. The reader i s r e f e r r e d to e x c e l l e n t reviews by R a i l and Sutherland (38, 39). S u f f i c e i t i s to say that adenosine - 3 1 , 5 '-monophosphate c l e a r l y has an a c t i o n not on phosphorylase d i r e c t l y but on phosphorylase kinase. Because of the minute amounts of t h i s compound a v a i l a b l e from n a t u r a l sources, the need.arose f o r s y n t h e t i c m a t e r i a l . Smith et a l . (i+0) accomplished the synthesis by a r e l a t i v e l y simple procedure which r e s u l t e d i n chromatographically pure m a t e r i a l i n h i g h y i e l d . R a i l and Sutherland (36) found that adenosine - 3 T , 5 '-mono-- 23 - •• - • -phosphate r a p i d l y l o s t i t s a c t i v i t y i n the presence of t i s s u e e x t r a c t s and t h i s l e d them to the d i s c o v e r y of an enzyme which degraded the compound s p e c i f i c a l l y to adenosine-5'-phosphate. This phosphodiesterase has been studied i n b r a i n by Drummond and Perrott-Yee (<L|!) and i n heart by Butcher and Sutherland (1+2). I t appears to be s p e c i f i c f o r the h y d r o l y s i s of nucleoside-3' ,5' -mo no phosphates and i s p a r t i c u l a r l y a c t i v e in. b r a i n . I n t e r e s t i n the metabolsim of adenosine-3 1 ,5 '-mono-phosphate i s i n c r e a s i n g r a p i d l y but much remains to be learned. The need has a r i s e n f o r c h e m i c a l l y s i m i l a r s t r u c t u r e s to a s s i s t i n . s p e c i f i c i t y and mechanistic i n v e s t i g a t i o n s . Con-s i d e r a b l e u s e f u l i n f o r m a t i o n has a l r e a d y been uncovered using the s e v e r a l other ribonucleoside-3' ,5 '-monophosphates which have been made a v a i l a b l e (1+0, 38). The second part of t h i s t h e s i s i s concerned w i t h attempts to synthesis a number of deoxyribonucleoside-3 1 ,5 '-monophosphates and to examine t h e i r a b i l i t y to act as substrate's f o r the phosphodiesterase enzyme from b r a i n . - 2k -- - EXPERIMENTAL METHODS AND RESULTS M a t e r i a l s A n h y d r o u s p y r i d i n e was p r e p a r e d b y a d d i n g c a l c i u m h y d r i d e t o c o m m e r c i a l p y r i d i n e . The h y d r i d e was k e p t s u s p e n d e d f r o m one t o two d a y s b y o c c a s i o n a l s h a k i n g . D r y n e s s was i n d i c a t e d b y t h e c e s s a t i o n o f h y d r o g e n e v o l u t i o n . !4.-Morpholino-N,N' - D i c y c l o h e x y l c a r b o x a m i d i n e was p r e p a r e d b y r e f l u x i n g 2 .08 g DGG and 780 mg m o r p h o l i n e f o r f i v e h o u r s w i t h a minimum v o l u m e ( a b o u t t h r e e m l ) o f t - b u t a n o l . The s o l u t i o n was e v a p o r a t e d t o d r y n e s s a n d t h e r e s i d u e d i s s o l v e d i n a minimum v o l u m e o f b o i l i n g n - h e p t a n e . T h i s s o l u t i o n was a l l o w e d t o c o o l s l o w l y t o r e f r i g e r a t o r t e m p e r a t u r e . C r y s t a l s were r e m o v e d b y f i l t r a t i o n and washed t w i c e w i t h c o l d h e p t a n e and d r i e d u n d e r vacuum. C o m m e r c i a l d i c y c l o h e x y l c a r b o d i i m i d e was u s e d t h r o u g h o u t . C h r o m a t o g r a p h i c S y s t e m s A l l p a p e r c h r o m a t o g r a p h y was c o n d u c t e d b y the d e s c e n d i n g m e t h o d a t ro o m t e m p e r a t u r e i n s e a l e d g l a s s j a r s , a p p r o x i m a t e l y s a t u r a t e d w i t h v a p o u r s o f t h e s o l v e n t . The s o l v e n t s u s e d w e r e : A I s o p r o p a n o l - c e n c e n t r a t e d a m m o n i a - w a t e r ( 7 : 1 : 2 ) B N o r m a l - b u t a n o l - g l a c i a l a c e t i c a c i d - w a t e r (5:2:3) G 95% e t h a n o l - 0 . 5 M ammonium a c e t a t e , pH 3 . 8 (5:2) D 95% e t h a n o l - 1 M ammonium a c e t a t e , pH 7.5 (5:2) E S a t u r a t e d a q u e o u s ammonium s u l f a t e - 1 M s o d i u m a c e t a t e - i s o p r o p a n o l ( 8 0 : 1 8 : 2 ) E l e c t r o p h o r e s i s A l l e l e c t r o p h o r e s i s was done w i t h a n E.-C. A p p a r a t u s C o . , w a t e r c o o l e d , p r e s s u r e - p l a t e t y p e i n s t r u m e n t . U s u a l l y 2|r b y l8-|-I n c h s h e e t s o f S c h l e i c h e r and S c h u e l l a n a l y t i c a l p a p e r were u s e d . - 25 -The i n s t r u m e n t was o p e r a t e d a t 1000 v o l t s o r 190 m i l l i a m p e r e s . The b u f f e r u s e d was 0 .05 M p o t a s s i u m p h o s p h a t e , pH 7 .5. Enzyme P r e p a r a t i o n s a n d I n c u b a t i o n C o n d i t i o n s a. B r a i n P h o s p h o d i e s t e r a s e The i n i t i a l p r e p a r a t i o n s o f t h e enzyme f r o m r a b b i t b r a i n were made b y t h e p r o c e d u r e o f Brummond and P e r r o t t - Y e e (!+!)• L a t e r , t h e a b o v e p r o c e d u r e was somewhat i m p r o v e d b y u s i n g . b e e f b r a i n a c e t o n e powder e x t r a c t s . B e e f b r a i n a c e t o n e powder was p r e p a r e d b y h o m o g e n i z i n g t h e b r a i n t i s s u e t h r e e t i m e s i n a n O m n i - M i x e r i n l a r g e v o l u m e s o f a c e t o n e a t - 1 5 ° , c o n c e n t r a t i n g t h e p r e c i p i t a t e e a c h t i m e b y c e n t r i f u g a t i o n a t - 1 5 ° , a n d f i n a l l y d r y i n g t h e p r e c i p i t a t e u n d e r vacuum. The powder was e x t r a c t e d t w i c e w i t h s i x v o l u m e s o f 0.01 M c i t r a t e , pH 6 .5, u s i n g a P o t t e r - E l v e h j e m h o m o g e n i z e r f o r t h e l a s t e x t r a c t i o n . The p r e c i p i t a t e s were r e m o v e d e a c h t i m e b y c e n t r i f u g a t i o n a t 23 ,500 x g f o r 10 m i n u t e s a t 0 ° . The e x t r a c t was t h e n f r a c -t i o n a t e d b y a d d i n g s a t u r a t e d ammonium s u l f a t e pH 7*4 i n t h e p r e s e n c e o f two ^ u l m e r c a p t o e t h a n o l p e r 10 m l . P r e c i p i t a t e s were r e m o v e d b y c e n t r i f u g a t i o n a s a b o v e . The p r e c i p i t a t e s were d i s s o l v e d i n 0.02 M T r i s - H C l , pH 7.5 and d i a l y s e d f o r f o u r h o u r s a g a i n s t 2 x 2 l i t e r s o f t h e same b u f f e r i n t h e c o l d room. S i n c e t h e 30-1+0$ s a t u r a t e d ammonium s u l f a t e p r e c i p i t a t e f r a c t i o n was b y f a r t h e m o s t a c t i v e i t was u s e d f o r h y d r o l y s i s s t u d i e s . S i n c e a . d e f i n i t e a s s a y s y s t e m and u n i t was n o t b e i n g u s e d , t h e y i e l d a n d t h e p u r i t y o f t h e p r e p a r a t i o n was n o t d e t e r m i n e d . However, i t was more a c t i v e t h a n p r e v i o u s p r e p a r a t i o n s . - 26 -The d i e s t e r a s e was incubated with about. 1 ^imole of the c y c l i c n u c l e o t i d e s ( c a l c u l a t e d using approximate e x t i n c t i o n c o - e f f i c i e n t s ) i n a r e a c t i o n mixture 20 mM with T r i s - H G l , pH 7.5; mM w i t h MgC^ and about 0.51-1-9 mg p r o t e i n , depending on the enzyme p r e p a r a t i o n . A f t e r i n c u b a t i o n at 30 degrees, u s u a l l y f o r 20 minutes, the r e a c t i o n was stopped by adding 12% p e r c h l o r i c a c i d to a f i n a l c o n c e n t r a t i o n of 3%>. The per-c h l o r i c a c i d was then p r e c i p i t a t e d w i t h KOH and the suspension c e n t r i f u g e d . A l i q u o t s of the supernates were spotted and run i n e l e c t r o p h o r e s i s as i n the preparative procedures. The decrease i n the amount of c y c l i c n u c l e o t i d e was measured by e l u t i n g the appropriate spot w i t h water, d i l u t i n g the e l u a t e to 1.5 ml and determing the o p t i c a l d e n s i t y at an appropriate wavelength on the Beckman, model DU, spectrophotometer. Per cent h y d r o l y s i s was determined by comparing the o p t i c a l d e n s i t y w i t h that of a standard sample which had been incubated without enzyme. b. Muscle Adenylate Deaminase This enzyme was prepared by the method of Lee (1+3) and stored at - 2 0 ° f o r about one year. The p r e p a r a t i o n was cent-r i f u g e d before use. C a r t e r (1+1+) found t h a t muscle ad e n y l i c a c i d deaminase would deaminate deoxyadenosine-5'-monophosphate as w e l l as adenosine-5"-monophosphate, but at an undetermined slower r a t e . Since the deaminase i s s p e c i f i c f o r the 5'-adenine n u c l e o t i d e , i t was decided that t h i s would form a method of i d e n t i f y i n g the product of the d i e s t e r a s e a c t i o n on deoxyadenosine - 3 '5 '-- 27 -monophosphate. Using authentic deoxyadenosine - 5 '-phosphate i t was found t h a t the r a t e of deamination of the above nu c l e o t i d e was o n e - t h i r t i e t h that of adenosine-^ 1-phosphate i n the presence of I4.3 mM c i t r a t e , pH 6.0, 0.1 M KC1 and 0.13 mM n u c l e o t i d e at 25° C. The muscle adenylic a c i d deaminase had been found to be I n a c t i v e on adenosine-3' ,5'-monophosphate (l+l). The present study found i t to be i n a c t i v e on the deoxyadenosine-3 1 , 5 ' -monophosphate under the above c o n d i t i o n s , c. Snake Venom The f r e e z e - d r i e d snake venom powder was a commercial p r e p a r a t i o n prepared from Grotalus adamanteus venom and purchased from Ross A l l e n ' s r e p t i l e farm, S i l v e r Springs, F l o r i d a , A p r i l 1962. A 1%> s o l u t i o n of the f r e e z e - d r i e d powder i n 0 .05 M T r i s - H C l , pH 8.15" was prepared. The s o l u t i o n was r a t h e r t u r b i d with some s o l i d m a t e r i a l which was not removed. Since the nucleotidase of snake venom has been reported to be s p e c i f i c f o r nucleoside-^'-phosphates (i+5), i t was evident that t h i s enzyme could be used to i d e n t i f y the p o s i t i o n of the phosphate on the mononucleotides produced from the deoxynucleoside-3' ,5'-monophosphate analogs by b r a i n d i e s t e r a s e a c t i o n . Hence, the nucleotides i s o l a t e d from the r e a c t i o n mixture of the d i e s t e r a s e and the cyclic-phosphate analogues;by e l e c t r o p h o r e s i s , were incubated w i t h a 0.1%, snake venom s o l u t i o n , 10 mM with magnesium acetate, and Ij, mM w i t h T r i s - H C l , pH 8.15 ( f i n a l volume 0.1 ml). A l i q u o t s (25 u l ) were spotted on Whatman No. 1 paper without d e p r o t e i n i z a t i o n at about 15" and 30 minutes a f t e r i n i t i a t i o n of i n c u b a t i o n . The chromatogram - 28 -was developed i n solvent A. Pre p a r a t i o n and P u r i f i c a t i o n of C y c l i c - 3 ' , 5 1 - D e o x y n u c l e o t i d e s . a. Deoxyadenosine - 3 ' ,5'-Monophosphate.:• The f o l l o w i n g procedure was e s s e n t i a l l y that used by Smith, e_t a l . (1+0). 0.5 mmole of the diammonium s a l t of deoxyadenosine - 5 1-phosphate was d i s s o l v e d i n water, converted to the p y r i d i n i u m s a l t with excess, f r e s h p y r i d i n i u m Dowex 50, and f r e e z e - d r i e d . The. f r e e z e - d r i e d powder was then co-evaporated i n a r o t a r y evaporator with 3 x 10 ml po r t i o n s of anhydrous p y r i d i n e . The pr e p a r a t i o n was f u r t h e r d r i e d by hig h vacuum from a vacuum pump. The dry residue was suspended, with warming to d i s s o l v e as much as p o s s i b l e , i n 50 ml of dry p r y i d i n e c o n t a i n i n g a 0.5 mmole l+-morpholino-N,N 1 -dicyclohexlcarboxamidine, and the suspension was added to a dry, heated dropping f u n n e l . The carboxamidinium s a l t of the nucleo t i d e was added dropwise (over two hours) i n t o 75 ml of anhydrous p y r i d i n e , c o n t a i n i n g 1+00 mg DCG, under r a p i d r e f l u x and w i t h r a p i d mechanical s t i r - r i n g . Anhydrous c o n d i t i o n s were maintained throughout. A f t e r the a d d i t i o n was complete, the dropping funnel was r i n s e d w e l l with hot anhydrous p y r i d -ine and the r i n s i n g s dropped i n t o the r e a c t i o n f l a s k , s t i l l under r e f l u x . When the a d d i t i o n was f i n a l l y complete the he a t i n g was stopped and the r e a c t i o n mixture evaporated to dryness. The dry re s i d u e was extracted a l t e r n a t e l y with water and ether, and the e x t r a c t s were f i l t e r e d . The ether e x t r a c t was ext r a c t e d once with water. The combined water e x t r a c t s were treated three times w i t h about one-third volume of ether - 29 -and f i n a l l y the combined ether l a y e r s were ext r a c t e d once wit h water. The water e x t r a c t s were concentrated to a small volume wi t h the r o t a r y evaporator, streaked on Whatman No. 3 MM paper and subjected to chromatography u s i n g solvent A. A f t e r d r y i n g the chromatograms, the appropriate bands were e x t r a c t e d o f f the paper by p u l v e r i z i n g three times i n glass d i s t i l l e d water and f i l t e r i n g . Since t r a c e u l t r a - v i o l e t absorbing contaminants were s t i l l present the concentrated water e x t r a c t was rechromatographed as above. The appropriate bands of the second set of chromatograms were extracted as before. The concentrated water e x t r a c t was c e n t r i f u g e d and f r e e z e - d r i e d . The r e s u l t a n t powder was stored i n a deep-fr e e z e . The product at pH 5.5 had an a b s o r p t i o n maximum at 258 mu and minimum at 225 mu. Test samples moved as a s i n g l e u l t r a - v i o l e t absorbing spot on chromatography i n solvent A and during e l e c t r o p h o r e s i s . b. N-Benzoyl-Deoxyadenosine -3 ' ,5 '-Monophosphate The b e n z o y l a t i o n procedure followed here was e s s e n t i a l l y that of Smith, et a l . (1+0). 183 mg of diammonium deoxy-adenosine - 5 1-phosphate were converted to the p y r i d i n i u m s a l t as before. The f r e e z e - d r i e d p y r i d i n i u m s a l t was suspended i n dry p y r i d i n e (10 ml), and 1.5 ml of benzoyl c h l o r i d e added. The f l a s k was s w i r l e d to suspend a l l the m a t e r i a l and the l i g h t yellow-pink s o l u t i o n allowed to stand, w i t h o c c a s i o n a l s w i r l i n g , one and one-half hours at room temperature i n the dark. Jj.0 ml of glass d i s t i l l e d water were added to the f i n a l red s o l u t i o n . P r e c i p i t a t i o n of a white s o l i d increased as the - 30 -water was added. A f t e r 5 minutes at 25°, the suspension was ex t r a c t e d w i t h 3 x 1+0 and 1 x 20 ml chloroform, and the chloroform was back e x t r a c t e d w i t h water. The chloroform l a y e r was then f l a s h evaporated•to an o i l . Water (6.6 ml) and p y r i d i n e (13.2: ml) were added, to the o i l producing a yellow s o l u t i o n . Then, 20 ml o f 2 N NaOH were added, with s w i r l i n g . Some t u r b i d i t y formed. The suspension was allowed to stand f o u r minutes at 25°, w i t h a s o l u t i o n colour change from yellow to orange, and with increased p r e c i p i t a t i o n . At four minutes, 57 g of b l o t t e d , moist, f r e s h p y r i d i n i u m IR 120 was added to decrease the pH to 7. S o l u b i l i z a t i o n of•the p r e c i p i t a t e occurred and the s o l u t i o n changed to l i g h t yellow as the f i n a l pH was reached. The r e s i n was removed and washed by f i l t r a t i o n . The combined f i l t r a t e and washings were concentrated, and ext r a c t e d twice with ether to remove some of the copious p r e c i p i t a t e which formed during f l a s h evaporation. A f t e r ether e x t r a c t i o n the concentrate was a p p l i e d to a 6olumn of f r e s h p y r i d i n i u m IR 120 (2.5 cm x 30 cm) at a f l o w rate of about f o u r ml per minute. The column was washed with g l a s s d i s t i l l e d water u n t i l a l l of the u l t r a - v i o l e t absorbing m a t e r i a l had been removed (900 m l ) . The eluate was then concentrated, extracted s e v e r a l times with ether and the water l a y e r evaporated to dryness. The residue was r e d i s s o l v e d i n water and f r e e z e - d r i e d . Chromatography of a sample i n solvent B i n d i c a t e d some contamination, c h i e f l y from f r e e deoxyadenosine - 5 '-phosphate. However, since the contain:ination was r e l a t i v e l y minor the product was not p u r i f i e d . - 31 -The b e n z o y l a t i o n product was converted to the nucleoside-3',5'-monophosphate by the procedure described f o r deoxy-adenylate. Since the f i n a l water e x t r a c t of the d r i e d r e a c t i o n mixture contained a r a t h e r l a r g e amount of a ye l l o w contamin-ant, the nucle o t i d e was p r e c i p i t a t e d w i t h BaJ^ and two volumes of acetone. The washed p r e c i p i t a t e was converted to the potassium s a l t with excess potassium IR 120 and p u r i f i e d by chromatography i n solvent B. The m a t e r i a l streaked badly i n other chromatographic systems (A, 0, D and E) so the p u r i t y could not be checked by paper chromatography. E l e c t r o -phoresis showed considerable contamination. Therefore, a small amount (20 mg of the above product) was p u r i f i e d of other u l t r a - v i o l e t absorbing m a t e r i a l by e l e c t r o p h o r e s i s , using the c o n d i t i o n s described under "procedures", and wit h Whatman No. 3 I'M paper. Three bands r e s u l t e d . T V J O of the Bands ( I and I I ) could have been the c y c l i c - 3 1 , 5 ' - n u c l e o t i d e . P u r i f i c a t i o n of another sample of the c y c l i z a t i o n product was attempted u s i n g DEAE-cellulose w i t h a 0.02 to 0.1 M gradient of triethylammonium carbonate. The column chromatography f a i l e d to separate two of the u l t r a - v i o l e t absorbing compounds under those c o n d i t i o n s . c. Deoxyinosine -3 ' ,5 '-Monophosphate The deamination procedure reported was an adaptation of the procedure report e d by K l e i n z e l l e r (i+6). 95 mg of d i a -mmonium deoxyadenosine-5'-phosphate were d i s s o l v e d i n three ml of water, and 0.323 g anhydrous sodium acetate, 1.0 ml g l a c i a l a c e t i c a c i d and 1.2 g sodium n i t r a t e i n about 2.0 ml water, were added. The s o l u t i o n was t i t r a t e d to pH 1+ (paper) with g l a c i a l a c e t i c and allowed to stand f i v e and one-half hours at 25°. A f t e r deamination the 250 rap. absorbing m a t e r i a l formed was adsorbed on a one and one-half f o l d excess of a c i d -and water-washed Nuchar C190 N (about 1 g). The e x t r a c t i o n charcoal was washed with 3 x 5 nil 0.05 M sodium acetate, pH 1+.0 and 2 x 10 ml glass d i s t i l l e d water. The nucle o t i d e was eluted with 7 x 5 ml isopropanol - water (1:1), pH 8.5 (paper) i-oLth ammonium hydroxide. The e x t r a c t s were concentrated and chromatographed i n solvent A. The only u l t r a - v i o l e t absorbing band was the n u c l e o t i d e , but some s a l t was evident. The nucleotide was e l u t e d from the paper, converted to the p y r i d i n i u m s a l t , and f r e e z e - d r i e d ; producing a gummy product. The product was then converted to the nucleoside - 3 1 , 5 '-monophosphate by the procedure usexi; f o r producing deoxyadenosine - 3 ' , 5 ' -monophosphate. The nucleotide was markedly i n s o l u a b l e i n anhydrous p y r i d i n e , even with heating i n the presence of the carboxamidine. Hence, excess carboxamidine and dry p y r i d i n e were used to permit the t r a n s f e r of most of the m a t e r i a l to the dropping f u n n e l . S o l u b i l i z a t i o n occurred i n the r e f l u x -i n g r e a c t i o n mixture i n the presence of a large excess of DCC. The c y c l i c n u c l e o t i d e was p u r i f i e d , u s i n g solvent A and B chromatography c o n s e c u t i v e l y . The p u r i f i e d m a t e r i a l appeared uniform i n solvents A and B, and by e l e c t r o p h o r e s i s . The p u r i f i e d m a t e r i a l at pH 5.5 had an u l t r a - v i o l e t a bsorption maximum at 21+7-5-280 mu and minimum at 221+ mu. - - 33 -d. Deoxyuridine - 3 1 , 5 '-Monophosphate 100 mg (0.28 mmole) of disodium deoxyuridine - 5 '-phosphate was converted to the cyclic-phosphate e s t e r by the method described p r e v i o u s l y f o r deoxyadenosine - 5 '-phosphate. The p y r i d i n i u m s a l t d i s s o l v e d r e a d i l y i n anhydrous p y r i d i n e i n the presence of the carboxamidine. The d e o x y n u c l e o s i d e - 3 ' , 5 ' -monphosphate was p u r i f i e d using solvent A chromatography. In solvent A and i n e l e c t r o p h o r e s i s , the m a t e r i a l moved as a s i n g l e u l t r a - v i o l e t absorbing spot, and also displayed the c h a r a c t e r i s t i c s of a c y c l i c - 3 5 ' - n u c l e o t i d e . The u l t r a -v i o l e t absorption maximum was at 259-260 mu at pH 7«5 and 259 at pH 5.5- The minimum was at 230 and 229 mu at pH 7.5 and 5 .5 , r e s p e c t i v e l y . Enzymatic I d e n t i f i c a t i o n of the C y c l i c - 3 ' , 5 ' - D e o x y n u c l e o t i d e s The cyclic - 3 ' , 5 '-monophosphates of deoxyadenosine, deoxy-ino s i n e and deoxyuridine were t e n t a t i v e l y i d e n t i f i e d by paper chromatography and by e l e c t r o p h o r e s i s . To complete the i d e n t i f i c a t i o n , small q u a n t i t i e s of each c y c l i c - 3 ' , 5 ' - d e o x y -nucleotide were incubated w i t h b r a i n d i e s t e r a s e . E l e c t r o -phoresis of samples of the d e p r o t e i n i z e d i n c u b a t i o n mixtures showed that the c y c l i c - 3 ' , 5 ' - d e o x y n u c l e o t i d e s were s l o w l y con-verted to new compounds. These new compounds moved durin g e l e c t r o p h o r e s i s i n a manner c h a r a c t e r i s t i c of n u c l e o s i d e - 5 ' -(3 ' -) phosphates. To e s t a b l i s h that the new nucleotides produced by the d i e s t e r a s e a c t i o n were the 5 1-phosphates, two i d e n t i f i c a t i o n methods were used. The f i r s t of these i n v o l v e d the p r e p a r a t i o n of a pure sample of product of d i e s t e r a s e a c t i o n on deoxy-- 3 1 + -adenosine-3' , 5'-monophosphate.: The d e p r o t e i n i z e d r e a c t i o n mixture of 10 mg of the semi-p u r i f i e d deoxyadenosine-3' , 5 '-monophosphate ( a f t e r i n c u b a t i o n w i t h an appropriate amount of the d i e s t e r a s e , ) was streaked on Whatman No. 3 MM paper and chromatographed i n solvent A. Pour bands r e s u l t e d . The two darkest and slowest running bands (I and I I ) were e l u t e d w i t h water. Samples of the concentrated eluates were then incubated w i t h adenylic deam-inase. The sample from band I was slowly converted to a new compound, with about J+0/£ of the o r i g i n a l o p t i c a l d e n s i t y at 265 mu, a f t e r 27 minutes at room temperature, and with a spectrum apparently i d e n t i c a l to that of i n o s i n i c a c i d . The band I I sample was unaffected by the deaminase. The deaminase produced neither- a change i n spectrum of the band I I sample, nor decrease i n o p t i c a l d e n s i t y at 265 mu a f t e r 1+6 . minutes. Hence, band I must have been the 5'-deoxynucleotide and band I I was probably unreacted c y c l i c - 3 5 ' - n u c l e o t i d e . Bands I I I and IV were not i d e n t i f i e d but were l i k e l y deoxyadenosine and adenine. These u n i d e n t i f i e d products were always found a f t e r prolonged i n c u b a t i o n with the crude b r a i n d i e s t e r a s e preparation, but d i d not appear u n t i l a f t e r appreciable amount of the deoxynucleoside - 5 1-phosphate had been formed. Hence, they are probably formed from the n u c l e o s i d e - 5 1 -phosphate. In a d d i t i o n , these two compounds behave i n solvent A as the nucleoside and free base, moving much more r a p i d l y than e i t h e r the 5 ' - or c y c l i c - 3 ' , 5 ' - d e o x y n u c l e o t i d e s . 1 The second method i d e n t i f y i n g the d i e s t e r a s e h y d r o l y s i s - 35 - • products as the nucleoside-5 '-phosphates was to incubate an e l e c t r o p h o r e t i c a l l y p u r i f i e d sample of each w i t h crude snake venom. Samples of the snake venom r e a c t i o n mixture were then chromatographed i n solvent A, (see Figure I I ) . A l l of the product nucleotides (of the d i e s t e r a s e a c t i o n ) were converted to new compounds which moved i n the solvent as the nucleosides. Although no authentic deoxynucleosides were available, the r a t e s of movement were compared to that of adenosine, and found to be approximately the same (see Figure I I ) . . Samples of the d i e s t e r a s e product nu c l e o t i d e s were compared with a u t h e n t i c deoxynucleoside-5'-phosphate samples of each analogue, and were found to move i n i d e n t i c a l manners i n solvent A (see 1 Figure I ) . Since the product deoxynucleoside-5 '-phosphate moved as the 5' ooiipounds i n e l e c t r o p h o r e s i s and solvent A chromatography, and were changed to new compounds by snake venom, (the new compounds a c t i n g i n solvent A as the deoxy-nucleosides) the products of d i e s t e r a s e h y d r o l y s i s were the appropriate deoxynucleoside-5'-phosphates. The i d e n t i f i c a t i o n of N-benzoyl-deoxyadenosine-3' ,5 ' l -monophosphate by b r a i n d i e s t e r a s e h y d r o l y s i s was incomplete. The samples of both bands, i s o l a t e d by e l e c t r o p h o r e s i s ( w i t h phosphate contamination), were incubated w i t h d i e s t e r a s e under the described c o n d i t i o n s , no h y d r o l y s i s occurred. This l a c k of h y d r o l y s i s was taken to i n d i c a t e that probably the d i e s t e r a s e was i n a c t i v e on the N-benzoyl compound. However, t h i s could not be considered c o n c l u s i v e . The small amount of m a t e r i a l that could be i s o l a t e d by the electrophoresis technique made de-- 36 -FIGURE I A Chromatogram of the (5'-) N u c l e o t i d e Products o f ' B r a i n  Phosphodiesterase A c t i o n - o n the D e o x y n u c l e o s i d e - 3 ' , 5 ' -Mo no pho s pha t e s Spots 1, 3 and 5 were the a u t h e n t i c 5'-phosphates of deoxyadenosine, deoxyinosine and d e o x y u r i d i n e . Spots 2, 1+ and 6 were the (5'-) n u c l e o t i d e products of b r a i n phospho-d i e s t e r a s e a c t i o n on the cyclic-3' , 5 '-monophosphates of deoxyadenosine, deoxyinosine and d e o x y u r i d i n e , r e s p e c t i v e l y . The product samples had been p r e v i o u s l y separated from the c y c l i c - p h o s p h a t e es:ter s t a r t i n g m a t e r i a l by e l e c t r o p h o r e s i s . 1 2 o 3 h 5" 6 o - 37 -FIGURE I I A C h r o m a t o g r a m o f t h e P r o d u c t s o f Snake Venom A c t i o n S p o t s 1, 2, 5> and 6 r e p r e s e n t a u t h e n t i c a d e n o s i n e , d e o x y a d e n o s i n e - 5 ' - p h o s p h a t e , d e o x y i n o s i n e - 5 ' - p h o s p h a t e and d e o x y u r i d i n e - 5 ' - p h o s p h a t e , r e s p e c t i v e l y . S a m p l e s 3 , 6 and 9 r e p r e s e n t t h e p r o d u c t s o f ' s n a k e venom a c t i o n on u n a u t h e n t i c d e o x y a d e n o s i n e - 5 ' - p h o s p h a t e , d e o x y i n o s i n e - 5 1 - p h o s p h a t e and d e o x y u r i d i n e - 5 ' - p h o s p h a t e f o r 1$ m i n u t e s . S a m p l e s [(., 7 and 10 r e p r e s e n t e q u i v a l e n t p r o d u c t s a f t e r 30 m i n u t e s o f i n c u b -a t i o n . The u n a u t h e n t i c s a m p l e s were o b t a i n e d b y e l e c t r o -p h o r e s i s o f t h e d i e s t e r a s e i n c u b a t i o n m i x t u r e s c o n t a i n i n g t h e p a r e n t c y c l i c - 3 ' , 5 ' - d e o x y n u c l e o t i d e s ( s e e t e x t ) . 6 D 7 9 10 - 38 - . b e n z o y l a t i o n I m p r a c t i c a l . Hence, the i d e n t i t y of the e l c t r o -phoresis bands I and I I were not e s t a b l i s h e d . Chromatography of a sample of the crude N-benzoyl-deoxyadenosine-3' ,5 '-monophosphate on DEAE-cellulose d i d not produce a sample pure enough f o r enzymatic s t u d i e s . R e l a t i v e H y d r o l y s i s Rates of the C y c l i c - ^ ' , 5 ' - deoxynucleotides by B r a i n Diesterase A f t e r the .compounds synthesized had been e s t a b l i s h e d as the cyclic-3' , 5'-deoxynucleotides, I t was decided to determine the approximate r a t e s at which they were hydrolysed by the b r a i n phosphodiesterase. Since the cyclic-3',5'-deoxynucleo-t i d e s had not yet been produced i n a b s o l u t e l y pure form, the degree of h y d r a t i o n and hence the e x t i n c t i o n c o - e f f i c i e n t s were not known. However, us i n g approximate e x t i n c t i o n co-e f f i c i e n t s determined from those of the nearest analogues f o r which c o - e f f i c i e n t s were a v a i l a b l e , approximate concentrations could be c a l c u l a t e d . The e x t i n c t i o n c o - e f f i c i e n t s used were 9.7 cm mM \ 260 mu, f o r deoxyuridin.e-3' ,5 ' -monophosphate ; 13.2 cm mM \ 2l|9 mu , f o r deoxyinosine-3' ,5 '-monophosphate and l£.2 cm mM \ 260 mu, f o r deoxyadenosine-3 1 ,5'-monophosphate. C a l c u l a t e d samples of the cyclic-3' ,5 '-monophosphates of deoxyuridine, deoxyinosine and deoxyadenosine, under the s p e c i f i e d c o n d i t i o n s , were incubated f o r the s p e c i f i e d time wi t h the b r a i n phosphodiesterase. The r a t e of decrease i n o p t i c a l d e n s i t y of the s t a r t i n g m a t e r i a l was p l o t t e d against the i n c u b a t i o n time, (Figure I I I ) . Examination of the graph f o r the r a t e of b r a i n d i e s t e r a s e h y d r o l y s i s of the c y c l i c - 39 -FIGURE I I I Time R a t e o f B r a i n P h o s p h o d i e s t e r a s e H y d r o l y s i s o f D e o x y n u c l e o s i d e -3 1 > 5'-Monophosphates The o p t i c a l d e n s i t y o f c y c l i c - 3 ' , 5 ' - m o n o p h o s p h a t e s o f d e o x y a d e n o s i n e and d e o x y u r i d i n e were m e a s u r e d a t 260 mu-. T h a t o f d e o x y i n o s i n e - 3 ' , 5 ' - m o n o p h o s p h a t e was m e a s u r e d a t 21+8 rap,. O p t i c a l d e n s i t i e s f o r e a c h , a t t i m e z e r o , were d e t e r m i n e d f r o m a s t a n d a r d s a m p l e . The enzyme p r e p a r a t i o n u s e d was p r e p a r e d b y t h e s e c o n d p r o c e d u r e f o r a c e t o n e powder p r e p a r a t i o n , e x t r a c t i o n and e x t r a c t f r a c t i o n a t i o n . 0.51+9 mg o f t h e 30-1+0$ s a t u r a t e d ammonium s u l f a t e f r a c t i o n p r o t e i n was u s e d p e r s a m p l e , e x c e p t f o r t h e i n c u b a t i o n o f d e o x y u r i d i n e - 3 ' , 5 ' - m o n o p h o s p h a t e , when d o u b l e t h a t amount was r e q u i r e d . - ko - - • • -depxynucle'otides showed that the r a t e of h y d r o l y s i s of each was l i n e a r with r e s p e c t to time. Comparison to the slopes of each (-AO.D./at) i n d i c a t e d t h a t the c y c l i c - 3 ' ,5' -deoxy-n u c l e o t i d e s were h y d r o l y s e d a t 100, 72.5 and 1 + . 6 f o r the cyclic - 3 ' , 5 '-monophosphates of deoxyadenosine, deoxyinosine and deoxyuridine, r e s p e c t i v e l y . A r b i t r a r i l y , the r a t e f o r deoxyadenosine - 3 ' ,5 '-monophosphate h y d r o l y s i s was e s t a b l i s h e d as 100. Measurement o f the r a t e of appearance of the deoxy-nucleoside-5'-phosphate products was not p r a c t i c a l as. the b r a i n d i e s t e r a s e p r e p a r a t i o n contained a p p r e c i a b l e amounts of phosphatase and, presumably, n u c l e o s i d a s e a c t i v i t y . The c y c l i c - 3 ' , 5 ' - d e o x y n u c l e o t i d e h y d r o l y s i s r a t e s were not compared to those of the analogous r i b o n u c l e o t i d e s because the r e s u l t s obtained here are s t i l l too imprecise to y i e l d a ccurate data. In a d d i t i o n , N a i r (kl) has i n d i c a t e d that deoxy-adenosine - 3 ' ,5'-monophosphate i s h y d r o l y s e d more r a p i d l y than adenosine - 3 ' ,5 '-monophosphate by a p u r i f i e d ( 190-fold) dog heart d i e s t e r a s e , the s p e c i f i c i t y of which was e s s e n t i a l l y the same as that r e p o r t e d f o r the b r a i n enzyme by Drummond and P e r r o t t - Y e e (1+1). - m -DISCUSSION The method o f f o r m i n g the c y c l i c n u c l e o t i d e s was e s s e n t -i a l l y t h a t developed by Smith, e t a l . (1+0). Only s m a l l m o d i f i c a t i o n s were made t o the above p r o c e d u r e . A l l c y c l i z a t i o n s i n v o l v e d the s l o w a d d i t i o n of the d i l u t e s o l u t i o n o f the c a r b o x -a m i d i n i u m s a l t of the d e o x y n u c l e o s i d e - 5 1 - p h o s p h a t e t o a d i l u t e , r a p i d l y r e f l u x i n g s o l u t i o n o f DCC i n d r y p y r i d i n e . The r e a c t i o n m i x t u r e was s t i r r e d r a p i d l y t o keep the c o n c e n t r a t i o n o f the added n u c l e o t i d e as low as p o s s i b l e u n t i l c y c l i z a t i o n c o u l d o c c u r . The one hour r e f l u x mentioned i n the above r e f -e r e n c e , was not used as p r e v i o u s work i n t h i s l a b o r a t o r y i n d i c a t e d t h a t t h i s r e s u l t e d i n the i n c r e a s e d f o r m a t i o n o f an unknown d e r i v a t i v e . I t was found t h a t by f o l l o w i n g t h i s s l i g h t l y m o d i f i e d procedure s i g n i f i c a n t amounts o f two unchar-a c t e r i z e d compounds x<rere formed. These compounds d i d not n o r m a l l y i n t e r f e r e i n the i s o l a t i o n o f the c y c l i c - p h o s p h a t e p r o d u c t s , but d i d p r e v e n t t h e p u r i f i c a t i o n o f N - b e n z o y l -deoxyadenosine - 3 ' , 5 '-monophosphate. Y i e l d s o f the compounds s y n t h e s i z e d i n t h i s s t u d y were r a t h e r l ow as compared t o th o s e a c h i e v e d by S m i t h , e_t a l . (1|0). However, s i n c e o n l y s m a l l amounts o f the m a t e r i a l s were r e q u i r e d f o r the p r e s e n t s t u d y the p r o c e d u r e s were adequate. The use o f paper chromatography f o r the i s o l a t i o n of the c y c l i c p r o d u c t s was d e s i r a b l e because the method i s r a p i d and s i m p l e . However, paper chromatography produces contaminated, p r o d u c t s and has the added d i s a d v a n t a g e o f low c a p a c i t y . The ; • - 4 2 - - - ----- -contaminations r e s u l t from small amounts of the suspended paper and a yellow pigment e l u t e d with the n u c l e o t i d e . The y e l l o w pigment could not be removed by washing the papers w i t h the chromatographic solvents (solvent B was p a r t i a l l y s u c c e s s f u l ) . Hence, before very pure products could be pro-duced, the m a t e r i a l s p u r i f i e d by paper chromatography would have to be r e p u r i f i e d by some other method. For the p r e p a r a t i o n of large amounts of these compounds column chromatography (e.g.,DEAE-cellulose) would be most s a t i s f a c t o r y . Three of the compounds which were synthesized were i d e n t i f i e d c o n c l u s i v e l y as the d e o x y r i b o n u c l e o s i d e ^ ' j ^ ' -monophosphates. They were the cyclic-3 ' , 5'-monophosphates of deoxyadenosine, deoxyinosine and deoxyuridine. The enzymatic h y d r o l y s i s , by an enzyme s p e c i f i c f o r nucleoside-3 ' , 5 '-mono-phosphates (Lj!) would lend support to t h e i r i d e n t i t y . The products of the d i e s t e r a s e h y d r o l y s i s were i d e n t i f i e d as the 5'-analogues of the s t a r t i n g m a t e r i a l s by e l e c t r o p h o r e s i s , chromatography i n solvent A and by snake venom 5 '-nucleotidase a c t i o n , thus concluding the i d e n t i f i c a t i o n . That the product of the d i e s t e r a s e h y d r o l y s i s was i n f a c t the n u c l e o s i d e - 5 ' -phosphate was a l s o supported by the f a c t that the major product of the b r a i n d i e s t e r a s e a c t i o n on d e o x y a d e o n o s i n e - 3 1 , 5 > ' -monophosphate was deaminated by p u r i f i e d muscle adenylic a c i d deaminase, producing a new compound w i t h an ab s o r p t i o n spectrum i d e n t i c a l to that of deoxyinosine - 5 '-phosphate. As mentioned p r e v i o u s l y , the p r e p a r a t i o n of N-benzoyl-deoxyadenosine-3' ,5 '-monophosphate was not s u c c e s s f u l . 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