UBC Theses and Dissertations

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UBC Theses and Dissertations

Studies on adenyl cyclase in heart and skeletal muscle Severson, David Lester 1972

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STUDIES ON AD ENYL CYCLASE IN HEART AND SKELETAL MUSCLE by D a v i d L e s t e r S e v e r s o n B . Sc . ( H o n s . ) , U n i v e r s i t y o f A l b e r t a , 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e D e p a r t m e n t o f Pharma c o1o gy We a c c e p t t h i s t h e s i s as 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 p r i l , 19 7 2 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C olumbia, I agree 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 agree 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 urposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Pharmacology The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date A p r i l 8, 1972 ( i ) ABSTRACT The k i n e t i c p r o p e r t i e s of a d e n y l c y c l a s e i n h e a r t and s k e l e t a l m u s c l e were examined to d e t e r m i n e the mechanisms by w h i c h hormones and o t h e r r e g u l a t o r y a g e n t s s t i m u l a t e the f o r m a t i o n of a d e n o s i n e 3 ' , 5 * - c y c l i c monophosphate ( c y c l i c AMP). E x p e r i m e n t s were p e r f o r m e d w i t h washed p a r t i c l e s and l i t h i u m b r o m i d e - e x t r a c t e d p a r t i c u l a t e enzyme p r e p a r a t i o n s f r o m g u i n e a p i g or r a b b i t h e a r t . M y o c a r d i a l a d e n y l c y c l a s e was s t i m u l a t e d by c o n c e n t r a t i o n s of m e t a l i o n s i n e x c e s s of the ATP c o n c e n t r a t i o n . M e t a l i o n s a p p a r e n t l y b i n d t o some s e c o n d s i t e on the enzyme i n a d d i t i o n t o the c a t a l y t i c s i t e and as a c o n s e q u e n c e i n c r e a s e r e a c t i o n v e l o c i t y . Manganese was more e f f e c t i v e t h a n magnesium i n a c t i v a t i n g the c a r d i a c enzyme; c o b a l t was l e s s e f f e c t i v e . The K g f o r manganese was 0.7 mM i n c o m p a r i s o n to 3 mM f o r magnesium. The K m f o r ATP was 0.1 mM; m e t a l i o n s i n c r e a s e d maximal v e l o c i t y w i t h o u t a l t e r i n g the s u b s t r a t e a f f i n i t y o f the enzyme. C o n c e n t r a t i o n s o f ATP i n e x c e s s of the m e t a l i o n c o n c e n t r a t i o n r e s u l t e d i n i n h i b i t i o n of m y o c a r d i a l a d e n y l c y c l a s e due t o c o m p e t i t i o n w i t h t h e enzyme f o r m e t a l i o n s . A d e n y l c y c l a s e was a l s o s t u d i e d i n r a b b i t s k e l e t a l m u s c l e p l a s m a membrane p r e p a r a t i o n s . I s o l a t i o n of p l a s m a membranes r e s u l t e d i n a t e n to t w e n t y - f o l d i n c r e a s e i n s p e c i f i c enzyme a c t i v i t y w i t h a y i e l d of a p p r o x i m a t e l y 30 p e r c e n t of the a d e n y l c y c l a s e a c t i v i t y of the whole homogenate. Phase c o n t r a s t m i c r o s c o p y as w e l l as the a n a l y s i s of the c h e m i c a l c o m p o s i t i o n and v a r i o u s enzyme a c t i v i t i e s s u g g e s t e d t h a t the membrane ( i i ) p r e p a r a t i o n s c o n s i s t e d of h i g h l y p u r i f i e d plasma membranes. The K f o r magnesium was 3 to 5 mM and the K f o r ATP was a m approximately 0.3 mM. Magnesium i n c r e a s e d the r e a c t i o n v e l o c i t y at a l l c o n c e n t r a t i o n s of s u b s t r a t e , presumably by b i n d i n g to some second s i t e . The s t i m u l a t i o n of s k e l e t a l muscle adenyl c y c l a s e by f l u o r i d e was markedly temperature s e n s i t i v e and p a r t i a l l y i r r e v e r s i b l e . F l u o r i d e - s t i m u l a t e d enzyme a c t i v i t y was p a r t i c u l a r l y s e n s i t i v e to i n h i b i t i o n by pyrophosphate; pyrophosphate i n h i b i t i o n was c o m p e t i t i v e with r e s p e c t to ATP (K^, 0.45 mM). The r e l a t i v e p o t e n c i e s of catecholamines i n s t i m u l a t i n g enzyme a c t i v i t y and the i n h i b -i t i o n of the ep i n e p h r i n e s t i m u l a t i o n by p r o p r a n o l o l i n d i c a t e d some r e l a t i o n s h i p between adenyl c y c l a s e and the beta-adren-e r g i c r e c e p t o r . The s t i m u l a t i o n by ep i n e p h r i n e was enhanced by the a d d i t i o n of low c o n c e n t r a t i o n s of GTP or GDP; guanyl n u c l e o t i d e s had l i t t l e or no e f f e c t on b a s a l or f l u o r i d e -s t i m u l a t e d adenyl c y c l a s e a c t i v i t y . The k i n e t i c nature of the s t i m u l a t i o n by f l u o r i d e and epi n e p h r i n e was i d e n t i c a l . Both e p i n e p h r i n e and f l u o r i d e i n c r e a s e d maximal v e l o c i t y w i t h -out a f f e c t i n g the a f f i n i t y of the enzyme f o r magnesium or ATP. Thus, the r e g u l a t i o n of adenyl c y c l a s e i n heart and s k e l e t a l muscle can be d e s c r i b e d as a "V" a l l o s t e r i c system. The p r o p e r t i e s of adenyl c y c l a s e are d i s c u s s e d i n r e l a t i o n to the involvement of the enzyme i n a hormone i n f o r m a t i o n a l t r a n s f e r u n i t . ( i i i ) TABLE OF CONTENTS Page I . INTRODUCTION 1 I I . EXPERIMENTAL PROCEDURE A. M a t e r i a l s 13 B. Methods 15 1. Enzyme p r e p a r a t i o n s from heart 15 2. Enzyme p r e p a r a t i o n s from s k e l e t a l 16 musele 3. Enzyme assays 17 4. Chemical assays 24 I I I . RESULTS A. Adenyl c y c l a s e i n heart 26 1. P r e l i m i n a r y 26 2. S u b c e l l u l a r d i s t r i b u t i o n 30 3. K i n e t i c p r o p e r t i e s of myo c a r d i a l 32 adenyl c y c l a s e B. Adenyl c y c l a s e i n s k e l e t a l muscle 43 1. P r e l i m i n a r y 44 2. S u b c e l l u l a r d i s t r i b u t i o n and p r e p a r a t i o n 44 plasma membranes 3. Assay c o n d i t i o n s 54 4. K i n e t i c p r o p e r t i e s of s k e l e t a l muscle 62 adenyl c y c l a s e IV. DISCUSSION 94 V. BIBLIOGRAPHY 118 ( i v ) LIST OF TABLES No. Page I D e t e r m i n a t i o n of adenyl c y c l a s e a c t i v i t y by 29 three methods. I I S u b c e l l u l a r d i s t r i b u t i o n of myocardial adenyl 31 c y c l a s e . I I I Adenyl c y c l a s e i n s k e l e t a l muscle from s e v e r a l 45 s p e c i e s . IV S u b c e l l u l a r d i s t r i b u t i o n of adenyl c y c l a s e 47 i n s k e l e t a l muscle t i s s u e . V Chemical composition of s k e l e t a l muscle 55 plasma membranes. VI E f f e c t i v e n e s s of the ATP-regenerating system. 57 VII E f f e c t of u n l a b e l l e d c y c l i c AMP and t h e o p h y l - 58 l i n e on the d e g r a d a t i o n and formation of c y c l i c AMP. V I I I I r r e v e r s i b i l i t y of f l u o r i d e a c t i v a t i o n of 71 s k e l e t a l muscle adenyl c y c l a s e . IX A d d i t i v i t y of ep i n e p h r i n e and f l u o r i d e 76 a c t i v a t i o n . X E f f e c t of GTP and ITP on s k e l e t a l muscle adenyl 88 eyelase . XI E f f e c t of n u c l e o t i d e s on e p i n e p h r i n e - s t i m u 1 - 89 ated adenyl c y c l a s e . (v) LIST OF FIGURES No. P a g e 1. Paper chromatographic s e p a r a t i o n of adenine 20 n u c l e o t i d e s and adenosine. 2. E f f e c t of metal ions on myocardial adenyl 34 eyelase . 3. E f f e c t of ATP c o n c e n t r a t i o n on my o c a r d i a l adenyl 38 c y c l a s e . 4. R e v e r s a l of ATP i n h i b i t i o n by manganese. 41 5. Phase c o n t r a s t photomicrographs of f r a c t i o n s 50 obtained d u r i n g p r e p a r a t i o n of s k e l e t a l muscle plasma membranes. 6. Dependence of s k e l e t a l muscle adenyl c y c l a s e 61 a c t i v i t y on time and p r o t e i n 7. E f f e c t of metal ions on s k e l e t a l muscle adenyl 64 c y c l a s e . 8. E f f e c t of ATP c o n c e n t r a t i o n on s k e l e t a l muscle 67 adenyl c y c l a s e . 9. E f f e c t of f l u o r i d e and temperature on s k e l e t a l 70 muscle adenyl c y c l a s e . 10. E f f e c t of catecholamines on s k e l e t a l muscle 75 adenyl c y c l a s e . 11. E f f e c t s of f l u o r i d e and epi n e p h r i n e on magnesium 79 a c t i v a t i o n of s k e l e t a l muscle adenyl c y c l a s e . 12. E f f e c t of f l u o r i d e and e p i n e p h r i n e on ATP s a t u r - 81 a t i o n of s k e l e t a l muscle adenyl c y c l a s e . 13. E f f e c t of pyrophosphate on s k e l e t a l muscle adenyl 84 c y c l a s e . 14. E f f e c t of pyrophosphate on ATP s a t u r a t i o n of 86 s k e l e t a l muscle adenyl c y c l a s e . 15. E f f e c t of GTP and GDP on s k e l e t a l muscle adenyl 92 c y c l a s e . 16. Schematic r e p r e s e n t a t i o n of hormone a c t i o n on 111 adenyl c y c l a s e as an i n f o r m a t i o n a l t r a n s f e r u n i t . ( v i ) ABBREVIATIONS ATP cc,|5 -methylene ATP ^ ,JC -methylene ATP ADP 5'-AMP c y c l i c AMP c y c l i c 2*,3'-AMP CTP GTP GDP GMP ITP UTP ACTH EDTA EGTA adenosine 5 1 - t r i p h o s p h a t e ac, p -methylene adenosine 5 ' - 1 r ip hosp ha t e t ¥ -methylene adenosine 5'-triphosphate adenosine 5 *-diphosphate adenosine 5 1-monophosphate adenosine 3 ' , 5 ' - c y c l i c monophosphate adenosine 2 , , 3 ' - c y c l i c monophosphate c y t i d i n e 5 1 - t r i p h o s p h a t e guanosine 5 1 - t r i p h o s p h a t e guanosine 5 1-diphosphate guanosine 5'-monophosphate i n o s i n e 5 '-triphosphate u r i d i n e 5 ' - t r i p h o s p h a t e a d r e n o c o r t i c o t r o p i c hormone ethylenediamine t e t r a a c e t a t e ethylene g l y c o l b i s (j3-amino e t h y l e t h e r ) N , N ' - t e t r a a c e t a t e T r i s t r i s (hydroxymethyl) aminomethane ( v i i ) ACKNOWLEDGEMENTS I t i s a p l e a s u r e t o t h a n k D r . George I . Drumraond f o r h i s g u i d a n c e and e n c o u r a g e m e n t d u r i n g the c o u r s e of t h i s work and f o r p r o v i d i n g a s u p e r b example of s c i e n t i f i c d e d i c a t i o n and e x c e l l e n c e . The a u t h o r would l i k e to e x p r e s s h i s g r a t i t u d e t o Mrs. L o v e r n e Duncan f o r h e r t e c h n i c a l i n s t r u c t i o n and p e r s o n a l e n c o u r a g e m e n t d u r i n g the i n i t i a l p a r t of t h i s work. The a u t h o r a l s o w i s h e s t o thank D r . M.C. S u t t e r and D r . D. G o d i n f o r many u s e f u l d i s c u s s i o n s , and a l l o t h e r s who have c o n t r i b u t e d t o my e d u c a t i o n and the p r e p a r a t i o n of t h i s t h e s i s . The f i n a n c i a l s u p p o r t o f t h e M e d i c a l R e s e a r c h C o u n c i l of Canada i s g r a t e f u l l y a c k n o w l e d g e d . 1 -INTRODUCTION Hormones are i n t i m a t e l y i n v o l v e d i n the r e g u l a t i o n and i n t e g r a t i o n of the complex p h y s i o l o g i c a l and metabolic processes i n l i v i n g organisms. However, the mechanisms by which hormones e x e r t t h e i r e f f e c t s on t a r g e t t i s s u e s have remained a mystery u n t i l r e c e n t l y . In the e a r l y 1950's, Sutherland i n i t i a t e d h i s b r i l l i a n t s e r i e s of i n v e s t i g a t i o n s i n t o the mechanisms by which ep i n e p h r i n e and a p a n c r e a t i c h y p e r g l y c e m i c - g l y c o g e n o l y t i c f a c t o r (glucagon) caused i n c r e a s e d h e p a t i c g l y c o g e n o l y s i s and hyper-g l y c e m i a . A n a l y s i s of t i s s u e l e v e l s of gl u c o s e , g l u c o s e - 1 -phosphate , and glucose-6-phosphate i n d i c a t e d that the phos-ph o r y l a s e r e a c t i o n was r a t e - 1 i m i t i n g and that the hyperglycemia produced by hormones was due to i n c r e a s e d glucose p r o d u c t i o n r a t h e r than an i n c r e a s e i n glucose t r a n s p o r t out of the c e l l ( 1 , 2 ) . I t was a l s o shown that hormones i n c r e a s e d phosphorylase a c t i v i t y i n l i v e r s l i c e s (2,3) and that t h i s was accompanied by the cov a l e n t i n c o r p o r a t i o n of phosphate i n t o l i v e r phos-ph o r y l a s e ( 3 ) . Of great importance was the d i s c o v e r y that the a c t i v a t i o n of phosphorylase by glucagon and epi n e p h r i n e observed i n l i v e r s l i c e s could a l s o be demonstrated by the hormones i n l i v e r homogenates ( 4 ) . The a b i l i t y to respond to hormones r e q u i r e d the presence of the p a r t i c u l a t e f r a c t i o n of the l i v e r homogenate, and was mediated by the p r o d u c t i o n of a h e a t - s t a b l e f a c t o r which was able to a c t i v a t e l i v e r phosphorylase i n the supernatant f r a c t i o n of the homogenate ( 4 ) . This h e a t - s t a b l e f a c t o r was i s o l a t e d and c h a r a c t e r i z e d as adenosine 3 ' , 5 ' - c y c l i c - 2 -monophosphate ( c y c l i c AMP) ( 5 , 6 ) . The f o r m a t i o n of c y c l i c AMP was a l s o observed i n b r a i n , and i t s accumulation i n s k e l e t a l muscle and heart was i n f l u e n c e d by e p i n e p h r i n e ( 7 ) . These o b s e r v a t i o n s r e s u l t e d i n the p r o p o s a l of the second messenger concept f o r hormone a c t i o n ( 8 , 9 ) . A c c o r d i n g to t h i s concept, the hormone (the f i r s t messenger) i n t e r a c t s at the membrane of the t a r g e t c e l l r e s u l t i n g i n the s t i m u l a t i o n of the i n t r a c e l l u l a r formation of a second messenger ( c y c l i c AMP) which then r e g u l a t e s the p h y s i o l o g i c a l or m e t a b o l i c processes w i t h i n the c e l l . C y c l i c AMP i s the only second messenger i d e n t i f i e d as yet and has been i m p l i c a t e d i n the m e d i a t i o n of a wide v a r i e t y of c e l l u l a r processes by a m u l t i -tude of hormones ( f o r recent reviews and monographs, see r e f e r e n c e s 10-17). The f o r m a t i o n of c y c l i c AMP from ATP i s c a t a l y z e d by adenyl c y c l a s e (7,18). Pyrophosphate was shown to be the other product of the enzyme r e a c t i o n ( 1 9 ) . I n t r a c e l l u l a r l e v e l s of c y c l i c AMP are a l s o r e g u l a t e d by degradat ion of the c y c l i c n u c l e o t i d e to 5'-AMP by r i b o n u c l e o s i d e 3 ' , 5 ' - c y c l i c phosphate d i e s t e r a s e ( 2 0 ) . Adenyl c y c l a s e i s w i d e l y d i s t r i b u t e d i n n a t u r e . Enzyme a c t i v i t y has been det e c t e d i n mammals, b i r d s , amphibians, f i s h , i n s e c t s , segmented worms and l i v e r f l u k e s , as w e l l as i n u n i c e l -l u l a r organisms such as b a c t e r i a and slime molds (14). Adenyl c y c l a s e a c t i v i t y has not been observed i n p l a n t s , although r e c e n t l y i t was r e p o r t e d that c y c l i c AMP l e v e l s i n p l a n t s are i n c r e a s e d by i n d o l e - 3 - a c e t i c a c i d (21,22). - 3 -A d e n y l c y c l a s e a c t i v i t y was o r i g i n a l l y o b s e r v e d t o be r e s t r i c t e d t o the p a r t i c u l a t e f r a c t i o n s e d i m e n t e d a t low g r a v i t a t i o n a l f o r c e s i n l i v e r homogenates ( 4 ) . The s u b c e l l u l a r l o c a l i z a t i o n of a d e n y l c y c l a s e i n p a r t i c u l a t e f r a c t i o n s of l i v e r was c o n f i r m e d ( 1 8 ) . In a d d i t i o n , a d e n y l c y c l a s e was l o c a l i z e d i n p a r t i c u l a t e f r a c t i o n s when examined i n b r a i n ( 2 3 ) , k i d n e y ( 2 4 ) , t h y r o i d ( 2 5 ) , mammary g l a n d and myometrium ( 2 4 ) , brown and w h i t e a d i p o s e t i s s u e ( 2 6 , 2 7 ) , h e a r t ( 2 8 ) , t o a d b l a d d e r ( 2 9 ) , f r o g e r y t h r o c y t e s ( 3 0 ) , s k e l e t a l m u s c l e (31,32) and i n n o r m a l a d r e n o c o r t i c a l t i s s u e (33-35) and a d r e n o c o r t i c a l tumors ( 3 5 , 3 6 ) . In most, but n o t a l l , of the t i s s u e s c i t e d a b o v e , the h i g h e s t a c t i v i t y of a d e n y l c y c l a s e was o b s e r v e d i n s u b c e l l u l a r f r a c t i o n s c o n t a i n i n g f r a g -ments of the p l a s m a membrane. A p o r t i o n of a d e n y l c y c l a s e i n s almon t e s t e s was s o l u b l e ( 3 7 ) . A d e n y l c y c l a s e i n v a r i o u s s t r a i n s of b a c t e r i a was e i t h e r s o l u b l e (38,39) o r p a r t i c u l a t e (39) a l t h o u g h the p a r t i c u l a t e enzyme was e a s i l y s o l u b i l i z e d (40) . A c u r i o u s f e a t u r e of a d e n y l c y c l a s e a c t i v i t y i s the a b i l i t y of f l u o r i d e i o n t o s t i m u l a t e the enzyme ( 1 8 ) . The s t i m u l a t i o n due t o f l u o r i d e i s n o n - s p e c i f i c s i n c e i t o c c u r s i n a l l mammalian t i s s u e s , a l t h o u g h h i g h l y s e l e c t i v e f o r f l u o r i d e i n c o m p a r i s o n w i t h o t h e r a n i o n s . In c o n t r a s t t o f l u o r i d e , hormones p r o d u c e a s e l e c t i v e s t i m u l a t i o n of a d e n y l c y c l a s e a c t i v i t y i n b r o k e n c e l l p r e p a r a t i o n s f r o m v a r i o u s t i s s u e s . A d e n y l c y c l a s e f r o m l i v e r was s t i m u l a t e d by g l u c a g o n and e p i n e p h r i n e ( 4 , 7 , 4 1 - 4 3 ) ; f r o m e r y t h r o c y t e s by c a t e c h o l a m i n e s - 4 -( 3 0 , 4 4 , 4 5 ) ; f r o m t h y r o i d by t h y r o i d - s t i m u 1 a t i n g hormone ( 2 5 , 4 6 ) ; and i n k i d n e y and t o a d b l a d d e r by n e u r o h y p o p h y s e a l h o r -mones ( 2 9 , 4 7 - 5 0 ) . In a d d i t i o n , p a r a t h y r o i d hormone s t i m u l -a t e d a d e n y l c y c l a s e i n s k e l e t a l t i s s u e (51,52) and ACTH s t i m u l a t e d enzyme a c t i v i t y i n a d r e n a l c o r t e x ( 3 3 , 3 6 ) . A d e n y l c y c l a s e i n a d i p o s e t i s s u e was s t i m u l a t e d by a wide v a r i e t y of l i p o l y t i c hormones i n c l u d i n g ACTH, g l u c a g o n , e p i n e p h r i n e , t h y r o i d - s t i m u l a t i n g hormone, l u t e i n i z i n g hormone and s e c r e t i n ( 2 7 , 5 3 - 5 6 ) . I t i s of i n t e r e s t t h a t i n c o n t r a s t to the s e l e c t i v e s t i m u l a t i o n of a d e n y l c y c l a s e by hormones as o u t -l i n e d a b o v e , the a d e n y l c y c l a s e of a r a t a d r e n o c o r t i c a l c a r c i n o m a was s t i m u l a t e d n o t o n l y by ACTH but a l s o by c a t e c h o l a m i n e s , t h y r o i d - s t i m u l a t i n g hormone, l u t e i n i z i n g hormone and f o l l i c l e -s t i m u l a t i n g hormone ( 3 5 , 5 7 ) . The r e g u l a t i o n of a d e n y l c y c l a s e a c t i v i t y i n c a r d i a c t i s s u e has been of p a r t i c u l a r i n t e r e s t w i t h r e s p e c t t o the p o s s i b i l i t y t h a t hormones may i n f l u e n c e b o t h m e c h a n i c a l ( i n o -t r o p i c ) and m e t a b o l i c ( g l y c o g e n m e t a b o l i s m ) p r o c e s s e s by a l t e r i n g t i s s u e l e v e l s of c y c l i c AMP ( s e e r e f e r e n c e s 58-60 f o r r e c e n t r e v i e w s ) . The a d m i n i s t r a t i o n of e p i n e p h r i n e i n c r e a s e d c y c l i c AMP l e v e l s i n i s o l a t e d , p e r f u s e d r a t and r a b b i t h e a r t s (61-64) as w e l l as i n r a t h e a r t s i n v i v o ( 6 5 ) . I n c r e a s e s i n t i s s u e l e v e l s of c y c l i c AMP i n r e s p o n s e t o e p i n e -p h r i n e p r e c e d e d the i n c r e a s e i n c o n t r a c t i l e f o r c e ( 6 1 , 6 2 , 6 4 ) ; b e t a - a d r e n e r g i c b l o c k i n g a e e n t s p r o n e t h a l o l and d i c h l o r o i s o -p r o t e r e n o l p r e v e n t e d the i n c r e a s e i n c y c l i c AMP l e v e l s and the i n o t r o p i c r e s p o n s e ( 6 2 , 6 3 , 6 5 ) . C a t e c h o l a m i n e s a l s o i n c r e a s e d c y c l i c AMP i n s l i c e s f r o m r a t h e a r t ( 6 6 , 6 7 ) . The e l e v a t i o n of m y o c a r d i a l c y c l i c AMP i n s l i c e s due to i s o p r o p y l -- 5 -n o r e p i n e p h r i n e was b l o c k e d by p r o p r a n o l o l , a be t a - a d r e n e r g i c b l o c k e r ( 6 7 ) . An i n c r e a s e i n a d e n y l c y c l a s e a c t i v i t y a f t e r a d d i t i o n of e p i n e p h r i n e t o b r o k e n - c e l l homogenates f r o m dog h e a r t was f i r s t o b s e r v e d i n 1958 ( 6 , 7 ) . T h i s s t i m u l a t i o n by c a t e c h o l a m i n e s has been c o n f i r m e d i n p a r t i c u l a t e p r e p a r a t i o n s f r o m r a t ( 6 8 ) , c a t ( 6 9 ) , and dog h e a r t ( 4 1 ) . The a c t i v a t i o n of a d e n y l c y c l a s e by c a t e c h o l a m i n e s i n a l l t h e s e c a r d i a c t i s s u e s was b l o c k e d by b e t a - a d r e n e r g i c b l o c k i n g a g e n t s s u c h as d i c h l o r o i s o p r o t e r e n o l and p r o p r a n o l o l ( 4 1 , 6 8 , 6 9 ) . F u r t h e r e v i d e n c e f o r the i n v o l v e m e n t of c y c l i c AMP i n the i n o t r o p i c r e s p o n s e t o c a t e c h o l a m i n e s was p r o v i d e d by t h e o b s e r v a t i o n t h a t the r e l a t i v e p o t e n c i e s of i s o p r o p y l n o r e p i n e p h r i n e , e p i n e p h r i n e and n o r e p i n e p h r i n e i n a c t i v a t i n g m y o c a r d i a l a d e n y l c y c l a s e (41) were v e r y s i m i l a r t o t h o s e o b s e r v e d f o r the i n c r e a s e i n c o n t r a c t i l e f o r c e i n dog h e a r t s i n v i v o ( 7 0 ) . G l u c a g o n has an e f f e c t on m y o c a r d i a l c o n t r a c t i l i t y s i m i l a r to t h a t o b s e r v e d f o r c a t e c h o l a m i n e s ( 5 9 , 6 0 ) . G l u c a g o n i n c r e a s e d t i s s u e l e v e l s of c y c l i c AMP 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 , a l t h o u g h the i n c r e a s e i n c y c l i c AMP d i d n o t p r e c e d e the i n c r e a s e i n c o n t r a c t i l e f o r c e ( 7 1 ) . C y c l i c AMP f o r m a t i o n was i n c r e a s e d by g l u c a g o n i n r a t h e a r t s l i c e s (67) and i n p a r t i c u l a t e p r e -p a r a t i o n s f r o m r a t ( 6 8 ) , and f r o m c a t and human h e a r t ( 6 9 ) . Combined maximal dos e s of g l u c a g o n and c a t e c h o l a m i n e s d i d n o t r e s u l t i n a d d i t i v e i n c r e a s e s i n c y c l i c AMP f o r m a t i o n i n s l i c e s (67) or b r o k e n c e l l p r e p a r a t i o n s ( 6 8 , 6 9 ) , s u g g e s t i n g t h a t the hormones ar e a c t i v a t i n g a s i n g l e a d e n y l c y c l a s e i n - 6 -c a r d i a c t i s s u e . G l u c a g o n d o e s n o t i n t e r a c t w i t h t h e b e t a -a d r e n e r g i c r e c e p t o r s i n c e b e t a - a d r e n e r g i c b l o c k i n g a g e n t s s u c h a s d i c h l o r o i s o p r o t e r e n o l , p r o n e t h a l o l o r p r o p r a n o l o l d i d n o t b l o c k t h e i n c r e a s e i n c y c l i c AMP l e v e l s d u e t o g l u c a -g o n i n t h e i s o l a t e d , p e r f u s e d r a t h e a r t ( 7 1 ) a n d r a t h e a r t s l i c e ( 6 7 ) , o r t h e i n c r e a s e i n a d e n y l c y c l a s e a c t i v i t y i n b r o k e n c e l l p r e p a r a t i o n s ( 6 8 , 6 9 ) . A n u m b e r o f o t h e r h o r m o n e s h a v e b e e n s h o w n t o a c t i v a t e m y o c a r d i a l a d e n y l c y c l a s e . T h y r o x i n e a n d t r i i o d o t h y r o n i n e i n c r e a s e d c y c l i c AMP f o r m a t i o n i n c a t h e a r t h o m o g e n a t e s ( 7 2 ) . A d e n y l c y c l a s e a c t i v i t y i n p a r t i c u l a t e p r e p a r a t i o n s o f c a t , g u i n e a p i g , a n d h u m a n h e a r t w a s i n c r e a s e d b y h i s t a m i n e ( 7 3 ) . S t i m u l a t i o n o f a d e n y l c y c l a s e a c t i v i t y b y h i s t a m i n e was n o t b l o c k e d b y p r o p r a n o l o l b u t w a s i n h i b i t e d b y t h e a n t i h i s t a m i n e , d i p h e n h y d r a m i n e . I n c o n t r a s t t o t h e r e s u l t s w i t h g l u c a g o n , c o m b i n e d m a x i m a l c o n c e n t r a t i o n s o f h i s t a m i n e a n d n o r e p i n e p h r i n e p r o d u c e d a n a d d i t i v e s t i m u l a t i o n o f a d e n y l c y c l a s e , s u g g e s t i n g t h a t h i s t a m i n e a n d n o r e p i n e p h r i n e may a c t o n s e p a r a t e a d e n y l c y c l a s e s y s t e m s ( 7 3 ) . M y o c a r d i a l a d e n y l c y c l a s e a c t i v i t y c a n a l s o be s t i m u l a t e d b y p r o s t a g l a n d i n s ( P G E j ^ , P G E 2 , a n d P G A ^ ) ( 7 4 ) a n d b y t o l b u t a m i d e ( 7 5 ) . S t i m u l a t i o n o f a d e n y l c y c l a s e b y PGEj_ w a s n o t b l o c k e d b y p r o p r a n o l o l a n d c o m b i n e d m a x i m a l c o n c e n t r a t i o n s o f P G E ^ a n d n o r e p i n e p h r i n e w e r e n o t a d d i t i v e ( 7 4 ) . I n a d d i t i o n t o t h e w e l l k n o w n a d r e n e r g i c s t i m u l a t i o n o f a d e n y l c y c l a s e i n c a r d i a c t i s s u e , c h o l i n e r g i c a g e n t s a l s o i n f l u e n c e e n z y m e a c t i v i t y . C a r b a c h o l i n h i b i t e d a d e n y l c y c l a s e - 7 -i n p a r t i c u l a t e p r e p a r a t i o n s f r o m d o g h e a r t ( 4 1 ) . C a r b a c h o l d e c r e a s e d c y c l i c AMP l e v e l s i n a t r i a l s l i c e s , d e p r e s s e d a d e n y l c y c l a s e a c t i v i t y i n p a r t i c u l a t e p r e p a r a t i o n s f r o m a t r i u m , a n d p r o d u c e d a n e g a t i v e i n o t r o p i c e f f e c t i n a t r i a l s t r i p s ( 7 6 ) . T h e e f f e c t s o f c a r b a c h o l o n c y c l i c AMP l e v e l s a n d a d e n y l c y c l a s e a c t i v i t y i n a t r i u m w e r e b l o c k e d b y a t r o -p i n e , a c h o l i n e r g i c b l o c k i n g a g e n t . I n c o n t r a s t t o t h e s e e f f e c t s i n t h e a t r i u m , c a r b a c h o l h a d l i t t l e o r n o e f f e c t o n a d e n y l c y c l a s e a c t i v i t y , t i s s u e l e v e l s o f c y c l i c AMP, o r on i s o m e t r i c t e n s i o n i n t h e v e n t r i c l e ( 7 6 ) . N o r e p i n e p h r i n e i n c r e a s e d c y c l i c AMP l e v e l s a n d a d e n y l c y c l a s e a c t i v i t y i n b o t h a t r i u m a n d v e n t r i c l e , a n d p r o d u c e d a p o s i t i v e i n o t r o p i c r e s p o n s e i n t h e a t r i u m a n d v e n t r i c l e ( 7 6 ) . T h u s , a d e n y l c y c l a s e may be r e g u l a t e d b y b o t h a d r e n e r g i c a n d c h o l i n e r g i c a c t i v i t y i n t h e a t r i u m , w h e r e a s a d r e n e r g i c i n f l u e n c e p r e d o m -i n a t e s i n t h e v e n t r i c l e . A d e n y l c y c l a s e a c t i v i t y i s n o t a l t e r e d i n t h e c h r o n i c a l l y d e n e r v a t e d c a t h e a r t ( 7 7 ) a n d h y p e r t h y r o i d i s m i n c a t s d i d n o t a l t e r t h e s e n s i t i v i t y o f t h e e n z y m e f o r n o r e p i n e p h r i n e ( 7 8 , 7 9 ) . A d e n y l c y c l a s e a c t i v i t y h a s a l s o b e e n e x a m i n e d i n f a i l i n g h e a r t s . I t was o r i g i n a l l y r e p o r t e d t h a t f l u o r i d e -s t i m u l a t e d e n z y m e a c t i v i t y w a s d e p r e s s e d i n t h e f a i l i n g g u i n e a p i g h e a r t ( 8 0 ) ; t h i s was n o t c o n f i r m e d i n a l a t e r s t u d y ( 8 1 ) . T h e a c t i v a t i o n o f a d e n y l c y c l a s e b y n o r e p i n e p h r i n e was n o t a l t e r e d i n c a t h e a r t s i n c a r d i a c f a i l u r e , h o w e v e r g l u c a g o n d i d n o t a c t i v a t e t h e e n z y m e i n p a r t i c u l a t e f r a c t i o n s o f f a i l i n g h e a r t h o m o g e n a t e s ( 5 9 , 5 0 , 8 2 ) . T h i s l a c k o f r e s p o n s i v e n e s s o f a d e n y l c y c l a s e t o g l u c a g o n i n f a i l i n g h e a r t s w a s s p e c i f i c - 8 -s i n c e p a r t i c u l a t e enzyme p r e p a r a t i o n s f r o m the l i v e r o f c a t s i n c a r d i a c f a i l u r e s t i l l r e s p o n s e d n o r m a l l y t o g l u c a g o n . These o b s e r v a t i o n s on the l o s s of r e s p o n s i v e n e s s of m yocar-d i a l a d e n y l c y c l a s e t o g l u c a g o n s u g g e s t a b i o c h e m i c a l b a s i s f o r the o b s e r v e d i n e f f e c t i v e n e s s of g l u c a g o n t o i n c r e a s e m y o c a r d i a l c o n t r a c t i l i t y i n human p a t i e n t s w i t h c h r o n i c c a r d i a c f a i l u r e ( 5 9 , 6 0 ) . A d e n y l c y c l a s e i n c a t h e a r t p a r t -i c u l a t e p r e p a r a t i o n s can be s o l u b i l i z e d by h o m o g e n i z a t i o n i n a b u f f e r c o n t a i n i n g L u b r o l - P X , a n o n - i o n i c d e t e r g e n t ( 8 3 ) . The s o l u b i l i z e d enzyme was s t i m u l a t e d by f l u o r i d e b u t n o t by h ormones. D e t e r g e n t c o u l d be removed by D E A E - c e l l u l o s e c h r o m a t o g r a p h y ( 8 4 ) . A d d i t i o n of p h o s p h a t i d y l s e r i n e t o the s o l u b i l i z e d enzyme i n c r e a s e d b a s a l a c t i v i t y and t o t a l l y r e s -t o r e d g l u c a g o n r e s p o n s i v e n e s s ( 8 4 ) . P h o s p h a t i d y l i n o s i t o l r e s t o r e d the a b i l i t y of n o r e p i n e p h r i n e t o s t i m u l a t e the s o l u b i l i z e d enzyme; the s o l u b i l i z e d a d e n y l c y c l a s e i n the p r e -s e n c e of p h o s p h a t i d y l i n o s i t o l was 1 0 0 - f o l d more s e n s i t i v e t o n o r e p i n e p h r i n e t h a n was the p a r t i c u l a t e enzyme ( 8 5 ) . C a t e c h o l a m i n e s a r e p r i m a r i l y r e s p o n s i b l e f o r r e g u l a t i n g m e t a b o l i c r a t h e r t h a n m e c h a n i c a l p r o c e s s e s i n s k e l e t a l m u s c l e ( 8 6 ) . The m e c h a n i c a l e f f e c t s of c a t e c h o l a m i n e s a r e s m a l l and depend on the t y p e of s k e l e t a l m u s c l e ; h i g h d o s e s of e p i n e -p h r i n e i n c r e a s e d the t w i t c h t e n s i o n of f a s t - c o n t r a c t i n g ( w h i t e ) s k e l e t a l m u s c l e , whereas e p i n e p h r i n e c a u s e d a d e c r e a s e i n t w i t c h t e n s i o n of s 1 ow-c on t r c t i n g ( r e d ) s k e l e t a l m u s c l e ( 8 7 ) . The most i m p o r t a n t r o l e f o r c a t e c h o l a m i n e s i n s k e l e t a l m u s c l e i s the r e g u l a t i o n of g l y c o g e n m e t a b o l i s m , w i t h c y c l i c AMP as - 9 -the p h y s i o l o g i c a l m e d i a t o r of h o r m o n a l e f f e c t s . The admin-i s t r a t i o n of e p i n e p h r i n e i n c r e a s e d t i s s u e l e v e l s of c y c l i c AMP i n r a t , f r o g and mouse s k e l e t a l m u s c l e ( 8 8-90). In a d d i t i o n , p h o s p h o r y l a s e b k i n a s e was a c t i v a t e d and p h o s p h o r -y l a s e b was c o n v e r t e d t o p h o s p h o r y l a s e a_ ( 8 8,90). The mechan-i s m i n v o l v e d i n t h e s e a c t i v a t i o n s has been e s t a b l i s h e d by the e l e g a n t s t u d i e s of K r e b s and h i s a s s o c i a t e s . P h o s p h o r y l a s e b k i n a s e f r o m r a b b i t s k e l e t a l m u s c l e c o u l d be a c t i v a t e d , p a r t i c u l a r l y a t p h y s i o l o g i c a l pH v a l u e s , by i n c u b a t i o n w i t h ATP and magnesium in_ v i t r o ( 9 1 , 9 2 ) . The a d d i t i o n of v e r y low c o n c e n t r a t i o n s of c y c l i c AMP i n c r e a s e d the r a t e of t h i s a c t i v a t i o n ( 9 1 , 9 2 ) . The a c t i v a t i o n of p h o s p h o r y l a s e b^  k i n a s e was a c c o m p a n i e d by p h o s p h o r y l a t i o n of the enzyme ( 9 3 ) . A c t i v a t i o n by c y c l i c AMP d e c r e a s e d w i t h p r e p a r a t i o n s of p h o s -p h o r y l a s e b^  k i n a s e of i n c r e a s i n g p u r i t y (92) and b i n d i n g of c y c l i c AMP to the p u r i f i e d , n o n - a c t i v a t e d enzyme c o u l d n o t be d e m o n s t r a t e d ( 9 3 ) . T h i s s u g g e s t e d t h a t c y c l i c AMP was i n t e r -a c t i n g w i t h a n o t h e r enzyme, a p h o s p h o r y l a s e b k i n a s e k i n a s e , w h i c h was removed d u r i n g the p u r i f i c a t i o n p r o c e d u r e . A k i n a s e f r a c t i o n was i s o l a t e d f r om s k e l e t a l m u s c l e t h a t was a c t i v a t e d by e x t r e m e l y low c o n c e n t r a t i o n s o f c y c l i c AMP and t h a t c a t a l -y z e d the A T P - d e p e n d e n t p h o s p h o r y l a t i o n and a c t i v a t i o n of p h o s p h o r y l a s e b_ k i n a s e ( 9 4 , 9 5 ) . S i n c e the k i n a s e a l s o i n c r e a s e d the A T P-dependent p h o s p h o r y l a t i o n o f o t h e r p r o t e i n s s u c h as c a s e i n and p r o t a m i n e , i t i s r e f e r r e d to by the g e n e r a l name of p r o t e i n k i n a s e ( 9 4 ) . R e c e n t e v i d e n c e i n d i c a t e s t h a t p r o t e i n k i n a s e i n s k e l e t a l m u s c l e c o n s i s t s of r e g u l a t o r y and - 10 -c a t a l y t i c s u b u n i t s ( 9 6 ) . The b i n d i n g of c y c l i c AMP t o the r e g u l a t o r y s u b u n i t d i s s o c i a t e s the enzyme and t h u s removes the i n h i b i t o r y i n f l u e n c e of the r e g u l a t o r y s u b u n i t and a l l o w s t h e e x p r e s s i o n of enzyme a c t i v i t y by the c a t a l y t i c s u b u n i t . I t must be e m p h a s i z e d t h a t mechanisms o t h e r t h a n t h o s e i n v o l v i n g c y c l i c AMP e x i s t f o r the r e g u l a t i o n of g l y c o g e n -o l y s i s . E l e c t r i c a l s t i m u l a t i o n of s k e l e t a l m u s c l e r e s u l t e d i n the c o n v e r s i o n o f p h o s p h o r y l a s e b_ to p h o s p h o r y l a s e a_ w i t h o u t i n c r e a s i n g c y c l i c AMP l e v e l s or a c t i v a t i n g p h o s p h o r -y l a s e b^  k i n a s e ( 9 0 ) . A l s o , the f o r m a t i o n of p h o s p h o r y l a s e a i n r a b b i t g r a c i l u s m u s c l e i n r e s p o n s e to low d o s e s of i s o p r o p y l n o r e p i n e p h r i n e was d i s s o c i a t e d f r o m i n c r e a s e d l e v e l s of c y c l i c AMP and a c t i v a t i o n of p h o s p h o r y l a s e b k i n a s e ( 9 7 ) . The p r e c i s e r e g u l a t i o n of g l y c o g e n o l y s i s may i n v o l v e mechanisms c o n c e r n e d w i t h i n t r a c e l l u l a r c a l c i u m l e v e l s as w e l l as c y c l i c AMP l e v e l s . In a d d i t i o n to r e g u l a t i n g g l y c o g e n m e t a b o l i s m i n s k e l e t a l m u s c l e by s t i m u l a t i n g g l y c o g e n o l y s i s as o u t l i n e d a b o v e , c a t e -c h o l a m i n e s a l s o i n f l u e n c e t h e r a t e of g l y c o g e n s y n t h e s i s ( 8 6 ) . G l y c o g e n s y n t h e t a s e e x i s t s i n two forms i n s k e l e t a l m u s c l e ; one f o r m i s d e p e n d e n t on g l u c o s e - 6 - p h o s p h a t e f o r a c t i v i t y ( g l y c o g e n s y n t h e t a s e - D ) whereas the o t h e r f o r m i s i n d e p e n d e n t of g l u c o s e - 6 - p h o s p h a t e ( g l y c o g e n s y n t h e t a s e - I ) . I n t e r c o n v e r -s i o n f r o m g l y c o g e n s y n t h e t a s e - I to g l y c o g e n s y n t h e t a s e - D i n v o l v e s an A T P-dependent p h o s p h o r y l a t i o n of the enzyme ( 9 8 ) . The i n t e r c o n v e r s i o n i s s i m i l a r t o t h a t o c c u r i n g d u r i n g p h o s -- 11 -p h o r y l a s e a c t i v a t i o n , e x c e p t t h a t p h o s p h o r y l a t i o n r e s u l t s i n a n i n a c t i v e f o r m o f g l y c o g e n s y n t h e t a s e r a t h e r t h a n a n a c t i v e f o r m a s w i t h p h o s p h o r y l a s e . The a d m i n i s t r a t i o n o f e p i n e p h r i n e r e s u l t e d i n a d e c r e a s e i n t h e p e r c e n t a g e o f g l y c o g e n s y n t h e -t a s e - I i n s k e l e t a l m u s c l e ( 9 9 , 1 0 0 ) . C y c l i c AMP l e v e l s a r e i n c r e a s e d i n s k e l e t a l m u s c l e t i s s u e f o l l o w i n g e p i n e p h r i n e a d m i n i s t r a t i o n ( 8 8 - 9 0 ) a n d the r a t e o f t h e c o n v e r s i o n o f g l y c o g e n s y n t h e t a s e - I t o g l y c o g e n s y n t h e t a s e - D i n v i t r o was i n c r e a s e d b y c y c l i c AMP ( 1 0 1 ) . A k i n a s e s t i m u l a t e d b y c y c l i c AMP c a t a l y z e d t h e i n a c t i v a t i o n o f g l y c o g e n s y n t h e t a s e ( 1 0 2 ) a n d h a s b e e n s h o w n t o be i d e n t i c a l w i t h t h e p r o t e i n k i n a s e i n v o l v e d i n t h e 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 b k i n a s e ( 1 0 3 , 1 0 4 ) . T h u s t h e r e g u l a t i o n o f g l y c o g e n m e t a b o l i s m i n s k e l e t a l m u s c l e b y c a t e c h o l a m i n e s i s a c c o m p l i s h e d b y a c o o r d -i n a t e d i n c r e a s e i n g l y c o g e n d e g r a d a t i o n a n d d e c r e a s e i n g l y c o -g e n s y n t h e s i s . A d e n y l c y c l a s e i n s k e l e t a l m u s c l e h a s n o t b e e n s t u d i e d a s e x t e n s i v e l y a s t h e c a r d i a c e n z y m e . E n z y m e a c t i v i t y w a s f i r s t o b s e r v e d i n h o m o g e n a t e s o f d o g s k e l e t a l m u s c l e ( 1 8 ) . T h e f o r m a t i o n o f c y c l i c AMP i n a m i c r o s o m a l f r a c t i o n o f r a b b i t s k e l e t a l m u s c l e was r e p o r t e d b y S e r a y d a r i a n a n d M o m m a e r t s ( 1 0 5 ) , a n d the d i s t r i b u t i o n o f a d e n y l c y c l a s e i n s k e l e t a l m u s c l e homo-g e n a t e s was s h o w n t o be c o n f i n e d t o t h e m i t o c h o n d r i a l a n d m i c r o s o m a l f r a c t i o n s ( 3 1 , 3 2 ) . E p i n e p h r i n e c a u s e d a n i n c r e a s e i n c y c l i c AMP f o r m a t i o n i n d o g s k e l e t a l m u s c l e h o m o g e n a t e s ( 7 , 1 0 6 ) a n d i n p a r t i c u l a t e p r e p a r a t i o n s f r o m r a b b i t s k e l e t a l - 12 -mus c l e ( 3 2 ) . D e s p i t e the wide r a n g e of t i s s u e s i n w h i c h a d e n y l c y c l a s e has been f o u n d and the l a r g e number of hormones t h a t s t i m u l a t e enzyme a c t i v i t y , v e r y l i t t l e i s known about the k i n e t i c p r o p e r t i e s of a d e n y l c y c l a s e . In p a r t , t h i s i s b e c a u s e of the p a r t i c u l a t e n a t u r e o f the enzyme. P r e p a r a t i o n s of a d e n y l c y c l a s e are c o n t a m i n a t e d by numerous o t h e r enzyme a c t i v i t i e s , i n p a r t i c u l a r ATPase and c y c l i c n u c l e o t i d e p h o s -p h o d i e s t e r a s e . T h i s has r e q u i r e d the c a r e f u l e x a m i n a t i o n of the methods and c o n d i t i o n s f o r the a s s a y of a d e n y l c y c l a s e . I n f o r m a t i o n on the k i n e t i c p r o p e r t i e s of a d e n y l c y c l a s e i s r e q u i r e d t o e s t a b l i s h the mechanisms by w h i c h hormones o r o t h e r r e g u l a t o r y a g e n t s i n f l u e n c e enzyme a c t i v i t y . I t i s r e a s o n a b l e t o assume t h a t t h e s e a g e n t s w i l l a c t by e i t h e r i n c r e a s i n g s u b s t r a t e a f f i n i t y , i n c r e a s i n g maximal v e l o c i t y , or by a c o m b i n a t i o n of b o t h t h e s e e f f e c t s . B i r n b a u m e r , P o h l and R o d b e l l (107) have s t u d i e d the k i n e t i c p r o p e r t i e s of a d e n y l c y c l a s e i n r a t f a t c e l l g h o s t s and the k i n e t i c n a t u r e o f ACTH and f l u o r i d e s t i m u l a t i o n . A d e n y l c y c l a s e was s t i m u l a t e d by c o n c e n t r a t i o n s of magnesium i n e x c e s s of the ATP s u b s t r a t e c o n c e n t r a t i o n . I t was s u g g e s t e d t h a t the enzyme i n f a t c e l l s has two b i n d i n g s i t e s f o r magnesium, one of w h i c h was at the c a t a l y t i c s i t e c o mplexed w i t h ATP, and the o t h e r at some s e c o n d s i t e . The enzyme c o u l d n o t be s a t u r a t e d w i t h magnesium and h i g h c o n c e n t r a t i o n s of magnesium (80 mM) c o u l d s u b s t i t u t e a l m o s t c o m p l e t e l y f o r ACTH or f l u o r i d e i n s t i m u l a t i n g enzyme - 13 -a c t i v i t y (107). ACTH and f l u o r i d e i n c r e a s e d the v e l o c i t y of the enzyme r e a c t i o n at a l l c o n c e n t r a t i o n s of ATP with no e f f e c t on the Kffl f o r ATP. In c o n t r a s t , the apparent a f f i n i t y of a d e n y l c y c l a s e f o r magnesium was i n c r e a s e d by f i v e - f o l d . I t was concluded that ACTH and f l u o r i d e a c t i v a t e d the same enzyme and the same k i n e t i c parameters but by d i f f e r e n t mechanisms (107). Some of the k i n e t i c p r o p e r t i e s of adenyl c y c l a s e i n heart have a l s o been s t u d i e d . Drummond and Duncan (28) a l s o found that magnesium bound to two s i t e s on the myo-c a r d i a l enzyme. However i n c o n t r a s t to the f a t c e l l enzyme, myocardial adenyl c y c l a s e was s a t u r a t e d by magnesium concen-t r a t i o n s ranging from 10 to 15 mM. F l u o r i d e had no e f f e c t on the a f f i n i t y f o r ATP or magnesium; the primary e f f e c t of f l u o r i d e was to i n c r e a s e the c a t a l y t i c r e a c t i v i t y and maximal v e l o c i t y (28). This t h e s i s d e s c r i b e s a f u r t h e r i n v e s t i g a t i o n of the k i n e t i c p r o p e r t i e s of myocardial adenyl c y c l a s e . These s t u d i e s were c a r r i e d out i n c o n j u n c t i o n with s t u d i e s by Drummond, Severson and Duncan (108) of the k i n e t i c nature of the s t i m u l -a t i o n by epi n e p h r i n e and f l u o r i d e . Adenyl c y c l a s e was a l s o s t u d i e d i n another c o n t r a c t i l e t i s s u e , s k e l e t a l muscle. The k i n e t i c p r o p e r t i e s of the enzyme i n a plasma membrane prep-a r a t i o n were examined with p a r t i c u l a r emphasis upon the mech-anisms by which hormones and f l u o r i d e s t i m u l a t e enzyme a c t i v i t y , EXPERIMENTAL PROCEDURE A. M a t e r i a l s Uniformly labe 1 led [ 1 4c]-ATP (418-462 mCi per mmole) and - 14 -£3H] - c y c l i c - A M P (24.1 C i p e r mmole) were o b t a i n e d f r o m New E n g l a n d N u c l e a r , B o s t o n . E t h a n o l was removed f r o m the commer-c i a l s o l u t i o n s u nder vacuum. £c£-3 2pj ATP was p u r c h a s e d f r o m New E n g l a n d N u c l e a r o r I n t e r n a t i o n a l C h e m i c a l and N u c l e a r C o r p o r a t i o n , I r v i n e , C a l i f o r n i a , w i t h s p e c i f i c a c t i v i t i e s r a n g i n g f r o m 1.0 t o 10.9 C i p e r mmole, - c y c l i c AMP (33.6 mCi p e r mmole) was p u r c h a s e d f r o m Schwarz B i o R e s e a r c h I n c . , Mt. V e r n o n , New Y o r k . S o l u t i o n s of r a d i o a c t i v e m a t e r i a l were d i l u t e d w i t h u n l a b e l l e d n u c l e o t i d e to t h e d e s i r e d s p e c i f i c a c t i v i t y and w i t h w a t e r t o the d e s i r e d c o n c e n t r a t i o n and s t o r e d a t - 1 8 ° . U n l a b e l l e d n u c l e o t i d e s were p u r c h a s e d f r o m C a l b i o c h e m , Los A n g e l e s , and Sigma C h e m i c a l Co., S t . L o u i s . P y r u v a t e k i n a s e ( r a b b i t m u s c l e ) and 2 - p h o s p h o e n o l p y r u -v a t e ( t r i s o d i u m s a l t ) were o b t a i n e d f r o m C a l b i o c h e m . V a r i o u s p r e p a r a t i o n s of p y r u v a t e k i n a s e , when a s s a y e d by t h e method of Bttcher and P f l e i d e r e r ( 1 0 9 ) , c a t a l y z e d the p h o s p h o r y l a t i o n of ADP w i t h s p e c i f i c a c t i v i t i e s r a n g i n g f r o m 64 t o 219 umoles p e r min p e r mg B Hormones and d r u g s were o b t a i n e d f r o m the f o l l o w i n g s o u r c e s : p h e n y l e p h r i n e h y d r o c h l o r i d e and o u a b a i n ( s t r o p h a n t h i n G ) , C a l b i o c h e m ; e p i n e p h r i n e ( L - a d r e n a 1 i n e b i t a r t r a t e ) , K and K L a b o r a t o r i e s , P l a i n v i e w , New Y o r k ; i s o p r o p y l n o r e p i n e p h r i n e h y d r o c h l o r i d e , W i n t h r o p L a b o r a t o r i e s , New Y o r k ; n o r e p i n e p h r i n e ( L - a r t e r n o 1 - D - b i t a r t r a t e ) and c a r b a c h o l ( c a r b a m y l c h o l i n e c h l o r i d e ) , Mann L a b o r a t o r i e s , New Y o r k ; c r y s t a l l i n e g l u c a g o n , Sigma; e p h e d r i n e s u l p h a t e , B r i t i s h Drug Houses L t d . , L o n d o n , E n g l a n d ; D , L - p r o p r a n o 1 o 1 , A y e r s t L a b o r a -t o r i e s , M o n t r e a l ; and i n s u l i n ( b o v i n e p a n c r e a s , 24 i n t e r n a t i o n a l u n i t s p e r mg), Sigma. Amadac-F r e a g e n t was p u r c h a s e d f r o m - 15 -B u r d i c k and J a c k s o n L a b o r a t o r i e s , Muskegon, M i c h i g a n ; p r o s t a g l a n d i n s E 2 , F l c C > F 2 o t * a n d A 2 w e r e s u P P l i e d t h r o u g h the c o u r t e s y of D r . John P i k e of the Upjohn Company, K a l a -mazoo, M i c h i g a n . Dowex AG 50 W-X4, 200 t o 400 mesh ( H + f o r m ) was p u r c h a s e d f r o m B i o - R a d L a b o r a t o r i e s , Richmond, C a l i f o r n i a . The r e s i n was washed w i t h 1 N NaOH, t h e n w i t h w a t e r f o l l o w e d by 1 N HC1. I t was t h e n washed e x t e n s i v e l y w i t h w a t e r t o remove the f i n e s , and s t o r e d as a 50% ( v / v ) s u s p e n s i o n i n w a t e r . B. Methods 1. Enzyme p r e p a r a t i o n s f r o m h e a r t Washed p a r t i c l e S - - A 1 1 p r o c e d u r e s were c a r r i e d out a t 4 ° . F r e s h or f r o z e n v e n t r i c u l a r t i s s u e f r o m g u i n e a p i g or r a b b i t h e a r t was w e i g h e d , m i n c e d w i t h s c i s s o r s and h o m o g e n i z e d i n 10 v o l u m e s of 10 mM T r i s - H C l , pH 7.5 f o r 10 s e c o n d s w i t h a S o r v a l l Omni-mixer ( r h e o s t a t at maximum). The whole homo-ge n a t e was s t r a i n e d t h r o u g h a c o a r s e n y l o n s i e v e ( p o r e s i z e 2 1 ram ) t o remove c o n n e c t i v e t i s s u e , and c e n t r i f u g e d at 600-1000 x g f o r 10 m i n . The p r e c i p i t a t e was washed t w i c e by s u s p e n d i n g to the o r i g i n a l volume w i t h 10 mM T r i s , pH 7.5 and c e n t r i f u g i n g . The washed p a r t i c l e s were s u s p e n d e d i n 5 v o l u m e s of b u f f e r ( b a s e d on o r i g i n a l t i s s u e w e i g h t ) u s i n g a g l a s s h o m o g e n i z e r w i t h a l o o s e - f i t t i n g t e f l o n p e s t l e . Washed p a r t i c u l a t e p r e p a r a t i o n s were a s s a y e d i m m e d i a t e l y or l y o p h i l i z e d . - 16 -L i t h i u m bromide-extracted p a r t i c l e s - - A l 1 procedures were c a r r i e d out at 4 ° . Guinea p i g or r a b b i t v e n t r i c u l a r t i s s u e was homogenized i n 10 volumes of e i t h e r 1 mM T r i s , pH 6.8 or 10 mM T r i s , pH 7.5, f o r 7 to 10 seconds i n the Omni-mixer. The whole homogenate was s t r a i n e d through a coarse nylon s i e v e . Washed p a r t i c l e s were c o l l e c t e d by c e n t r i f u g a t i o n and washing with 10 mM T r i s , pH 7.5 as d e s c r i b e d above. The f i n a l p e l l e t was suspended i n 5 volumes (based on i n i t i a l t i s s u e weight) of 10 mM T r i s , pH 8.2 and l i t h i u m bromide (4 M) was added to a f i n a l c o n c e n t r a t i o n of 0.4 M. The s o l u t i o n was s t i r r e d u s i n g a magnetic s t i r r e r f o r 45 min, d i l u t e d with 10 mM T r i s , pH 7.5 and c e n t r i f u g e d at 1500 x g f o r 15 min. The p r e c i p i t a t e was washed by suspending i n 10 mM T r i s , pH 7.5 f o l l o w e d by c e n t r i f u g a t i o n . The l i t h i u m b r o m i d e - e x t r a c t e d p a r t i c l e s were suspended i n 5 volumes of 10 mM T r i s , pH 7.5, assayed immediately and l y o p h i l i z e d . L y o p h i l i z e d p r e p a r a t i o n s were s t o r e d at - 1 8 ° . Before use, s u i t a b l e amounts were homo-genized i n an a p p r o p r i a t e volume of 10 mM T r i s , pH 7.5 u s i n g a g l a s s homogenizer with a t i g h t - f i t t i n g t e f l o n p e s t l e . 2. Enzyme p r e p a r a t i o n s from s k e l e t a l muscle Washed p a r t i c l e s - - A l 1 procedures were c a r r i e d out at 4 ° . Fresh or f r o z e n r a b b i t s k e l e t a l muscle (hind limb) was minced with s c i s s o r s and homogenized i n 5 volumes of 10 mM T r i s , pH 8 f o r 10 to 15 seconds i n a S o r v a l l Omni-mixer at maximal v e l o c i t y . Connective t i s s u e was removed by s t r a i n i n g the whole homogenate through a coarse nylon s i e v e (pore s i z e 1 o mm*-). The f i l t r a t e was c e n t r i f u g e d at 2000 x g f o r 10 min - 17 -and the p e l l e t was washed t w i c e by s u s p e n d i n g t o the o r i g i n a l volume w i t h 10 mM T r i s , pH 8 and c e n t r i f u g i n g . The washed p e l l e t was s u s p e n d e d i n 5 volume s of 10 mM T r i s , pH 8 u s i n g a g l a s s h o m o g e n i z e r w i t h a l o o s e - f i t t i n g t e f l o n p e s t l e and i s r e f e r r e d t o as a washed p a r t i c l e p r e p a r a t i o n . 3. Enzyme a s s a y s A d e n y l c y c l a s e — T h e a s s a y f o r a d e n y l c y c l a s e i n c a r d i a c t i s s u e c o n s i s t e d of the f o l l o w i n g components i n a t o t a l volume of 150 p i : 40 mM T r i s , pH 7.5; 20 mM p h o s p h o e n o l p y r u v a t e ; a p p r o x i m a t e l y 4 i n t e r n a t i o n a l u n i t s p y r u v a t e k i n a s e ; 6 mM K C l ; 8 mM t h e o p h y l l i n e ; 2 mM c y c l i c AMP; 18 mM MgSO^ 0.3 mM C^c] -ATP (20 u C i p e r umole) and enzyme (100-400 ug p r o t e i n ) . A s s a y t u b e s were e q u i l i b r a t e d at 3 7 ° and the r e a c t i o n s t a r t e d by the a d d i t i o n o f e i t h e r the enzyme p r e p a r a t i o n o r r a d i o -a c t i v e s u b s t r a t e . I n c u b a t i o n s were c a r r i e d o ut at 3 7 ° f o r 15 t o 20 min and were t e r m i n a t e d by p l a c i n g the t u b e s i n a b o i l i n g w a t e r b a t h f o r 3 m i n . C o n t r o l t u b e s c o n t a i n e d w a t e r i n p l a c e o f the enzyme p r e p a r a t i o n o r t h e ^ 4 c ] - A T P s u b s t r a t e was added a f t e r b o i l i n g the enzyme p r e p a r a t i o n . The t u b e s were c e n t r i f u g e d at 8000 x g f o r 5 min t o remove d e n a t u r e d p r o t e i n and 100 p i of the s u p e r n a t a n t was s t r e a k e d o v e r 2 cm on Whatman 3 MM c h r o m a t o g r a p h y p a p e r . D e s c e n d i n g p a p e r chrom-a t o g r a m s were d e v e l o p e d f o r 18 t o 22 hr at room t e m p e r a t u r e u s i n g IM ammonium a c e t a t e : 957. e t h a n o l (15:35) as the d e v e l -o p i n g s o l v e n t . A t y p i c a l c h r o m a t o g r a p h i c s e p a r a t i o n of a d e n i n e n u c l e o t i d e s and a d e n o s i n e i s shown i n f i g . 1. T h e o p h y l l i n e - 18 -m i g r a t e d a p p r o x i m a t e l y 5 cm f u r t h e r t h a n a d e n o s i n e . A f t e r d r y i n g , t h e a r e a c o r r e s p o n d i n g t o c y c l i c AMP was v i s u a l i z e d u n d e r u l t r a v i o l e t l i g h t , c u t o u t and p l a c e d i n 18 ml o f s c i n t i l l a t i o n f l u i d (4g o f 2, 5 - d i p h e n y 1 o x a z o l e and 50 mg of 1, 4 - b i s - [ 2 - ( 5 - p h e n y l o x a z o l y 1 ) ] - b e n z e n e d i s s o l v e d i n 1 l i t r e o f t o l u e n e ) and t h e r a d i o a c t i v i t y was m e a s u r e d i n a N u c l e a r C h i c a g o s c i n t i l l a t i o n s p e c t r o m e t e r . The amount of r a d i o -a c t i v e c y c l i c AMP f o r m e d was c a l c u l a t e d f r o m t h e s p e c i f i c a c t i v i t y o f t h e ^ ^ C ^ - A T P s u b s t r a t e a f t e r c o r r e c t i o n f o r r a d i o -a c t i v i t y p r e s e n t i n t h e c y c l i c AMP a r e a o f t h e c o n t r o l . T h i s method w i l l be r e f e r r e d t o as a s s a y A. The a s s a y f o r a d e n y l c y c l a s e i n s k e l e t a l m u s c l e was i d e n t i c a l t o t h a t d e s -c r i b e d above e x c e p t t h a t t h e pH o f t h e i n c u b a t i o n was 8.5 and t h e o p h y l l i n e was o m i t t e d . I n a d d i t i o n , 1 mM EGTA was p r e s e n t i n t h e a s s a y o f c r u d e h o m o g e n a t e s and washed p a r t i c l e p r e p a r a t i o n s . The a c t i v i t y o f a d e n y l c y c l a s e i s r o u t i n e l y e x p r e s s e d as p m o l e s c y c l i c AMP f o r m e d p e r min p e r mg p r o t e i n . F o r e x p e r i m e n t s r e q u i r i n g i j b s t r a t e c o n c e n t r a t i o n s g r e a t e r t h a n 0.3 mM, jj£-3 2 P ] - ATP was u s e d as s u b s t r a t e i n p l a c e o f [ 1 4c]-ATP b e c a u s e [ c C - 3 2 p ] - A T P c o u l d be o b t a i n e d i n much h i g h e r s p e c i f i c a c t i v i t y . A s s a y A c o u l d n o t be u s e d u n d e r t h e s e c o n d i t i o n s b e c a u s e o f h i g h b a c k g r o u n d c o u n t s i n t h e c y c l i c AMP a r e a o f c h r o m a t o g r a m s o f c o n t r o l t u b e s . T h e r e f o r e , two a d d i t i o n a l a s s a y s were e m p l o y e d when £?C-3 2p] -ATP was u s e d as s u b s t r a t e . A s s a y B i s a m o d i f i c a t i o n o f a s s a y A. F o l l o w i n g t e r m i n a t i o n o f the r e a c t i o n by b o i l i n g , 50 p i 0.25 M ZnSO^ - 19 -F i g u r e 1: Paper c h r o m a t o g r a p h i c s e p a r a t i o n of a d e n i n e n u c l e o -t i d e s and a d e n o s i n e . S o l u t i o n s of ATP, 5'-AMP, c y c l i c AMP and a d e n o s i n e (100 p i ; 0.05 p m o l e s ) were a p p l i e d t o the c h r o m a t o g r a m i n p o s i t i o n s one t o f o u r r e s p e c t i v e l y . At p o s i t i o n f i v e , 100 p i of the s u p e r n a t a n t of the a d e n y l c y c l a s e r e a c t i o n m i x t u r e was a p p l i e d f o l l o w i n g b o i l i n g and c e n t r i f u g a t i o n . P a p e r c h r o m a t o -grams were d e v e l o p e d as d e s c r i b e d i n methods w i t h ammonium a c e t a t e : e t h a n o l (15:35) as d e v e l o p i n g s o l v e n t , d r i e d , and v i s u a l i z e d u n der u l t r a v i o l e t l i g h t . ( S c a l e : 1cm- 3cm) F i g u r e 1 - 21 -was added t o e a c h tube f o l l o w e d by 50 p i 0.25 M BaCOH^. The t h i c k , w h i t e 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 a t 8000 x g f o r 10 min and 150 p i of e a c h c l e a r s u p e r n a t a n t was s u b j e c t e d t o p a p e r c h r o m a t o g r a p h y as d e s c r i b e d i n a s s a y A. B a r i u m - z i n c p r e c i p i t a t i o n removed more t h a n 95% of the ATP, ADP and AMP p r e s e n t ; r e c o v e r y of c y c l i c AMP r a n g e d f r o m 97 t o 100% as d e t e r m i n e d by the a d d i t i o n of known amounts of [ 8 - 1 4 c ] - c y c l i c AMP t o c o n t r o l t u b e s . A s s a y C i s e s s e n t i a l l y the method of K r i s h n a , Weiss and B r o d i e ( 1 1 0 ) . A s s a y c o n d i t i o n s r e m a i n e d as i n a s s a y A e x c e p t t h a t f j C C - 3 2 P ] -ATP r e p l a c e d [ 1 4 c ] - A T P as s u b s t r a t e and u n l a b e l l e d c y c l i c AMP was o m i t t e d . F o l l o w i n g t e r m i n a t i o n of t h e r e a c t i o n by b o i l i n g and c e n t r i f u g a t i o n , 0.4 ml w a t e r and 50 p i (1.0 pmole) o f c y c l i c AMP was added t o e a c h t u b e . The s u p e r n a t a n t f l u i d was c h r o m a t o g r a p h e d on Dowex (Dowex AG 50 W-X4, 200 to 400 mesh, H + f o r m ) columns p r e p a r e d by p i p e t t i n g 2 ml o f a 50% ( v / v ) s u s p e n s i o n of the r e s i n i n t o s m a l l g l a s s columns ( o u t e r d i a m e t e r 7 mm). Columns were e l u t e d w i t h w a t e r and the f i f t h and s i x t h ml were c o l l e c t e d i n c a l i -b r a t e d c e n t r i f u g e t u b e s . To t h i s f r a c t i o n , 0.2 ml of 0.25 M ZnS04 and 0.2 ml o f 0.25 M B a ( 0 H ) 2 s o l u t i o n s were a d d e d , mixed and c e n t r i f u g e d . The s u p e r n a t a n t was t r a n s f e r r e d t o a n o t h e r tube by d e c a n t a t i o n and the b a r i u m - z i n c p r e c i p i t a t i o n was r e p e a t e d . F o l l o w i n g c e n t r i f u g a t i o n , a 0.5 ml a l i q u o t of the s u p e r n a t a n t was added t o 15 ml B r a y ' s s o l u t i o n (111) and the r a d i o a c t i v i t y was measured i n a N u c l e a r C h i c a g o s c i n t i l l a t i o n - 22 -s p e c t r o m e t e r . The r e c o v e r y of c y c l i c AMP was c a l c u l a t e d f r o m measurements of the o p t i c a l d e n s i t y a t 259 nm of the s u p e r n a t a n t f r a c t i o n s ; r e c o v e r i e s r a n g e d f r o m 40 t o 707.. O t h e r enzymes — Mg + + - d e p e n d a n t ATPase a c t i v i t y was measured by i n c u b a t i n g a p p r o p r i a t e q u a n t i t i e s of p r o t e i n ( 5 0 -100 ug) w i t h 40 mM T r i s , pH 7.5, 4 mM ATP and 4 mM M g S 0 4 i n a t o t a l volume of 150 u l . The t u b e s were i n c u b a t e d f o r 10 min at 3 7 ° and the r e a c t i o n was t e r m i n a t e d by the a d d i t i o n of 1 ml of c o l d 37. t r i c h l o r o a c e t i c a c i d . A f t e r c e n t r i f u g a t i o n , i n o r g a n i c p h o s p h a t e was d e t e r m i n e d i n the s u p e r n a t a n t by the method of F i s k e and SubbaRow ( 1 1 2 ) . A 0.5 m l a l i q u o t of the s u p e r n a t a n t was added t o 0.43 ml wa t e r and 0.05 ml 2.57. ammonium m o l y b d a t e i n 5 N H2SO4. C o l o r was d e v e l o p e d by the a d d i t i o n of 0.02 ml r e d u c i n g s o l u t i o n (250 mg of a powder c o n s i s t i n g of 7.77. 1-amino-2-nap h t ho 1-4-su 1 f on i c a c i d , 46.15% NaHS03 and 46.15% N a £ S 0 3 on a d r y w e i g h t b a s i s i n 10 ml w a t e r ) , and was d e t e r m i n e d a f t e r 10 min i n a s p e c t r o p h o t o m e t e r at 720 nm. 5 1 - N u c l e o t i d a s e was a s s a y e d by i n c u b a t i n g m u s c l e p r o t e i n w i t h 40 mM T r i s , pH 7.5, 8 mM 5'-AMP and 18 mM MgS0 4, i n a f i n a l volume o f 250 u l f o r 10 min a t 3 7 ° . R e a c t i o n s were t e r m i n a t e d w i t h the a d d i t i o n of 1 ml c o l d , 3% t r i c h l o r o a c e t i c a c i d and p h o s p h a t e was d e t e r m i n e d as d e s c r i b e d a b o v e . A c i d p h o s p h a t a s e was e s t i m a t e d by d e t e r m i n i n g the i n o r g a n i c p h o s p h a t e r e l e a s e d f o l l o w i n g the i n c u b a t i o n of m u s c l e p r o t e i n w i t h 25 mM ^ - g l y c e r o l p h o s p h a t e b u f f e r e d a t pH 5 w i t h 50 mM sod i u m a c e t a t e i n a f i n a l volume of 200 jil f o r 20 min a t 3 7 ° . P y r o p h o s p h a t a s e was a s s a y e d a c c o r d i n g t o the method of - 23 -N o r d l i e and A r i o n ( 1 1 3 ) . The r e l e a s e of i n o r g a n i c p h o s p h a t e was e s t i m a t e d f o l l o w i n g i n c u b a t i o n of 40 mM c a c o d y l a t e , pH 5.5, 20 mM s o d i um p y r o p h o s p h a t e , and p r o t e i n i n a volume of 150 p i f o r 10 min a t 3 7 ° . C y c l i c 2 1 , 3 ' - n u c l e o t i d e p h o s p h o d i e s t e r a s e was a s s a y e d by t h e method of Drummond, Eng and M c i n t o s h ( 1 1 4 ) . I n c u b a t i o n s i n c l u d e d ( i n a f i n a l volume of 250 p i ) : 40 mM T r i s , pH 7.5 c o n t a i n i n g 6.25 mg p e r ml of egg a l b u m i n , 4 mM a d e n o s i n e 2', 3 ' - c y c l i c p h o s p h a t e , and an e x c e s s (30 p i ) of E s c h e r i c h i a  c o l i a l k a l i n e p h o s p h a t a s e (0.3 mg; 6 u n i t s ) . I n o r g a n i c p h o s -p h a t e r e l e a s e d was e s t i m a t e d as d e s c r i b e d a b o v e . A c i d m a l t a s e was e s t i m a t e d a c c o r d i n g t o the method of Hers and von Hoof (115) by d e t e r m i n i n g g l u c o s e r e l e a s e d by the g l u c o s t a t r e a g e n t f r o m i n c u b a t i o n s of 25 mM a c e t a t e , pH 4, 1 .257.. m a l t o s e , and p r o t e i n f o r 4 hr a t 3 7 ° i n a t o t a l volume of 200 p i . C ytochrome c o x i d a s e was d e t e r m i n e d by the p r o c e d u r e of C o o p e r s t e i n and Lazarow ( 1 1 6 ) . A p p r o p r i a t e q u a n t i t i e s of p r o t e i n were i n c u b a t e d w i t h c y t o c h r o m e c ( r e d u c e d by d i t h i o n -i t e ) i n 0.1 M p h o s p h a t e b u f f e r , pH 7. Cytochrome c o x i d a s e a c t i v i t y was d e t e r m i n e d by the r a t e of the d e c r e a s e i n a b s o r -bance a t 554 nm measured s p e c t r o p h o t o m e t r i c a 1 l y . A c e t y l c h o l i n e s t e r a s e was measured s p e c t r o p h o t o m e t r i c a 1 l y by t h e method of E l l r a a n e_t a_l ( 1 1 7 ) . The r a t e of h y d r o l y s i s of a c e t y l t h i o c h o l i n e was d e t e r m i n e d i n i n c u b a t i o n s c o n t a i n i n g 0,1 M p h o s p h a t e , pH 7, 0.5 mM a c e t y l t h i o c h o l i n e , 0.4 mM - 24 -d i t h i o b i s - n i t r o b e n z o a t e and p r o t e i n , by m e a s u r i n g the i n c r e a s e i n a b s o r b a n c e a t 412 nm. The h y d r o l y s i s of c y c l i c AMP by c y c l i c 3' , 5 ' - n u c l e o t i d e p h o s p h o d i e s t e r a s e was measured by i n c u b a t i n g 5 mM £3rfJ-cyclic AMP (2 p C i p e r umole) w i t h 25 mM T r i s , pH 7.5, 2.5 mM M g S 0 4 and p r o t e i n f o r 20 min a t 3 0 ° i n a t o t a l volume of 200 u l . R e a c t i o n s were t e r m i n a t e d by i m m e r s i n g the t u b e s i n a b o i l i n g w a t e r b a t h a n d , f o l l o w i n g r e m o v a l of d e n a t u r e d p r o t e i n by c e n t r i f u g a t i o n , 150 p i of the s u p e r n a t a n t f l u i d was chroma-t o g r a p h e d as d e s c r i b e d i n a s s a y A f o r a d e n y l c y c l a s e . A r e a s of t h e ch r o m a t o g r a m c o r r e s p o n d i n g t o 5'-AMP were c u t out and r a d i o a c t i v i t y was d e t e r m i n e d by s c i n t i l l a t i o n s p e c t r o m e t r y . A r e a s c o r r e s p o n d i n g to a d e n o s i n e were r o u t i n e l y m o n i t o r e d t o d e t e r m i n e p o s s i b l e i n t e r f e r e n c e i n the a s s a y by 5 ' - n u c l e o -t i d a s e a c t i v i t y . No c o n v e r s i o n of 5'-AMP t o a d e n o s i n e c o u l d be d e t e c t e d under t h e s e a s s a y c o n d i t i o n s . 4. C h e m i c a l a s s a y s The c o n t e n t of c h o l e s t e r o l i n s k e l e t a l m u s c l e sarcolemma was d e t e r m i n e d by the method of Zak e t a l ( 1 1 8 ) . Membrane p r o t e i n i n g l a c i a l a c e t i c a c i d was added t o the Zak r e a g e n t (a one i n h u n d r e d d i l u t i o n i n c o n c e n t r a t e d H2SO4 of a s o l u t i o n o f 2.5 g F e C l 3 . 6 H 2 0 i n 25 ml g l a c i a l a c e t i c a c i d ) and the a b s o r b a n c e a t 565 nm was r e a d a f t e r 30 min of c o l o r d e v e l o p m e n t . L i p i d p h o s p h o r o u s was d e t e r m i n e d by the B a r t l e t t v a r i a t i o n of the Fi s k e - S u b b a R o w method (119) f o l l o w i n g h y d r o l y s i s of the membrane p r e p a r a t i o n w i t h 10 N H2SO4 a t - 25 -1 5 0 ° . The c o n t e n t of p h o s p h o l i p i d was e s t i m a t e d by m u l t i -p l y i n g the l i p i d p h o s p h o r o u s v a l u e by t w e n t y - f i v e . S i a l i c a c i d ( a s s a y e d as N - a c e t y l n e u r a m i n i c a c i d ) was d e t e r m i n e d by the t h i o b a r b i t u r i c a c i d method of Warren (120) f o l l o w i n g m i l d h y d r o l y s i s of the sarcolemma p r e p a r a t i o n w i t h 0.1 N H2SO4 at 80° by the p r o c e d u r e of S v e n n e r h o l m ( 1 2 1 ) . F l u o r i d e was e s t i m a t e d w i t h the Amadac-F r e a g e n t ( 1 2 2 ) . P r o t e i n d e t e r m i n a t i o n s were made by t h e method of Lowry e t a l (123) u s i n g b o v i n e serum a l b u m i n as s t a n d a r d . - 26 -RESULTS A. ADENYL CYCLASE IN HEART P r e v i o u s i n v e s t i g a t i o n s i n t h i s l a b o r a t o r y by Drummond and Duncan (28) had examined the e f f e c t s of magnesium, c a l c i u m and f l u o r i d e on m y o c a r d i a l a d e n y l c y c l a s e . The r e s u l t s p r e s e n t e d i n s e c t i o n A r e p r e s e n t a p o r t i o n of t h e f u r t h e r i n v e s t i g a t i o n by Drummond, S e v e r s o n and Duncan (108) of the e f f e c t s of o t h e r d i v a l e n t c a t i o n s and the e x a m i n a t i o n of the e f f e c t s of e p i n e p h r i n e and f l u o r i d e s t i m u l a t i o n on the k i n e t i c p r o p e r t i e s of the c a r d i a c enzyme. 1. P r e l i m i n a r y V a l i d i t y of the a s s a y and a s s a y c o n d i t i o n s — T h e p a p e r c h r o m a t o g r a p h i c a s s a y f o r a d e n y l c y c l a s e a c t i v i t y i n p a r t i -c u l a t e f r a c t i o n s of g u i n e a p i g h e a r t and the a s s a y c o n d i t i o n s have been d i s c u s s e d by Drummond and Duncan ( 2 8 ) . In p a r t i c u l a r , the p r e s e n c e of ATPase a c t i v i t y (0.6 umole p e r min p e r mg) n e c e s s i t a t e d the use of an A T P - r e g e n e r a t i n g s y s t e m ( p h o s p h o e n o l p y r u v a t e and p y r u v a t e k i n a s e ) t o p r e s e r v e the s u b s t r a t e f o r a d e n y l c y c l a s e . T h i s A T P - r e g e n e r a t i n g s y s t e m was e f f e c t i v e u n d e r a wide v a r i e t y of c o n d i t i o n s ; however e x p e r i m e n t a l c o n d i t i o n s s u c h as h i g h c o n c e n t r a t i o n s of c a l c i u m o r the o m i s -s i o n o f magnesium were d e s c r i b e d i n w h i c h ATP was e x t e n s i v e l y d e g r a d e d d e s p i t e the p r e s e n c e o f the r e g e n e r a t i n g s y s t e m ( 2 8 ) . The a c t i v i t y of 3 ' , 5 f - c y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e was i n h i b i t e d by the i n c l u s i o n of 8 mM t h e o p h y l l i n e and 2 mM u n l a b e l l e d c y c l i c AMP i n the i n c u b a t i o n m i x t u r e . The f o r m a t i o n - 27 -of c y c l i c AMP was l i n e a r w i t h r e s p e c t t o t i m e and p r o t e i n (28) P r o o f t h a t the r a d i o a c t i v i t y p r e s e n t i n the c y c l i c AMP a r e a of the p a p e r c h r o m a t o g r a m c o r r e s p o n d e d t o c y c l i c AMP s y n t h e s i z e d d u r i n g the i n c u b a t i o n was p r o v i d e d by two e x p e r -i m e n t s ( 2 8 ) . The a r e a c o r r e s p o n d i n g t o c y c l i c AMP on the p a p e r c h r o m a t o g r a m was e l u t e d and s u b j e c t e d t o h i g h v o l t a g e e l e c t r o p h o r e s i s . G r e a t e r t h a n 97% o f the r a d i o a c t i v i t y was s t i l l r e c o v e r e d i n the a r e a c o r r e s p o n d i n g t o c y c l i c AMP a f t e r e l e c t r o p h o r e s i s . In a d d i t i o n , e l u t i o n of t h e c y c l i c AMP a r e a o f p a p e r chromatograms and i n c u b a t i o n w i t h a c r u d e b r a i n p h o s p h o d i e s t e r a s e p r e p a r a t i o n r e s u l t e d i n the d e s t r u c -t i o n o f 98% of the r a d i o a c t i v i t y ; the r a d i o a c t i v i t y was r e c o v e r e d as 5'-AMP and a d e n o s i n e ( a d e n o s i n e f o r m a t i o n was due t o the p r e s e n c e of 5 1 - n u c l e o t i d a s e a c t i v i t y i n the c r u d e p h o s p h o d i e s t e r a s e p r e p a r a t i o n ) ( 2 8 ) . In o r d e r t o i n v e s t i g a t e f u r t h e r the a p p l i c a b i l i t y of the p a p e r c h r o m a t o g r a p h i c t e c h n i q u e f o r the a s s a y of a d e n y l c y c l a s e , a p r e p a r a t i o n of g u i n e a p i g h e a r t washed p a r t i c l e s was a s s a y e d i n t r i p l i c a t e i n the a b s e n c e and p r e s e n c e of 8 mM f l u o r i d e . The f o r m a t i o n of c y c l i c AMP was d e t e r m i n e d by t h r e e m e t h o d s . The f i r s t method employed the p a p e r c h r o m a t o -g r a p h i c t e c h n i q u e d e s c r i b e d i n the me t h o d s as a s s a y A. The s e c o n d method was t h a t of K r i s h n a , Weiss and B r o d i e (110) w h i c h i s a l s o o u t l i n e d i n the me t h o d s ( a s s a y C ) . The t h i r d method i n v o l v e d c h r o m a t o g r a p h y on D E A E - c e l l u l o s e . F o l l o w i n g t e r m i n a t i o n of the r e a c t i o n by b o i l i n g and c e n t r i f u g a t i o n , - 28 -100 p i of the supernatant p l u s 1 pmole u n l a b e l l e d c y c l i c AMP was d i l u t e d to 1.0 ml with water and a p p l i e d to a 9 cm column of DE A E - c e l l u l o s e (outer diameter of the column was 7 mm). The column was washed with 5 ml of water which completely removed adenosine. The c y c l i c AMP was removed by washing the column with 5 ml of 0 .03 M NH^HCO-j. Under these c o n d i t i o n s , l e s s than 17. of the 5'-AMP was removed. This l a t t e r f r a c t i o n was l y o p h i l i z e d , r e d i s s o l v e d i n 1 ml water and the r a d i o a c t i v i t y was measured i n a 0 .5 ml a l i q u o t . The r e c o v e r y of c y c l i c AMP was c a l c u l a t e d from the absorb-ance at 259 nm of an a l i q u o t of the f r a c t i o n , and ranged from 60 to 847.. The comparison of the three assay methods i s shown i n Table I. I t i s ev i d e n t that a l l three methods are i n very c l o s e agreement, e s p e c i a l l y f o r those v a l u e s of a c t i v i t y measured i n the absence of f l u o r i d e . The paper chromatographic assay (assay A) has been used r o u t i n e l y except i n those experiments i n which ATP c o n c e n t r a t i o n s were e l e v a t e d and fc3 2 P ] -ATP was used as s u b s t r a t e . The paper chromatographic assay (assay A) has the advantage that ATP and i t s d e g r a d a t i o n products can be monitored r o u t i n e l y . In a d d i t i o n , a g r e a t e r p r o p o r t i o n ( t w o - t h i r d s ) of the product formed i s counted i n c o n t r a s t to the Krishna method (assay C) i n which a much lower percentage i s counted due to d i l u t i o n d u r i n g the Dowex column and Ba-Zn p r e c i p i t a t i o n s . Some questions have been r a i s e d c o n c erning the p o s s i b i l i t y of contamination by adenine, hypoxanthine , or adenosine f o l l o w i n g paper chromatographic - 29 TABLE I Determination of adenyl c y c l a s e a c t i v i t y by three methods. A l y o p h i l i z e d p r e p a r a t i o n of guinea p i g heart washed p a r t i c l e s was suspended i n 10 mM T r i s , pH 8 and assayed i n t r i p l i c a t e i n the absence and i n the presence of 8 raM f l u o r i d e . U n l a b e l l e d c y c l i c AMP was omitted from the i n c u b a t i o n . F o l l o w i n g t e r m i n a t i o n of the r e a c t i o n by b o i l i n g and c e n t r i -f u g a t i o n , the content of r a d i o a c t i v e c y c l i c AMP was determined by the three methods d e s c r i b e d i n the t e x t . T r i p l i c a t e blanks c o n t a i n e d water i n p l a c e of the enzyme p r e p a r a t i o n and were analyzed by the three d i f f e r e n t methods. As s ay DPM c y c l i c AMP/incubation -F~ + F" Paper chromatography (assay A) Krishna assay (assay C) DEAE-Ce1lu1ose chromatography 28,961 27,773 29 ,130 91,599 114,312 115 ,690 - 30 -t e c h n i q u e s ( 1 2 4 , 1 2 5 ) . However, e x c e l l e n t agreement has been o b t a i n e d when c o m p a r i n g £14c]-ATP and [cC3 2p] - ATP as s u b s t r a t e s , i n d i c a t i n g no c o n t a m i n a t i o n of the c y c l i c AMP a r e a of the c h r o m a t o g r a m by adenine, a d e n o s i n e or o t h e r d e g r a d a t i o n p r o d u c t s s u c h as i n o s i n e or h y p o x a n t h i n e . A l s o , s i g n i f i c a n t c o n t a m i n -a t i o n of the c y c l i c AMP a r e a of p a p e r c hromatograms c a n n o t be r e c o n c i l e d w i t h the c l o s e a greement of the t h r e e d i f f e r e n t a s s a y s shown i n T a b l e I. 2. S u b c e l l u l a r d i s t r i b u t i o n The s u b c e l l u l a r d i s t r i b u t i o n of a d e n y l c y c l a s e i n homogenates of g u i n e a p i g h e a r t s i s shown i n T a b l e I I . The v a s t m a j o r i t y of the a c t i v i t y was r e c o v e r e d i n the washed p a r t i c l e s s e d i m e n t i n g a t low g r a v i t a t i o n a l f o r c e s . A c t i v i t y was a l s o a s s o c i a t e d w i t h p a r t i c u l a t e m i t o c h o n d r i a l and m i c r o -somal f r a c t i o n s , however the a c t i v i t y o f t h e s e f r a c t i o n s a l w a y s c o n s t i t u t e d l e s s t h a n 107. of t h e o r i g i n a l a c t i v i t y i n the whole homogenate. The s p e c i f i c enzyme a c t i v i t y i n the m i c r o s o m a l f r a c t i o n was s i m i l a r t o the a d e n y l c y c l a s e a c t i v i t y r e p o r t e d t o be a s s o c i a t e d w i t h c a r d i a c s a r c o p l a s m i c r e t i c u l u m f r a g -ments ( 1 2 6 , 1 2 7 ) . S i n c e m y o c a r d i a l a d e n y l c y c l a s e a c t i v i t y was l a r g e l y r e s t r i c t e d t o washed p a r t i c l e f r a c t i o n s w h i c h p r e s u m a b l y c o n t a i n e d p l a s m a membranes, f u r t h e r a t t e m p t s were made t o i s o l a t e the p l a s m a membrane f r a c t i o n . However, p r o -c e d u r e s b a s e d on the method o f P o r t i u s and Repke (128) i n v o l v i n g l o n g term e x t r a c t i o n s w i t h h i g h s a l t c o n c e n t r a t i o n s were u n s u c c e s s f u l . A p a r t i a l p u r i f i c a t i o n was a c h i e v e d by a s h o r t t e r m e x t r a c t i o n of the washed p a r t i c l e s w i t h 0 .4 M - 31 -TABLE I I S u b c e l l u l a r d i s t r i b u t i o n of m y o c a r d i a l a d e n y l c y c l a s e . P a r t i c u l a t e , s u b c e l l u l a r f r a c t i o n s of g u i n e a p i g v e n t r i c l e were o b t a i n e d as d e s c r i b e d i n methods . A c t i v i t y of the f r a c t -i o n s was d e t e r m i n e d i n the p r e s e n c e of 8 mM f l u o r i d e by a s s a y A, F r a c t i o n P r o t e i n S p e c i f i c a c t i v i t y T o t a l a c t i v i t y u n i t s Y i e l d (mg) ( p m o l e s / min/mg) ( p m o l e s / min ) (%) Whole homogenate 157 73.5 11 ,540 100 Washed p a r t i c l e s 51 217.5 11 ,093 96 L i B r - e x t r a c t e d p a r t i c l e s 21 463 .3 9 ,729 84 L i B r ( T a b l e I I ) . T h i s p r o c e d u r e was o b s e r v e d by phase c o n t r a s t m i c r o s c o p y t o r e s u l t i n the e x t r a c t i o n of c o n t r a c -t i l e p r o t e i n , w i t h a r e s u l t a n t i n c r e a s e i n s p e c i f i c enzyme a c t i v i t y of 2 t o 4 - f o l d . The y i e l d o f a c t i v i t y f o l l o w i n g L i B r e x t r a c t i o n was g r e a t e r t h a n 80%. L i B r - e x t r a c t e d p a r t i c l e s or washed p a r t i c l e s c o u l d be l y o p h i l i z e d and s t o r e d o v e r d e s -s i c a n t a t -18° w i t h a m i n i m a l l o s s o f a c t i v i t y . P r e l i m i n a r y a t t e m p t s t o p u r i f y a d e n y l c y c l a s e by s o l u b i l i z a t i o n by v a r i o u s d e t e r g e n t s , s o n i c a t i o n o r o r g a n i c s o l v e n t s were u n s u c c e s s f u l due t o the e x t r e m e l a b i l i t y of the enzyme' -. 3. K i n e t i c p r o p e r t i e s of m y o c a r d i a l a d e n y l c y c l a s e E f f e c t of m e t a l i o n s on m y o c a r d i a l a d e n y l c y c l a s e — D r u m m o n d and Duncan (28) f o u n d t h a t magnesium c o u l d i n c r e a s e the r a t e of c y c l i c AMP f o r m a t i o n i n p a r t i c u l a t e p r e p a r a t i o n s f r o m c a r d i a c t i s s u e when p r e s e n t a t c o n c e n t r a t i o n s much i n e x c e s s of ATP. At pH 7.5, one magnesium i s bound to ATP and so i t i s l i k e l y t h a t the s u b s t r a t e i s a magnesium-ATP c o m p l e x . I t was a p p a r e n t t h e r e f o r e , t h a t the enzyme c o u l d b i n d a d d i t i o n a l magnesium ( K g 2-3 mM), p r e s u m a b l y at some s e c o n d s i t e . A com-p a r i s o n of the e f f e c t s of manganese and magnesium i s shown i n F i g . 2, p a n e l A. Manganese s t i m u l a t e d a d e n y l c y c l a s e a t much low e r c o n c e n t r a t i o n s t h a n magnesium; the a p p a r e n t K a was 0.7 mM. Maximal v e l o c i t i e s were o b t a i n e d w i t h manganese c o n c e n -t r a t i o n s of 2-4 mM and were s i g n i f i c a n t l y g r e a t e r t h a n the 1„ Drummond and Duncan, u n p u b l i s h e d o b s e r v a t i o n s . - 33 -F i g u r e 2: E f f e c t of m e t a l i o n s on m y o c a r d i a l a d e n y l c y c l a s e . P a n e l A: a l y o p h i l i z e d p r e p a r a t i o n of L i B r - e x t r a c t e d p a r t i c l e s (353 ug p r o t e i n ) f r o m g u i n e a p i g h e a r t was a s s a y e d i n the p r e s e n c e of v a r y i n g c o n c e n t r a t i o n s of magnesium and manganese by a s s a y A. P a n e l B: the e f f e c t of c o b a l t i n the p r e s e n c e of s e v e r a l f i x e d f l u o r i d e c o n c e n t r a t i o n s on the a c t i v i t y of a g u i n e a p i g L i B r - e x t r a c t e d p a r t i c u l a t e p r e p a r a t i o n (224 ug p r o t e i n ) . The c o n c e n t r a t i o n of f l u o r i d e (mM) i s i n d i c a t e d by the number on e a c h c u r v e . A c t i v i t y was d e t e r m i n e d by a s s a y A. 1.5 A B C a t i o n (mM) Figure 2 - 35 -s t i m u l a t i o n by t e n - f o l d g r e a t e r c o n c e n t r a t i o n s of magnesium. S i n c e the ATP c o n c e n t r a t i o n was 0.3 mM and the s u b s t r a t e i s l i k e l y to be an A T P - m e t a l i o n c o m p l e x , i t i s e v i d e n t t h a t t h e enzyme can a l s o b i n d e x c e s s manganese at some s e c o n d s i t e . C o n c e n t r a t i o n s of manganese g r e a t e r t h a n 4 mM were i n h i b i t o r y . T h i s i n h i b i t i o n c o u l d n o t be r e v e r s e d by a d d i t i o n of magnesium, m e t a l c h e l a t o r s s u c h as EDTA or EGTA, or a s c o r b i c a c i d . C o u n t i n g the a p p r o p r i a t e a r e a s of p a p e r chromatograms r e v e a l e d t h a t c o n c e n t r a t i o n s of manganese i n e x c e s s of 4 mM were n o t i n t e r f e r i n g w i t h the A T P - r e g e n e r a t i n g s y s t e m . F l u o r i d e r e s u l t s i n a p r o f o u n d s t i m u l a t i o n of m y o c a r d i a l a d e n y l c y c l a s e a c t i v i t y ( 2 8 ) . S t i m u l a t i o n by f l u o r i d e v a r i e d i n d i f f e r e n t p r e p a r a t i o n s b u t was u s u a l l y 4 to 1 0 - f o l d i n the p r e s e n c e o f magnesium and was maximal a t 8 mM. F l u o r i d e a l s o s t i m u l a t e d the enzyme i n the p r e s e n c e of manganese a l t h o u g h the a c t i v a t i o n t e n d e d t o be somewhat l e s s t h a n w i t h magnesium. S t i m u l a t i o n by f l u o r i d e v a r i e d f r o m 3 t o 5 - f o l d and was maximal at 8 mM and h a l f - m a x i m a l at 2 mM i n the p r e s e n c e of 1.8 mM manganese. F l u o r i d e s t i m u l a t e d the enzyme a t a l l c o n c e n t r a t i o n s of manganese. I n h i b i t i o n due to h i g h c o n c e n t r a t i o n s of man-ganese and the K f o r manganese were n o t a f f e c t e d by f l u o r i d e . a C o b a l t i s a l s o c a p a b l e o f a c t i v a t i n g m y o c a r d i a l a d e n y l , c y c l a s e ( F i g . 2, p a n e l B ) . C o b a l t was l e s s e f f e c t i v e t h a n magnesium i n a c t i v a t i n g the enzyme. The a p p a r e n t K, f o r c o b a l t was a p p r o x i m a t e l y 1 mM. The e f f e c t of s e v e r a l f i x e d f l u o r i d e c o n c e n t r a t i o n s on the a c t i v a t i o n by c o b a l t i s a l s o shown i n - 36 -Figo 2, p a n e l B. The p r i m a r y k i n e t i c e f f e c t o f f l u o r i d e was t o i n c r e a s e r e a c t i o n v e l o c i t y . The K f o r c o b a l t was n o t c h a n g e d a p p r e c i a b l y by t h e p r e s e n c e o f f l u o r i d e . The a b i l i t y o f o t h e r c a t i o n s t o a c t i v a t e m y o c a r d i a l a d e n y l c y c l a s e was n o t i n v e s t i g a t e d due t o t h e s p e c i f i c c a t i o n r e q u i r e m e n t s o f t h e A T P - r e g e n e r a t i n g s y s t e m . E f f e c t o f ATP c o n c e n t r a t i o n — T h e f o r ATP i n p a r t i c u l a t e a d e n y l c y c l a s e p r e p a r a t i o n s f r o m g u i n e a p i g h e a r t was d e t e r -m i n e d p r e v i o u s l y t o be a p p r o x i m a t e l y 0.1 mM ( 2 8 ) . M a g n e s i u m and f l u o r i d e i n c r e a s e d t h e r e a c t i o n v e l o c i t y w i t h o u t a f f e c t i n g t h e a f f i n i t y o f t h e enzyme f o r s u b s t r a t e . I n t h e s e e x p e r i m e n t s , £l^cQ- ATP was u s e d as s u b s t r a t e and c o n c e n t r a t i o n s o f magnesium were a l w a y s i n e x c e s s o f t h e s u b s t r a t e c o n c e n t r a t i o n . By u s i n g £c(.3 2p]-ATP as t h e s u b s t r a t e and a s s a y C, a much g r e a t e r r a n g e o f ATP c o n c e n t r a t i o n s c o u l d be e x a m i n e d . The e f f e c t o f v a r y i n g ATP c o n c e n t r a t i o n s a t two f i x e d manganese c o n c e n -t r a t i o n s i n t h e a b s e n c e and p r e s e n c e o f f l u o r i d e i s shown i n F i g . 3. The e f f e c t o f manganese and f l u o r i d e was t o i n c r e a s e m a x i m a l v e l o c i t y . N e i t h e r m e t a l i o n s n o r f l u o r i d e a p p r e c i a b l y c h a n g e d t h e a f f i n i t y f o r s u b s t r a t e ; t h e K m f o r ATP was a b o u t 0.1 mM. When manganese was p r e s e n t a t 0.6 mM, i n c r e a s i n g t h e ATP c o n c e n t r a t i o n t o g r e a t e r t h a n 0.7 mM r e s u l t e d i n d e c r e a s e d a c t i v i t y ( F i g . 3 ) . T h i s i n h i b i t i o n due t o ATP was e s p e c i a l l y n o t i c e a b l e i n t h e p r e s e n c e of f l u o r i d e . When t h e m e t a l i o n c o n c e n t r a t i o n was i n c r e a s e d t o 1.8 mM, a c t i v i t y was d e c r e a s e d by c o n c e n t r a t i o n s o f ATP g r e a t e r t h a n 1.5 mM. Thus - 37 -F i g u r e 3: E f f e c t of ATP c o n c e n t r a t i o n on myocardial adenyl c y c l a s e at two f i x e d manganese c o n c e n t r a t i o n s . The c o n c e n t r a -t i o n s of manganese (mM) are given by the numbers on each curve. A c t i v i t y i n the guinea p i g washed p a r t i c l e p r e p a r a t i o n (250 ug p r o t e i n ) was determined by assay C i n the absence ( ) and i n the presence ( ) of 8 mM f l u o r i d e . - 38 -ATP (mM) F i g u r e 3 - 39 -i t appears that ATP r e s u l t e d i n an i n h i b i t i o n of adenyl c y c l a s e a c t i v i t y when present i n c o n c e n t r a t i o n s i n excess of metal i o n s . S i m i l a r r e s u l t s were obtained by Drummond, Severson and Duncan (108) when ATP c o n c e n t r a t i o n s were i n excess of mag-nesium c o n c e n t r a t i o n s e i t h e r i n the absence or presence of f l u o r i d e . ATP c o n c e n t r a t i o n s were v a r i e d from 0.3 to 6.2 mM i n the presence of s e v e r a l f i x e d magnesium c o n c e n t r a t i o n s (1.0, 2.0, 3.6, 9.0 and 18 mM); ATP c o n c e n t r a t i o n s i n excess of magnesium c o n c e n t r a t i o n s r e s u l t e d i n i n h i b i t i o n of enzyme a c t i v i t y , p a r t i c u l a r l y i n the presence of 8 mM f l u o r i d e . I t i s apparent from the data i n F i g . 3 that the i n h i b i t i o n by ATP i s r e v e r s e d by i n c r e a s i n g the metal i o n c o n c e n t r a t i o n but not by the presence of f l u o r i d e . The a b i l i t y of manganese to r everse ATP i n h i b i t i o n was examined i n experiments i n which the decrease i n a c t i v i t y due to excess ATP was overcome by c a r e f u l l y t i t r a t i n g with i n c r e a s i n g c o n c e n t r a t i o n s of manganese ( F i g . 4 ) . When the c o n c e n t r a t i o n of ATP was i n c r e a s e d from 0.93 mM to 2.7 mM i n the presence of 1 mM manganese, the a c t i v i t y was decreased by one-half (compare hatched bars i n F i g . 4 ) . I n c r e a s i n g the c o n c e n t r a t i o n of manganese r e s u l t e d i n an i n c r e a s e i n r e a c t i o n v e l o c i t y ; ATP i n h i b i t i o n was over-come by 2 mM manganese. The i n h i b i t i o n by ATP c o n c e n t r a t i o n s i n excess of magnesium i n the presence of f l u o r i d e was a l s o shown by Drummond, Severson and Duncan (108) to be overcome by i n c r e a s e d magnesium c o n c e n t r a t i o n s . The i n h i b i t i o n observed when ATP was i n c r e a s e d to 7.9 mM from 0.7 mM i n the - 40 -F i g u r e 4 : R e v e r s a l of ATP i n h i b i t i o n by manganese. The e f f e c t of v a r y i n g c o n c e n t r a t i o n s of manganese at two f i x e d ATP concen-t r a t i o n s was determined by assay C with a washed p a r t i c u l a t e p r e p a r a t i o n ( 3 9 9 ug p r o t e i n ) from guinea p i g h e a r t . Hatched bars compare the e f f e c t of i n c r e a s i n g the ATP c o n c e n t r a t i o n at a s i n g l e , f i x e d manganese c o n c e n t r a t i o n . S t i p p l e d bars i n d i c a t e the e f f e c t of i n c r e a s i n g manganese c o n c e n t r a t i o n s at two f i x e d ATP c o n c e n t r a t i o n s . - 41 -Mn + + I 1.5 2 253 3.5 I 1.5 2 2.5 3 3.5 4 5 6 9 .93 mM ATP 2.7 mM ATP F i g u r e 4 - 42 -p r e s e n c e o f 3 mM magnesium was c o m p l e t e l y overcome when the magnesium c o n c e n t r a t i o n was i n c r e a s e d t o 6 mM. T h u s , ATP i n h i b i t i o n was overcome by magnesium c o n c e n t r a t i o n s l e s s t h a n t h a t r e q u i r e d to f o r m a one to one A T P - m e t a l i o n c o m p l e x , as was o b s e r v e d w i t h manganese i n F i g . 4. T h e r e f o r e t h e r e was not an a b s o l u t e s t o i c h i o m e t r y i n v o l v e d i n the a b i l i t y of m e t a l i o n s to overcome ATP i n h i b i t i o n . E f f e c t o f hormones on m y o c a r d i a l a d e n y l c y c l a s e - - E p i n e p h r i n e p r o d u c e d a v e r y modest ( 1 . 3 - f o l d ) b ut c o n s i s t e n t s t i m u l a t i o n of g u i n e a p i g washed p a r t i c l e p r e p a r a t i o n s of a d e n y l c y c l a s e i n the p r e s e n c e of manganese. However, i n the p r e s e n c e of magnesium, c a t e c h o l a m i n e s s t i m u l a t e d washed p a r t i c l e or L i B r -e x t r a c t e d p a r t i c u l a t e p r e p a r a t i o n s f r o m g u i n e a p i g o r r a b b i t h e a r t by a t l e a s t t w o - f o l d . H a l f - m a x i m a l a c t i v a t i o n was o b s e r v e d w i t h 5 x 10"^ M e p i n e p h r i n e ; the o r d e r of p o t e n c y of i s o p r o p y l n o r e p i n e p h r i n e , e p i n e p h r i n e and n o r e p i n e p h r i n e was i d e n t i c a l to t h a t r e p o r t e d by Murad e_t_ a l ( 4 1 ) . The s t i m u l a t i o n by e p i n e p h r i n e was b l o c k e d by p r o p r a n o l o l . G l u c a g o n , t e s t e d u nder a wide v a r i e t y of c o n d i t i o n s , d i d n o t a c t i v a t e a d e n y l c y c l a s e , i n c o n t r a s t t o o t h e r r e p o r t s ( 6 8 , 6 9 ) . T h i s same p r e p a r a t i o n of g l u c a g o n d i d s t i m u l a t e l i v e r a d e n y l c y c l a s e (108) and so why the hormone p r e p a r a t i o n was u n a b l e t o s t i m u l a t e m y o c a r d i a l a d e n y l c y c l a s e i s not c l e a r . The k i n e t i c n a t u r e of the s t i m u l a t i o n of m y o c a r d i a l a d e n y l c y c l a s e by e p i n e p h r i n e and f l u o r i d e was examined by Drummond, S e v e r s o n and Duncan ( 1 0 8 ) . B o t h e p i n e p h r i n e and f l u o r i d e s t i m u l a t e d the r e a c t i o n - 43 -v e l o c i t y a t a l l m a g n e s i u m a n d A T P c o n c e n t a t i o n s ; n e i t h e r e p i n e p h r i n e n o r f l u o r i d e s i g n i f i c a n t l y a l t e r e d t h e a p p a r e n t K a f o r m a g n e s i u m o r t h e f o r t h e A T P - m a g n e s i u m s u b s t r a t e . B . A D E N Y L C Y C L A S E I N S K E L E T A L M U S C L E F u r t h e r i n v e s t i g a t i o n s o f t h e k i n e t i c p r o p e r t i e s o f m y o c a r d i a l a d e n y l c y c l a s e w e r e l i m i t e d b y t h e p a r t i c u l a t e n a t u r e o f t h e e n z y m e p r e p a r a t i o n a n d t h e e x t r e m e l a b i l i t y o f t h e e n z y m e t o v a r i o u s p u r i f i c a t i o n t e c h n i q u e s . A l t h o u g h c y c l i c A M P w a s i n i t i a l l y d i s c o v e r e d d u r i n g t h e i n v e s t i g a t i o n o f t h e s t i m u l a t i o n o f h e p a t i c g l y c o g e n o l y s i s b y h o r m o n e s , t h e m o s t d e t a i l e d i n f o r m a t i o n c o n c e r n i n g t h e m e c h a n i s m o f a c t i o n o f t h e c y c l i c n u c l e o t i d e h a s b e e n o b t a i n e d f r o m s t u d i e s o f t h e h o r m o n a l r e g u l a t i o n o f g l y c o g e n m e t a b o l i s m i n s k e l e t a l m u s c l e . D e s p i t e t h e c o n s i d e r a b l e s u m o f k n o w l e d g e c o n c e r n i n g t h e . a c t i o n o f c y c l i c A M P i n s k e l e t a l m u s c l e , v e r y l i t t l e i n f o r m a t i o n i s a v a i l a b l e c o n c e r n i n g t h e p r o p e r t i e s o f a d e n y l c y c l a s e i n t h i s t i s s u e . T h e r e m a i n d e r o f t h i s t h e s i s d e s c r i b e s t h e i n v e s t i g a t i o n o f t h e k i n e t i c p r o p e r t i e s o f t h e s k e l e t a l m u s c l e . e n z y m e . T h e e f f e c t s o f f l u o r i d e a n d h o r m o n a l s t i m u l -a t i o n o n t h e k i n e t i c p r o p e r t i e s o f a d e n y l c y c l a s e w e r e e x a m i n e d t o i n v e s t i g a t e t h e m e c h a n i s m s i n v o l v e d i n t h e r e g u l -a t i o n o f c y c l i c A M P f o r m a t i o n . - 44 -1. P r e l i m i n a r y Adenyl c y c l a s e i n s k e l e t a l muscle t i s s u e s from s e v e r a l s p e c i e s — T h e a c t i v i t y of adenyl c y c l a s e i n whole homogenates of s k e l e t a l muscle from v a r i o u s s p e c i e s , measured i n the absence and presence of 8 mM f l u o r i d e , i s shown i n Table I I I . Enzyme a c t i v i t y i n the hind limb of r a b b i t and guinea p i g was gr e a t e r than i n r a t hind limb muscle, p a r t i c u l a r l y i n the presence of f l u o r i d e . Adenyl c y c l a s e i n mammalian s k e l e t a l muscle t i s s u e s was more a c t i v e than i n f r o g gast-rocnemius. Pigeon b r e a s t muscle was much more a c t i v e than ch i c k e n b r e a s t muscle. The lower a c t i v i t y i n f r o g g a s t r o -cnemius when compared with mammalian t i s s u e s and the d i f f e r e n c e s between b r e a s t muscle from pigeon and chi c k e n suggest that adenyl c y c l a s e a c t i v i t y may be g r e a t e r i n red muscle f i b e r s adapted f o r o x i d a t i v e metabolism than i n white s k e l e t a l muscle f i b e r s . S k e l e t a l muscle t i s s u e from the hind limb of r a b b i t s was s e l e c t e d f o r the remaining s t u d i e s because of ready a v a i l a b i l i t y and high a c t i v i t y . 2. S u b c e l l u l a r d i s t r i b u t i o n and p r e p a r a t i o n of plasma membranes The s u b c e l l u l a r l o c a l i z a t i o n of adenyl c y c l a s e was examined i n attempts to o b t a i n a p a r t i a l l y p u r i f i e d enzyme p r e p a r a t i o n . The s u b c e l l u l a r d i s t r i b u t i o n of enzyme a c t i v i t y i n r a b b i t s k e l e t a l muscle homogenates i s shown i n Table IV, p a r t A. Approximately 807. of the enzyme a c t i v i t y was present i n the washed p a r t i c l e s sedimented at 2000 x g. The m a j o r i t y of the a c t i v i t y present i n the 2000 x g supernatant could be sedimented at 37,000 x g. The high y i e l d of a c t i v i t y i n p a r t i c l e s s e d i -menting at low g r a v i t a t i o n a l f o r c e s suggested that adenyl c y c l a s e - 45 -TABLE I I I Adenyl c y c l a s e a c t i v i t y i n s k e l e t a l muscle from s e v e r a l s p e c i e s . P o r t i o n s of hind limb muscle from r a t , guinea p i g and r a b b i t , the gastrocnemius muscle from f r o g , and b r e a s t muscle from pigeon and chicken were homogenized i n 5 volumes 10 mM T r i s , pH8 f o r 10 sec i n a S o r v a l l omni-mixer. The whole homogenates were s t r a i n e d through a coarse nylon s i e v e and adenyl c y c l a s e a c t i v i t y was determined by assay A i n the presence of 1 mM EGTA and i n the absence or i n the presence of 8 mM f l u o r i d e . A c t i v i t y i s expressed as pmoles per min per mg p r o t e i n and as nmoles per min per g wet t i s s u e weight. Source Adenyl c y c l a s e a c t i v i t y -F" + F~ -F~ + F~ pmoles/min/mg nmoles/min/g(wet wt.) Frog l e g N.D .* 2.6 N.D. .05 Rat l e g 4.2 6.9 .42 .69 Guinea p i g l e g 8.9 39.6 .79 3.50 Rabbit l e g 5.0 37.4 .45 3 .30 Chicken b r e a s t N.D. 3.1 N.D . .25 Pigeon breast 2.2 19 .9 .29 j 2.53 -N.D . not d e t e c t a b l e - 46 -may r e s i d e i n the p l a s m a membrane ( s a r c o l e m m a ) of s k e l e t a l m u s c l e , and so a t t e m p t s were made to i s o l a t e a p l a s m a membrane f r a c t i o n f r o m t h i s t i s s u e . P r o c e d u r e s b a s e d upon the method of M c C o l l e s t e r (129) were u n s u c c e s s f u l b e c a u s e of l a r g e l o s s e s of a c t i v i t y . In p a r t i c u l a r , i n a c t i v a t i o n o c c u r r e d d u r i n g the 3 7 ° i n c u b a t i o n and the w a t e r washes; l e s s t h a n 107. of t h e a c t i v i t y i n the washed p a r t i c l e s was p r e s e n t i n the f i n a l membrane f r a c t i o n . The a d d i t i o n of e i t h e r f l u o r i d e or c r u d e l e c i t h i n p r e p a r a t i o n s d i d n o t p r o t e c t a g a i n s t t h i s i n a c t i v a t i o n . O t h e r e x p e r i m e n t s r e v e a l e d , however , t h a t washed p a r t i c u l a t e p r e p a r a t i o n s f r o m s k e l e t a l m u s c l e c o u l d be e x t r a c t e d w i t h 0.4 M L i B r f o r p e r i o d s of time f r o m 4 to 8 hr w i t h no l o s s of a c t i v i t y . A c c o r d i n g l y , p l a s m a membranes were i s o l a t e d f r o m r a b b i t s k e l e t a l m u s c l e by m o d i f i c a t i o n s of the method d e s c r i b e d by Kono and C o l o w i c k ( 1 3 0 ) . A l l p r o c e d u r e s were c a r r i e d o ut a t 4 ° . A p p r o x i m a t e l y 5 g p o r t i o n s of r a b b i t h i n d l i m b m u s c l e were m i n c e d w i t h s c i s s o r s and h o mogenized f o r 10 t o 15 sec i n 5 volumes of 50 mM c a l c i u m c h l o r i d e . The whole homogenate was p a s s e d t h r o u g h a c o a r s e 2 n y l o n s i e v e ( p o r e s i z e 1 mm ) and c e n t r i f u g e d f o r 10 min a t 2000 x g. The p r e c i p i t a t e was washed t w i c e by s u s p e n d i n g t o the o r i g i n a l volume w i t h 10 mM T r i s , pH 8 and c e n t r i f u g i n g a t 2000 x g. Washing removed s o l u b l e p r o t e i n and s u p e r f i c i a l l y bound c a l c i u m f r o m the washed p a r t i c l e p r e p a r a t i o n s . The v a r i o u s f r a c t i o n s o b t a i n e d d u r i n g the p u r i f i c a t i o n p r o c e d u r e were r o u t i n e l y examined by phase c o n t r a s t m i c r o s c o p y and - 47 -TABLE IV S u b c e l l u l a r d i s t r i b u t i o n of adenyl c y c l a s e i n s k e l e t a l musele t i s s u e . Adenyl c y c l a s e a c t i v i t y was measured i n s u b c e l l u l a r f r a c t i o n s of s k e l e t a l muscle i s o l a t e d as d e s c r i b e d i n the t e x t . Adenyl c y c l a s e a c t i v i t y was determined by assay A i n the presence of 8 mM f l u o r i d e and 1 mM EGTA. Frac t i o n P r o t e i n Spec i f i c ac t i v i t y T o t a l ac t i v i t y u n i t s Y i e l d (mg) (pmole s/ min/mg) (pmoles/ min) (%) A. Whole homogenate 368 51.4 18,915 100 Washed p a r t i c l e s 276 55.0 15,180 80 Supernatant 102 20.7 2,111 11 B. Washed p a r t i c l e s 391 57.1 22,326 100 L i B r - e x t r a c t e d p a r t i c l e s 191 91.2 17 ,419 78 "Membranes" 10 810.4 8,104 37 - 48 -t y p i c a l photomicrographs are shown i n F i g . 5. Cross s t r i a t i o n s are c l e a r l y v i s i b l e i n the whole homogenate and washed p a r t i c l e p r e p a r a t i o n s ( F i g . 5, p l a t e s A and B ) . Some of the c o n t r a c -t i l e p r o t e i n i n the s k e l e t a l muscle f i b e r s was e x t r a c t e d d u r i n g the washing of the 2000 x g p e l l e t with 10 mM T r i s , pH 8 f o l l o w i n g the i n i t i a l homogenization i n 50 mM c a l c i u m c h l o r i d e ( p l a t e C) . The washed p a r t i c l e s were suspended i n 5 volumes (based on i n i t i a l t i s s u e weight) of 10 mM T r i s , pH 8 and l i t h i u m bromide (4 M) was added to produce a f i n a l c o n c e n t r a t i o n of 0.4 M. The suspension was s t i r r e d with a magnetic s t i r r e r f o r 4 to 5 h r . The v i s c o u s suspension was d i l u t e d with 100 ml of 10 mM T r i s , pH 8 and c e n t r i f u g e d f o r 10 min at 2000 x g. The l i t h i u m bromide-extracted p a r t i c l e s d i d not sediment at 2000 x g without t h i s d i l u t i o n . E x t r a c -t i o n of the washed p a r t i c l e s with 0.4 M l i t h i u m bromide r e s u l t e d i n an i n c r e a s e i n s p e c i f i c a c t i v i t y of n e a r l y t w o -f o l d with a y i e l d of 787. (Table IV, p a r t B). A f t e r 10 min of e x t r a c t i o n with l i t h i u m bromide, c o n t r a c t i l e p r o t e i n was being extruded from the muscle f i b e rs ( F i g . 5, p l a t e D ) ; no cross s t r i a t i o n s were v i s i b l e a f t e r 30 min of e x t r a c t i o n ( p l a t e E ) . An e x t r a c t i o n time of approximately 4 hr was found to be op t i m a l ; i n c r e a s i n g the time of e x t r a c t i o n to 12 hr d i d not r e s u l t i n a f u r t h e r i n c r e a s e i n s p e c i f i c a c t i v i t y . L i t h i u m bromide-extracted p a r t i c l e s were washed by suspending i n 10 mM T r i s , pH 8 and c e n t r i f u g i n g at 2000 x g f o r 10 min. The p e l l e t was suspended i n 4 volumes (based on i n i t i a l t i s s u e weight) of 257. (w/w) potassium bromide ( d e n s i t y 1.21) and c e n t r i f u g e d at 25,000 x g f o r 30 min. Unextracted muscle - 49 -F i g u r e 5: Phase c o n t r a s t p h o t o m i c r o g r a p h s of f r a c t i o n s o b t a i n e d d u r i n g the p r e p a r a t i o n of s k e l e t a l m u s c l e p l a s m a membranes. P l a t e A: whole homogenate. P l a t e B: washed p a r t i c l e s . P l a t e C: washed p a r t i c l e s . P l a t e D: p a r t i c l e s a f t e r 5 min L i B r - e x t r a c t i o n . P l a t e E: p a r t i c l e s a f t e r 30 min L i B r -e x t r a c t i o n . P l a t e F: p l a s m a membranes. Figure 5 - 51 -f i b e r s remained i n the supernatant; the p e l l e t was washed by suspending i n 10 mM T r i s , pH 8 and c e n t r i f u g i n g f o r 10 min at 2000 x g. The p e l l e t (plasma membranes) was suspended in one ml 10 mM T r i s , pH 8 per gram of i n i t i a l t i s s u e weight, kept at 4° and assayed w i t h i n 30 min. This membrane p r e p a r a t i o n contained 3 7% of the adenyl c y c l a s e a c t i v i t y w ith an i n c r e a s e i n s p e c i f i c a c t i v i t y of f i f t e e n - f o l d (Table IV, p a r t B ) . The f i n a l membrane p r e p a r a t i o n i s shown i n F i g . 5, p l a t e F; the empty t u b u l a r s t r u c t u r e s resemble those observed by Kono and Colowick (130) and M c C o l l e s t e r (129). Membrane f r a c t i o n s were r o u t i n e l y obtained with i n c r e a s e s i n the s p e c i f i c a c t i v i t y of adenyl c y c l a s e of 10 to 2 0 - f o l d and with y i e l d s r a n g i n g from 15 to 40%. The i n c r e a s e i n s p e c i f i c enzyme a c t i v i t y measured i n the absence of f l u o r i d e p a r a l -l e l e d the i n c r e a s e d s p e c i f i c a c t i v i t y measured i n the presence of f l u o r i d e d u r i n g the p u r i f i c a t i o n procedure. A c r i t i c a l f e a t u r e of t h i s procedure f o r the p r e p a r a t i o n of plasma mem-branes from s k e l e t a l muscle t i s s u e i s the i n i t i a l homogen-i z a t i o n i n c a l c i u m c h l o r i d e . If the i n i t i a l homogenization was performed i n 10 mM T r i s , pH 8, the s p e c i f i c a c t i v i t y of adenyl c y c l a s e i n the f i n a l membrane p r e p a r a t i o n was lower than i n membranes which had been i s o l a t e d f o l l o w i n g an i n i t i a l homogenization i n 50 mM c a l c i u m c h l o r i d e . The i n i t i a l homogenization i n c a l c i u m c h l o r i d e seemed to be r e q u i r e d f o r the complete e x t r a c t i o n of c o n t r a c t i l e p r o t e i n , since membranes prepared without an i n i t i a l c a l c i um c h l o r i d e homogenization m i c r o s c o p i c a l l y d i d not appear as empty as d i d those prepared with the i n i t i a l c a l c i u m c h l o r i d e homogenization. The enzyme p r e p a r a t i o n was u n s t a b l e . Storage of membrane p r e p a r a t i o n s - 52 -f n r 18 h o u r s a t - 4 ° or - 8 0 ° d e c r e a s e d a c t i v i t y by 50%. From 50-80% of the a c t i v i t y c o u l d be r e c o v e r e d f o l l o w i n g l y o p h i l i z a t i o n , w i t h a f u r t h e r d e c r e a s e of o n e - h a l f d u r i n g s t o r a g e of the l y o p h i l i z e d powder a t - 1 8 ° f o r one week. Be c a u s e of t h i s i n s t a b i l i t y , a l l e x p e r i m e n t s were p e r f o r m e d w i t h f r e s h membrane p r e p a r a t i o n s and so e a c h e x p e r i m e n t u s e d a d i f f e r e n t membrane p r e p a r a t i o n . Membrane p r o p e r t i e s and c r i t e r i a f o r the p u r i t y of the  membrane s--A number of c r i t e r i a were examined to e s t a b l i s h t h a t the f i n a l f r a c t i o n c o n t a i n i n g a d e n y l c y c l a s e a c t i v i t y d i d i n d e e d c o n t a i n p l a s m a membranes and t o d e t e r m i n e the r e l a t i v e p u r i t y of t h i s membrane p r e p a r a t i o n . As m e n t i o n e d a b o v e , phase c o n t r a s t p h o t o m i c r o g r a p h s of the f i n a l membrane p r e p a r a t i o n r e v e a l e d the p r e s e n c e of empty, t u b u l a r s t r u c t u r e s w i t h no e v i d e n c e of c r o s s - s t r i a t i o n s and w i t h an i d e n t i c a l a p p e a r a n c e t o the p l a s m a membranes f r o m s k e l e t a l m u s c l e p r e p a r e d by o t h e r t e c h n i q u e s ( 1 2 9 , 1 3 0 ) . A s s a y s f o r a number of enzyme a c t i v i t i e s were p e r f o r m e d w i t h the membrane p r e p a r a t i o n . A magnesium-dependent ATPase was p r e s e n t i n a c t i v i t i e s r a n g i n g f r o m 8.4 t o 30 pmoles ATP h y d r o l y z e d p e r mg p e r hr and was i n h i b i t e d a p p r o x i m a t e l y 507. by the a d d i t i o n of 8 mM f l u o r i d e . The magnesium-dependent ATPase a c t i v i t y i n t h i s membrane p r e p a r a t i o n was shown by S e v e r s o n , Drummond and S u l a k h e (131) to be i n c r e a s e d by the a d d i t i o n of s o d i u m and p o t a s s i u m . In a t y p i c a l membrane p r e p -a r a t i o n , a magnesium-dependent ATPase a c t i v i t y of 16.1 pmoles p e r rag p e r hr was i n c r e a s e d t o 24.1 pmoles p e r mg p e r hr i n - 53 -the p r e s e n c e of 100 mM sodium and 20 mM p o t a s s i u m . S t i m u l a t i o n by s o d i um and p o t a s s i u m u s u a l l y r a n g e d f r o m 10 t o 50%. The s o d i u m , p o t a s s i u m - s t i m u l a t e d a c t i v i t y was p a r t i a l l y i n h i b i t e d by the a d d i t i o n of 0.4 mM o u a b a i n . The s p e c i f i c a c t i v i t y of the s o d i u m , p o t a s s i u m - s t i m u l a t e d , magnesium-dependent ATPase a c t i v i t y o b s e r v e d w i t h t h i s membrane p r e p a r a t i o n was g r e a t e r t h a n t h a t o b s e r v e d by P e t e r f o r r a t m u s c l e sarcolemma (132) b u t l e s s t h a n t h a t p r e s e n t i n hamster s k e l e t a l m u s c l e s a r c o -lemma ( 1 3 3 ) . The p r e s e n c e of c a l c i u m - s t i m u l a t e d ATPase a c t i v i t y was a l s o o b s e r v e d by S e v e r s o n , Drummond and S u l a k h e (131) w i t h t h i s membrane p r e p a r a t i o n . C a l c i u m - A T P a s e d i d n o t r e q u i r e magnesium f o r a c t i v i t y and was p r e s e n t i n a c t i v i t i e s (7.9 uraoles ATP h y d r o l y z e d p e r mg p e r h r ) s i m i l a r t o the magnesium-ATPase a c t i v i t y . In a d d i t i o n , S e v e r s o n , Drummond and S u l a k h e (131) have r e p o r t e d t h a t t h i s membrane f r a c t i o n was a b l e t o b i n d c a l c i u m i n an ATP-dependent manner w h i c h c o u l d be f u r t h e r e n h a n c e d by the a d d i t i o n of 2 mM p h o s p h a t e . 5 1 - N u c l e o t i d a s e i s commonly us e d as a marker f o r p l a s m a membranes; however, i n s k e l e t a l m u s c l e , the a c t i v i t y was s o l u b l e and c o u l d n o t be d e t e c t e d i n the membrane f r a c t i o n u s i n g the o p t i c a l a s s a y d e s c r i b e d i n methods . The a c t i v i t y of a c e t y l -c h o l i n e s t e r a s e i n the membrane f r a c t i o n was 37 nmoles a c e t y l -t h i o c h o l i n e h y d r o l y z e d p e r min p e r mg w h i c h r e p r e s e n t e d an i n c r e a s e i n s p e c i f i c , a c t i v i t y of s i x - f o l d and a y i e l d of 107. f r o m the whole homogenate. S i m i l a r a c t i v i t i e s of a c e t y l -c h o l i n e s t e r a s e i n r a b b i t m u s c l e sarcolemma have been r e p o r t e d by Ferdman e_t a _ l ( 1 3 4 ) . The a c t i v i t y of c y c l i c 3 ' , 5 '-nu c l e o-- 54 -t i d e phosphodiesterase was determined to be 0.3 nmoles of c y c l i c AMP hydrolyzed per min per mg i n a y i e l d of 1 to 37. of the a c t i v i t y i n the whole homogenate. C y c l i c 2',3,-AMP n u c l e o t i d e p h o s p h o d i e s t e r a s e , a p a r t i c u l a t e enzyme found p r i m a r i l y i n nerve t i s s u e (114), c a t a l y z e d the h y d r o l y s i s of 1.8 nmoles c y c l i c 2',3'-AMP per min per mg i n t h i s s k e l e t a l muscle membrane p r e p a r a t i o n . The f o l l o w i n g enzyme a c t i v i t i e s could not be detected i n the membrane f r a c t i o n : cytochrome c o x i d a s e , a c i d maltase, a c i d phosphatase or pyrophosphatase. The absence of these enzymes i n d i c a t e d minimal contamination of the plasma membrane f r a c t i o n by lysosomes or m i t o c h o n d r i a . I t has been suggested that a common p r o p e r t y of plasma membrane p r e p a r a t i o n s i s a high molar r a t i o of c h o l e s t e r o l to p h o s p h o l i p i d (135). The a n a l y s i s of three separate membrane p r e p a r a t i o n s f o r c h o l e s t e r o l , p h o s p h o l i p i d a n d s i a l i c a c i d i s shown i n Table V. The content of c h o l e s t e r o l and p h o s p h o l i p i d i s i n c l o s e agreement with the l i p i d d e t e r m i n a t i o n s of Fiehn e t a 1 (136). The molar r a t i o s of c h o l e s t e r o l to phospho-l i p i d range from 0.5 to 0.6 (Table V ) , which are c o n s i s t e n t with those of other plasma membranes (135). 3. Assay c o n d i t i o n s The c o n d i t i o n s under which enzyme a c t i v i t y c ould be r e l i a b l y determined had to be s t u d i e d p r i o r to the i n v e s t i g -a t i o n of the k i n e t i c p r o p e r t i e s of s k e l e t a l muscle adenyl c y c l a s e . The presence of ATPase a c t i v i t y i n the plasma mem-brane p r e p a r a t i o n r e s u l t e d i n the requirement f o r an ATP-- 55 -TABLE V C h e m i c a l c o m p o s i t i o n of s k e l e t a l m u s c l e p l a s m a membranes. The c h o l e s t e r o l , p h o s p h o l i p i d and s i a l i c a c i d c o n t e n t of t h r e e p l a s m a membrane p r e p a r a t i o n s were d e t e r m i n e d as d e s -c r i b e d i n m e t h o d s . M o l a r q u a n t i t i e s of c h o l e s t e r o l and p h o s p h o l i p i d were c a l c u l a t e d a s s u m i n g m o l e c u l a r w e i g h t s of 387 and 700 r e s p e c t i v e l y . Membrane P r e p a r -a t i o n C h o l e s t e r o l P hospho-1 i p i d S i a l i c a c i d M o l a r r a t i o nmoles/mg p r o t e i n nmole s/mg p r o t e i n nmole s/rag p r o t e i n c h o l e s t e r o l / p h o s p h o l i p i d A 270 490 5.8 .55 B 360 570 8.5 .63 C 390 730 9 . 1 .53 - 56 -r e g e n e r a t i n g s y s t e m t o p r e s e r v e t h e s u b s t r a t e f o r a d e n y l c y c l a s e . The e f f e c t i v e n e s s of the p h o s p h o e n o l p y r u v a t e -p y r u v a t e k i n a s e r e g e n e r a t i n g s y s t e m was examined by chroma-t o g r a p h i n g v a r i o u s i n c u b a t i o n m i x t u r e s i n an i s o b u t y r i c a c i d : 1 M NH 40H: 0.1 M EDTA ( 125: 75: 2) s o l v e n t s y s t e m . T h i s s o l v e n t s y s t e m s e p a r a t e s ADP fr o m ATP more e f f e c t i v e l y t h a n the 1 M ammonium a c e t a t e : 95% e t h a n o l s o l v e n t s y s t e m d e s c r i b e d i n a s s a y A and F i g . 1. In the a b s e n c e of the r e g e n e r a t i n g s y s t e m , t h e a d d i t i o n of 75 ug of membrane p r o t e i n r e s u l t e d i n the d e g r a d a t i o n o f 75% of the ATP, w i t h c o r r e s p o n d i n g i n c r e a s e s i n the amount of r a d i o a c t i v e ADP and 5'-AMP ( T a b l e V I ) . The a d d i t i o n of 8 mM f l u o r i d e d i d n o t a l t e r t h e p r e c e n t a g e of ATP d e g r a d e d but i t d i d d e c r e a s e the amount of 5'-AMP w i t h a c o r r e s p o n d i n g i n c r e a s e i n ADP. A d d i t i o n of the A T P - r e g e n e r a t i n g s y s t e m r e s u l t e d i n the r e c o v e r y of 90% of the ATP ( T a b l e V I ) . In a d d i t i o n t o p r e s e r v i n g the s u b s t r a t e f o r a d e n y l c y c l a s e , a s s a y c o n d i t i o n s must be c h o s e n t o a l l o w the c y c l i c AMP formed e n z y m a t i c a 1 l y t o be p r o t e c t e d f r o m d e g r a d a t i o n by the c y c l i c n u c l e o t i d e p h o s p h o d i e s t e r a s e . R e c o v e r y of c y c l i c AMP from r e a c t i o n m i x t u r e s was examined by the a d d i t i o n of 10,902 DPM of f_8- li*c]-eye 1 i c AMP to a s s a y t u b e s and c a r r y i n g o ut i n c u b a t i o n s i n the a b s e n c e of l a b e l l e d ATP and w i t h the a d d i t i o n s i n d i c a t e d i n T a b l e V I I , e x p e r i m e n t I . The r e c o v e r y of r a d i o a c t i v e c y c l i c AMP w i t h no a d d i t i o n s was 987.. The a d d i t i o n of 90 ug membrane p r o t e i n r e s u l t e d i n the d e g r a d a t i o n of 95% of the c y c l i c AMP; r a d i o a c t i v i t y on - 57 -TABLE VI E f f e c t i v e n e s s of t h e A T P - r e g e n e r a t i n g s y s t e m . f _ 1 4 c]-ATP (0.3 mM; 20 p C i p e r pmole) was i n c u b a t e d w i t h 40 mM T r i s , pH 7.5, 18 mM MgSO^ and the a d d i t i o n s as i n d i c a t e d f o r 20 min a t 3 0 ° i n a volume of 150 u l . I n c u b a t i o n m i x t u r e s were p l a c e d i n a b o i l i n g w a t e r b a t h f o r 3 m i n , a c i d i f i e d by the a d d i t i o n o f 30 u l of 127. t r i c h l o r o a c e t i c a c i d and c e n t r i -f u g e d . A l i q u o t s (100 p i ) of the s u p e r n a t a n t s were s t r e a k e d o v e r 2 cm on Whatman 3 MM p a p e r and d e s c e n d i n g p a p e r c h r o m a t o -grams were d e v e l o p e d f o r 18 hr w i t h i s o b u t y r i c a c i d : 1 M NH 40H: 0.1 M EDTA (125: 75: 2) as the d e v e l o p i n g s o l v e n t . A p p r o p r i a t e a r e a s of the chromatograms were c u t out and r a d i o -a c t i v e c o n t e n t was d e t e r m i n e d as d e s c r i b e d i n t h e m e t h o d s . Add i t i o n s D P M / i n c u b a t i o n ATP ADP AMP cAMP Adeno-s i n e None 1,456,834 49 ,438 5,977 2,615 7 ,264 Membrane p r o t e i n (75 pg) 345 ,457 771,472 321 ,267 4,053 12,254 Membrane p r o t e i n (75 pg) + 8 mM F~ 334,027 922,273 165,024 10,173 7,713 Membrane p r o t e i n (75 pg) + ATP r e g e n e r a t i n g s y s t e m 1 ,304,998 71,490 77,311 4 , 190 16 ,038 - 58 -TABLE V I I E f f e c t of u n l a b e l l e d c y c l i c AMP and t h e o p h y l l i n e on the d e g r a -d a t i o n and f o r m a t i o n of c y c l i c AMP. I n c u b a t i o n s c o n t a i n e d 40 mM T r i s , pH 8 . 5 , 8 mM f l u o r i d e , 18 mM MgSO and the p h o s p h o e n o l p y r u v a t e - p y r u v a t e k i n a s e 4 r e g e n e r a t i n g s y s t e m . R e c o v e r y of c y c l i c AMP was m o n i t o r e d by the a d d i t i o n , of 1 0 , 9 0 2 DPM of C 8 - 1 4 c ] - c y c l i c AMP w i t h o u t l a b e l l e d ATP ( e x p e r i m e n t I ) . The f o r m a t i o n of c y c l i c AMP was measured u s i n g 0 . 3 mM C 1 4 c j-ATP (20 u C i / u m o l e ) as s u b s t r a t e ( e x p e r i m e n t I I ) . In e a c h e x p e r i m e n t , membrane p r o -t e i n , u n l a b e l l e d c y c l i c AMP and t h e o p h y l l i n e was added as i n d i c a t e d . F o l l o w i n g i n c u b a t i o n s of 20 min, r e a c t i o n m i x t u r e s were b o i l e d , c e n t r i f u g e d and c h r o m a t o g r a p h e d as d e s c r i b e d i n a s s a y A. A r e a s c o r r e s p o n d i n g t o c y c l i c AMP were c u t out and the r a d i o a c t i v i t y d e t e r m i n e d as d e s c r i b e d i n methods. A d d i t i o n s E x p e r i m e n t I E x p e r i m e n t I I DPM (8-14 c ] - c y c l i c AMP r e c o v e r e d A d e n y l c y c l a s e a c t i v i t y pmole s/min/mg None 1 0 , 7 2 4 -Membrane p r o t e i n (92 ug) 395 29 8 Membrane p r o t e i n (92 ug) + 0.5 mM cAMP 1 0 , 2 6 6 708 Membrane p r o t e i n (92 ug) + 2 mM cAMP 1 0 , 4 7 5 664 Membrane p r o t e i n (92 pg) + 1 . 3 mM t h e o p h y l l i n e 4 , 7 0 3 467 Membrane p r o t e i n (92 pg) + 1 3 . 4 mM t h e o p h y l l i n e 9 , 4 5 5 387 - 59 -the c h r omatogram was r e c o v e r e d i n the a r e a s c o r r e s p o n d i n g t o 5'-AMP (4740 DPM) and a d e n o s i n e (6287 DPM). T h i s i n d i c a t e d the p r e s e n c e of t h e c y c l i c p h o s p h o d i e s t e r a s e m e n t i o n e d p r e v -i o u s l y as w e l l as 5 ' - n u c l e o t i d a s e a c t i v i t y w h i c h had not been o b s e r v e d p r e v i o u s l y u s i n g the l e s s s e n s i t i v e o p t i c a l a s s a y d e s c r i b e d i n the m e thods. R e c o v e r y of t h e l a b e l l e d c y c l i c AMP was i n c r e a s e d t o g r e a t e r t h a n 957. by the a d d i t i o n of u n l a b e l l e d c y c l i c AMP (0.5 and 2 mM) t o the i n c u b a t i o n m i x t u r e ( T a b l e V I I , e x p e r i m e n t I ) . The a d d i t i o n of t h e o p h y l l i n e (1.3 and 13,4 mM) a l s o d e c r e a s e d the d e g r a d a t i o n of l a b e l l e d c y c l i c AMP, but was l e s s e f f e c t i v e t h a n the a d d i t i o n of the u n l a b e l -l e d c y c l i c n u c l e o t i d e . The a d d i t i o n of u n l a b e l l e d c y c l i c AMP t o t h e i n c u b a t i o n m i x t u r e a l s o g r e a t l y i n c r e a s e d the s y n t h e s i s of r a d i o a c t i v e c y c l i c AMP f r o m [ [ l 4 c ] - A T P ( T a b l e V I I , e x p e r i m e n t I I ) . In c o n t r a s t , t h e a d d i t i o n o f t h e o p h y l l i n e d e c r e a s e d a d e n y l c y c l a s e a c t i v i t y i n e x p e r i m e n t I I . In s e p a r a t e e x p e r i m e n t s , i t was shown t h a t the a d d i t i o n of v a r y i n g amounts of t h e o p h y -l l i n e i n the p r e s e n c e of 2 mM c y c l i c AMP r e s u l t e d i n a c o n c e n t r a t i o n - d e p e n d e n t d e c r e a s e i n f l u o r i d e - s t i m u l a t e d a d e n y l c y c l a s e a c t i v i t y , w i t h 507. i n h i b i t i o n o c c u r r i n g at 20 mM t h e o p h y l l i n e . I n h i b i t i o n of a d e n y l c y c l a s e a c t i v i t y by t h e o -p h y l l i n e has a l s o been o b s e r v e d i n the t o a d b l a d d e r ( 2 9 ) , and mouse l i v e r ( 4 3 ) . In the s t a n d a r d a s s a y , the l a b e l l e d c y c l i c AMP f o r m e d was p r o t e c t e d a g a i n s t d e s t r u c t i o n by the p r e s e n c e of 2 mM u n l a b e l l e d c y c l i c n u c l e o t i d e . Under the c o n d i t i o n s f i n a l l y a d o p t e d , a d e n y l c y c l a s e - 60 -F i g u r e 6: Dependence of s k e l e t a l muscle adenyl c y c l a s e a c t i v i t y on time and p r o t e i n . Panel A: a c t i v i t y i n a membrane pr e p a r a -t i o n (108 pg p r o t e i n ) was measured by assay A at the times i n d i c a t e d i n the absence or i n the presence of 8 mM f l u o r i d e . Panel B: c o n d i t i o n s were the same as i n panel A except that the amount of p r o t e i n was v a r i e d and the i n c u b a t i o n time was 20 rain. Time (min) Protein(^xg) F i g u r e 6 - 62 -a c t i v i t y was l i n e a r w i t h time and p r o t e i n when measured i n t h e a b s e n c e and p r e s e n c e of 8 mM f l u o r i d e ( F i g . 6 ) . The s t a n d a r d a s s a y employed i n c u b a t i o n t i m e s of 20 min; p r o t e i n c o n t e n t r a n g e d f r o m 100 to 300 p g . The pH d e pendence was e x a m i n e d f r o m pH 6 to pH 10.5 u s i n g c o m b i n a t i o n s of p - g l y c e r o l -p h o s p h a t e , T r i s - H C l and 2-amino-2-methy 1-1 , 3 - p r o p a n e d i o 1 ( e a c h a t 40 mM) as b u f f e r . The pH optimum b o t h i n the a b s e n c e and p r e s e n c e of f l u o r i d e was 8.5. T h i s pH optimum i s s l i g h t l y more a l k a l i n e t h a n t h a t f o u n d f o r c a r d i a c t i s s u e (28) and most o t h e r mammalian t i s s u e s ( 1 8 ) . 4. K i n e t i c p r o p e r t i e s of s k e l e t a l m u s c l e a d e n y l c y c l a s e The p r e v i o u s r e s u l t s have d e s c r i b e d the i s o l a t i o n of r a b b i t s k e l e t a l m u s c l e p l a s m a membranes c o n t a i n i n g a good y i e l d of a d e n y l c y c l a s e , and the e x p e r i m e n t a l c o n d i t i o n s f o r the r e l i a b l e a s s a y of enzyme a c t i v i t y . The d e t e r m i n a t i o n of the mechanisms by w h i c h hormones s t i m u l a t e c y c l i c AMP f o r m a t i o n r e q u i r e s the knowledge of the k i n e t i c p r o p e r t i e s of a d e n y l c y c l a s e . The f o l l o w i n g r e s u l t s d e s c r i b e the k i n e t i c p r o p e r t i e s of the enzyme and the k i n e t i c p a r a m e t e r s i n f l u e n c e d by hormones and f l u o r i d e . E f f e c t of m e t a l i o n s on a d e n y l c y c l a s e i n s k e l e t a l m u s c l e — In a greement w i t h e a r l i e r s t u d i e s on a d e n y l c y c l a s e i n c a r d i a c t i s s u e ( 2 8 , 1 0 8 ) , m e t a l i o n s s t i m u l a t e d the s k e l e t a l m u s c l e enzyme at c o n c e n t r a t i o n s i n e x c e s s of ATP. The e f f e c t of magnesium, manganese and c o b a l t i n the p r e s e n c e of 0.3 mM ATP i s shown i n F i g . 7. The K g f o r magnesium was f r o m 3 to - 6 3 -F i g u r e 7: E f f e c t o f m e t a l i o n s o n s k e l e t a l m u s c l e a d e n y l c y c l a s e . A c t i v i t y w a s d e t e r m i n e d i n a m e m b r a n e p r e p a r a t i o n ( 9 1 p g p r o t e i n ) b y a s s a y A i n t h e p r e s e n c e o f t h e i n d i c a t e d m e t a l i o n c o n c e n t r a t i o n s . - 64 -F i g u r e 7 - 65 -5 mM; manganese and c o b a l t s t i m u l a t e d the enzyme a t lo w e r c o n c e n t r a t i o n s ( K . 1 t o 2 mM) and were i n h i b i t o r y a t c o n c e n -t r a t i o n s g r e a t e r t h a n 5 mM. I t i s a p p a r e n t t h a t the s k e l e t a l m u s c l e enzyme has the a b i l i t y t o b i n d m e t a l i o n s i n e x c e s s of t h a t r e q u i r e d at the c a t a l y t i c s i t e . The a d d i t i o n of c a l c i u m r e s u l t e d i n an i n h i b i t i o n of enzyme a c t i v i t y . H a l f - m a x i m a l i n h i b i t i o n i n the p r e s e n c e of 18 mM magnesium o c c u r r e d a t 0.5 mM c a l c i u m f o r b o t h b a s a l and e p i n e p h r i n e -s t i m u l a t e d a d e n y l c y c l a s e a c t i v i t y . The k i n e t i c n a t u r e of the i n h i b i t i o n by c a l c i u m was n o t d e t e r m i n e d due to the i n a b i l i t y of the A T P - r e g e n e r a t i n g s y s t e m t o m a i n t a i n s u b -s t r a t e l e v e l s when c o n c e n t r a t i o n s of magnesium were d e c r e a s e d i n the p r e s e n c e of c a l c i u m . T h i s c o m p l i c a t i o n i s due t o the p r e s e n c e of c a l c i u m - s t i m u l a t e d ATPase a c t i v i t y d i s c u s s e d p r e v i o u s l y and the i n h i b i t i o n of p y r u v a t e k i n a s e by c a l c i u m ( 1 3 7 ) . E f f e c t o f ATP and magnesium on s k e l e t a l m u s c l e a d e n y l  eye 1ase — The e f f e c t of v a r y i n g c o n c e n t r a t i o n s of ATP i n the p r e s e n c e of t h r e e f i x e d magnesium c o n c e n t r a t i o n s i s shown i n F i g . 8. I n c r e a s i n g the magnesium c o n c e n t r a t i o n i n c r e a s e d the r e a c t i o n v e l o c i t y at a l l c o n c e n t r a t i o n s of ATP. The K^ , f o r ATP can be e s t i m a t e d t o be a p p r o x i m a t e l y 0.3 mM i n the p r e s e n c e of e x c e s s magnesium. The d a t a i n F i g . 8 a l s o i n d i c a t e s t h a t i n c r e a s i n g ATP c o n c e n t r a t i o n s r e s u l t e d i n an i n h i b i t i o n of a d e n y l c y c l a s e a c t i v i t y s i m i l a r to t h a t o b s e r v e d w i t h the c a r d i a c enzyme ( F i g . 3 and r e f e r e n c e 1 0 8 ) , and t h a t t h i s i n h i b i t i o n c o u l d be overcome by i n c r e a s i n g the magnesium c o n -- 66 -F i g u r e 8: E f f e c t of ATP c o n c e n t r a t i o n on s k e l e t a l m u s c l e a d e n y l c y c l a s e . A c t i v i t y i n a membrane p r e p a r a t i o n (225 pg p r o t e i n ) was measured by a s s a y B i n the p r e s e n c e of t h r e e f i x e d magnesium c o n c e n t r a t i o n s . C o n c e n t r a t i o n s of magnesium (mM) a r e g i v e n by the numbers on e a c h c u r v e . - 67 -ATP (mM) F i g u r e 8 - 68 -c e n t r a t i o n . S t i m u l a t i o n of s k e l e t a l muscle adenyl c y c l a s e by f l u o r i d e - - T h e f l u o r i d e anion s t i m u l a t e s adenyl c y c l a s e from most mammalian sources (18) but the mechanism i n v o l v e d i n t h i s s t i m u l a t i o n i s unknown. S t i m u l a t i o n by f l u o r i d e i n v a r i o u s s k e l e t a l muscle membrane p r e p a r a t i o n s was observed to vary from 10 to 2 0 - f o l d . The e f f e c t of v a r i o u s concen-t r a t i o n s of f l u o r i d e on adenyl c y c l a s e a c t i v i t y at two temperatures i s shown i n F i g . 9, panel A. Maximal a c t i v i t y was achieved at 12 mM f l u o r i d e . Temperature had a much gr e a t e r e f f e c t on f l u o r i d e - s t i m u l a t e d a c t i v i t y than on b a s a l a c t i v i t y . The e f f e c t of v a r y i n g the temperature from 12° to 43° on b a s a l and f l u o r i d e - s t i m u l a t e d a c t i v i t y i s presented i n F i g . 9, panel B i n the form of an A r r h e n i u s p l o t . There i s a l i n e a r r e l a t i o n s h i p between a c t i v i t y and temperature except at 43° where presumably i n a c t i v a t i o n occurs due to d e n a t u r a t i o n . Energies of a c t i v a t i o n c a l c u l a t e d from the slopes are 7.8 k c a l per mole f o r b a s a l a c t i v i t y and 17.4 k c a l per mole f o r f l u o r i d e - s t i m u l a t e d a c t i v i t y . The s t i m u l a t i o n by f l u o r i d e of adenyl c y c l a s e from b r a i n (138), p a r o t i d gland (139) and adrenal (140,141) has been shown to be i r r e v e r s i b l e . F o l l o w i n g exposure of these enzyme p r e p a r a t i o n s to f l u o r i d e , enzyme a c t i v i t y remained at the s t i m u l a t e d value d e s p i t e d e c r e a s i n g the c o n c e n t r a t i o n of f l u o r i d e by d i l u t i o n , washing or d i a l y s i s . The e f f e c t of p r e i n c u b a t i o n of s k e l e t a l muscle plasma membranes with raagne-- 69 -F i g u r e 9: E f f e c t o f f l u o r i d e a n d t e m p e r a t u r e o n s k e l e t a l m u s c l e a d e n y l c y c l a s e . P a n e l A: e n z y m e a c t i v i t y w a s m e a s u r e d b y a s s a y A i n a m e m b r a n e p r e p a r a t i o n ( 9 5 u g p r o t e i n ) i n c u b a t e d w i t h 6 mM m a g n e s i u m a n d v a r y i n g f l u o r i d e c o n c e n t r a t i o n s a t 27° a n d 3 7 ° . P a n e l B: e f f e c t o f t e m p e r a t u r e ( 1 2 ° , 1 6 ° , 2 3 ° , 3 0 ° 3 8° a n d 4 3 ° ) o n e n z y m e a c t i v i t y i n a m e m b r a n e p r e p a r a t i o n ( 1 8 3 p g p r o t e i n ) d e t e r m i n e d b y a s s a y A i n t h e p r e s e n c e o f 6 mM m a g n e s i u m a n d i n t h e a b s e n c e (0~0) o r i n t h e p r e s e n c e ( A - A ) o f 12 mM f l u o r i d e . - oi -- 71 -TABLE V I I I I r r e v e r s i b i l i t y of f l u o r i d e a c t i v a t i o n of s k e l e t a l muscle adenyl c y c l a s e . A membrane p r e p a r a t i o n was pr e i n c u b a t e d under the c o n d i t i o n s i n d i c a t e d f o r 30 min at 4 ° . Samples (1.0 ml) were d i a l y z e d at 4° ag a i n s t 100 ml of 10 mM T r i s , pH 8 c o n t a i n i n g 2 mM mercaptoethanol f o r 4 hr with two b u f f e r changes. F o l l o w i n g d i a l y s i s , adenyl c y c l a s e a c t i v i t y was determined by assay A. Incubation mixtures c o n t a i n e d 9 mM MgS04 and 150 pg membrane p r o t e i n ; 12 mM f l u o r i d e was presen t as i n d i c a t e d . P r e i n c u b a t i o n a d d i t ions Assay A d d i t i o n , (12 mM F") Adenyl c y c l a s e a c t i v i t y pmoles/min/mg None - 34 + 636 9 mM Mg** - 28 9 mM Mg"*"*" + 12 mM F~ - 245 12 mM F" - 253 - 72 -s i u r a , w i t h f l u o r i d e , or w i t h magnesium p l u s f l u o r i d e , f o l l o w e d by d i a l y s i s i s shown i n T a b l e V I I I . The enzyme a c t i v i t y of t h i s membrane p r e p a r a t i o n p r i o r t o any p r e i n c u b a t i o n or d i a l y s i s was 678 pmoles p e r min p e r mg when a s s a y e d i n the p r e s e n c e of 12 mM f l u o r i d e . D i a l y s i s of c o n t r o l s a m p l e s p r e -i n c u b a t e d w i t h no a d d i t i o n s i n d i c a t e d the enzyme p r e p a r a t i o n d i d n o t l o s e a c t i v i t y d u r i n g t h i s p r o c e d u r e . A l l d i a l y z e d s a m p l e s were a s s a y e d i n the p r e s e n c e of 9 mM magnesium; sa m p l e s p r e i n c u b a t e d w i t h magnesium f o l l o w e d by d i a l y s i s were i n a c t i v e u n l e s s the c a t i o n was added t o the a s s a y , i n d i c a t i n g the com-p l e t e r e v e r s i b i l i t y of magnesium s t i m u l a t i o n . Membrane p r e p -a r a t i o n s t h a t were p r e i n c u b a t e d w i t h f l u o r i d e and d i a l y z e d s t i l l e x h i b i t e d an 8 - f o l d a c t i v a t i o n , c o r r e s p o n d i n g t o a p p r o x i m a t e l y 407. of t h e f l u o r i d e - s t i m u l a t e d c o n t r o l . In c o n -t r a s t t o r e s u l t s o b t a i n e d w i t h the r a t p a r o t i d g l a n d ( 1 3 9 ) , the a b s e n c e or p r e s e n c e of magnesium i n the p r e i n c u b a t i o n m i x t u r e had no e f f e c t on the i r r e v e r s i b i l i t y o f the f l u o r i d e -s t i m u l a t i o n ( T a b l e V I I I ) . The c h e m i c a l a n a l y s i s f o r f l u o r i d e w i t h the Amadac -F r e a g e n t i n d i c a t e d t h a t g r e a t e r t h a n 957. o f the f l u o r i d e had been removed by d i a l y s i s . E q u i v a l e n t r e s u l t s were o b t a i n e d when p r e i n c u b a t i o n s were c a r r i e d out f o r 10 min a t 25° . These r e s u l t s i n d i c a t e t h a t the f l u o r i d e s t i m u l a t i o n of s k e l e t a l m u scle p l a s m a membranes i s a t l e a s t p a r t i a l l y i r r e v e r s i b l e . S t i m u l a t i o n of s k e l e t a l m u s c l e a d e n y l c y c l a s e by h o r m o n e s — The s t i m u l a t i o n by c a t e c h o l a m i n e s of the f o r m a t i o n of c y c l i c AMP i s r e c o g n i z e d as an i m p o r t a n t f e a t u r e of the r e g u l a t i o n of g l y c o g e n m e t a b o l i s m i n s k e l e t a l m u s c l e . C a t e c h o l a m i n e s - 73 -a c t i v a t e d a d e n y l c y c l a s e by 2 t o 4 - f o l d i n s k e l e t a l m u s c l e membrane p r e p a r a t i o n s . D o s e - r e s p o n s e c u r v e s to i s o p r o p y l -n o r e p i n e p h r i n e , e p i n e p h r i n e and n o r e p i n e p h r i n e are shown i n F i g . 10. The c o n c e n t r a t i o n s r e q u i r e d f o r h a l f - m a x i m a l a c t i v i t y were 0.15, 0.5 and 10 pM, r e s p e c t i v e l y . P h e n y l -e p h r i n e (0.1 mM) a c t i v a t e d a d e n y l c y c l a s e by 2 . 1 - f o l d u n d e r c o n d i t i o n s i n w h i c h e p i n e p h r i n e (0.1 mM) a c t i v a t e d the same enzyme p r e p a r a t i o n by 3 . 4 - f o l d . E p h e d r i n e (0.1 mM) was i n a c t i v e . The a c t i v a t i o n due to 0.1 mM e p i n e p h r i n e was b l o c k e d by p r o p r a n o l o l (0.1 mM); p r o p r a n o l o l i t s e l f had no e f f e c t on b a s a l a c t i v i t y . C a r b a c h o l (0.1 mM) had no e f f e c t on b a s a l o r e p i n e p h r i n e - s t i m u l a t e d a d e n y l c y c l a s e . S i m i l a r l y , p r o s t a g l a n d i n s E ^ , E 2 , Ficc> F 2cc> a n d A, 2 i n c o n c e n t r a t i o n s of 0.01 mM had no e f f e c t on b a s a l enzyme a c t i v i t y . P r e i n c u b a t i o n of the membrane p r e p a r a t i o n f o r v a r i o u s t i m e s (10 t o 30 min) w i t h v a r y i n g c o n c e n t r a t i o n s of i n s u l i n (1 m i l l i u n i t p e r ml to 10 u n i t s p e r ml) d i d n o t a l t e r e i t h e r b a s a l or e p i n e p h r i n e -s t i m u l a t e d a d e n y l c y c l a s e a c t i v i t y . The e f f e c t s of e p i n e p h r i n e i n the p r e s e n c e of f l u o r i d e a r e shown i n T a b l e IX. The s t i m u l a t i o n by a maximal c o n c e n -t r a t i o n of f l u o r i d e i s much g r e a t e r t h a n w i t h a maximal c o n c e n t r a t i o n of e p i n e p h r i n e ( T a b l e IX, e x p e r i m e n t I ) . C o m b i n a t i o n of f l u o r i d e and e p i n e p h r i n e d i d not i n c r e a s e the s t i m u l a t i o n t o g r e a t e r t h a n t h a t a c h i e v e d by f l u o r i d e a l o n e . T h i s s u g g e s t s t h a t e p i n e p h r i n e and f l u o r i d e a c t on the same a d e n y l c y c l a s e enzyme i n s k e l e t a l m u s c l e p l a s m a membranes, - 74 -F i g u r e 10: E f f e c t of c a t e c h o l a m i n e s on s k e l e t a l m u s c l e a d e n y l c y c l a s e . Enzyme a c t i v i t y was d e t e r m i n e d by a s s a y A i n a mem-br a n e p r e p a r a t i o n (390 pg p r o t e i n ) i n c u b a t e d w i t h 6 mM magnesium and i s op r op y l n o r e p i n e p hr i n e ( 0 ~ 0 ^ » e p i n e p h r i n e ( A _ A ) o r n o r e p i n e p h r i n e ( Q - Q ) a t the c o n c e n t r a t i o n s i n d i c a t e d . - 75 -F i g u r e 10 - 76 -TABLE IX A d d i t i v i t y of epinephrine and f l u o r i d e a c t i v a t i o n . Adenyl c y c l a s e a c t i v i t y was measured by assay A i n the presence of 6 mM magnesium with the i n d i c a t e d a d d i t i o n s . A d i f f e r e n t membrane p r e p a r a t i o n was used i n each experiment. Experiment I, 118 pg membrane p r o t e i n ; experiment I I , 189 pg membrane p r o t e i n . Addi t ions Adenyl c y c l a s e a c t i v i t y pmoles/min/mg Experiment I: None 69.7 12 mM F" 1191.5 0.01 mM e p i 440.1 12 mM F~ and 0.01 mM e p i 1160.9 Experiment I I : None 55.0 1.2 mM F~ 100.1 0.01 mM e p i 208. 2 1.2 mM F~ and 0.01 mM e p i 256 .1 - 77 -w i t h f l u o r i d e r e s u l t i n g i n maximal c a t a l y t i c r e a c t i v i t y . However, c o m b i n a t i o n of submaximal c o n c e n t r a t i o n s of f l u o r i d e w i t h maximal e p i n e p h r i n e c o n c e n t r a t i o n s d i d p r o d u c e an a d d i t i v e s t i m u l a t i o n ( T a b l e IX, e x p e r i m e n t I I ) . The l a c k of any com-p e t i t i o n between maximal c o n c e n t r a t i o n s i n e x p e r i m e n t I and the a d d i t i v i t y of s t i m u l a t i o n by sub-maximal c o n c e n t r a t i o n s of f l u o r i d e and maximal c o n c e n t r a t i o n s of e p i n e p h r i n e s u g g e s t t h a t f l u o r i d e i o n and e p i n e p h r i n e a c t i v a t e a d e n y l c y c l a s e by d i f f e r e n t m e c h a n i s m s . K i n e t i c n a t u r e of e p i n e p h r i n e and f l u o r i d e s t i m u l a t i o n  of s k e l e t a l m u s c l e a d e n y l c y c l a s e — P r e v i o u s i n v e s t i g a t i o n s w i t h the c a r d i a c enzyme by Drummond and h i s c o - w o r k e r s (28, 108) r e v e a l e d t h a t b o t h f l u o r i d e and e p i n e p h r i n e i n c r e a s e d maximal v e l o c i t y ; a f f i n i t i e s f o r magnesium or s u b s t r a t e were n o t a p p r e c i a b l y a l t e r e d . The a c t i o n of f l u o r i d e (4 mM and 12 mM) and e p i n e p h r i n e (0.1 mM) on the a c t i v a t i o n of s k e l e t a l m u s c l e a d e n y l c y c l a s e by magnesium i s shown i n F i g . 11. B o t h f l u o r i d e ( p a n e l A) and e p i n e p h r i n e ( p a n e l B) i n c r e a s e d the r e a c t i o n v e l o c i t y a t a l l c o n c e n t r a t i o n s of magnesium. The p r e d o m i n a n t k i n e t i c e f f e c t was t o i n c r e a s e the maximal v e l o c i t y w i t h o u t s i g n i f i c a n t l y a l t e r i n g the K a f o r magnesium. The e f f e c t of f l u o r i d e (12 mM) and e p i n e p h r i n e (0.01 mM) on ATP s a t u r a t i o n i n the p r e s e n c e of two f i x e d magnesium c o n c e n t r a t i o n s i s shown i n F i g . 12. Once a g a i n , b o t h f l u o r i d e ( p a n e l A) and e p i n e p h r i n e ( p a n e l B) s t i m u l a t e d the enzyme a c t i v i t y a t a l l ATP c o n c e n t r a t i o n s . In the p r e s e n c e of 2 mM - 78 -F i g u r e 11: E f f e c t of f l u o r i d e and e p i n e p h r i n e on magnesium a c t i v a t i o n of s k e l e t a l muscle adenyl c y c l a s e . A c t i v i t y was determined by assay A i n a membrane p r e p a r a t i o n (100 ug p r o t e i n ) at v a r y i n g c o n c e n t r a t i o n s of magnesium. Panel A: e f f e c t of magnesium c o n c e n t r a t i o n at two f i x e d f l u o r i d e c o n c e n t r a t i o n s . F l u o r i d e c o n c e n t r a t i o n s (mM) are given by the numbers on each cu r v e . Panel B: e f f e c t of magnesium c o n c e n t r a t i o n i n the absence ( Q - O ) a n d presence ( A - A ) of 0.1 mM e p i n e p h r i n e . 5 10 15 5 10 15 MgS0 4 (mM) F i g u r e 11 - 80 -Fi g u r e 12: E f f e c t of f l u o r i d e and epi n e p h r i n e on the ATP s a t u r a t i o n of s k e l e t a l muscle adenyl c y c l a s e . Enzyme a c t i v i t y i n a membrane p r e p a r a t i o n (250 ug p r o t e i n ) was measured by assay B with v a r y i n g ATP c o n c e n t r a t i o n s at two f i x e d magnesium concen-t r a t i o n s . Magnesium c o n c e n t r a t i o n s (mM) are given by the numbers on each c u r v e . The same membrane p r e p a r a t i o n was used i n panels A and B. Panel A: a c t i v i t y measured i n the absence ( -} or i n the presence ( ) of 12 mM f l u o r i d e . Panel B: assay was performed i n the absence t\ •) or i n the presence ( ) of 0.01 mM e p i n e p h r i n e . F i g u r e 12 - 82 -magnesium, i n c r e a s i n g ATP c o n c e n t r a t i o n s became i n h i b i t o r y . The i n h i b i t i o n by ATP was n o t r e v e r s e d by f l u o r i d e or e p i n e -p h r i n e ; as p r e v i o u s l y shown i n F i g , 8, i n c r e a s i n g the magne-si u m c o n c e n t r a t i o n overcame the i n h i b i t i o n . The a c t i o n of b o t h f l u o r i d e and e p i n e p h r i n e was t o i n c r e a s e m aximal v e l o c i t y ; the a f f i n i t y of the enzyme f o r ATP was n o t s u b s t a n t i a l l y a l t e r e d . Thus the k i n e t i c n a t u r e o f the s t i m u l a t i o n of s k e l e t a l m u s c l e a d e n y l c y c l a s e by f l u o r i d e and e p i n e p h r i n e i s v e r y s i m i l a r t o t h a t f o r m y o c a r d i a l a d e n y l c y c l a s e . E f f e c t of P y r o p h o s p h a t e on s k e l e t a l m u s c l e a d e n y l c y c l a s e — P y r o p h o s p h a t e i s fo r m e d s t o i c h i o m e t r i c a 1 l y w i t h c y c l i c AMP d u r i n g the a d e n y l c y c l a s e r e a c t i o n ( 1 9 , 1 4 2 ) . The a d d i t i o n of c o n c e n t r a t i o n s of p y r o p h o s p h a t e f r o m 0.1 mM t o 2 mM t o the a s s a y r e s u l t e d i n an i n h i b i t i o n of b a s a l , e p i n e p h r i n e - s t i m u l -a t e d and f l u o r i d e - s t i m u l a t e d a d e n y l c y c l a s e a c t i v i t y ( F i g . 1 3 ) . The f l u o r i d e - s t i m u l a t e d a c t i v i t y was the most s e n s i t i v e t o p y r o p h o s p h a t e i n h i b i t i o n ; p y r o p h o s p h a t e i n h i b i t i o n was o b s e r v e d w i t h f l u o r i d e c o n c e n t r a t i o n s v a r y i n g f r o m 2 mM t o 16 mM. P y r o p h o s p h a t e (2 mM) a l s o i n h i b i t e d the a d e n y l c y c l a s e f r o m E s c h e r i c h i a c o l i (143) and the f l u o r i d e - s t i m u l a t e d enzyme a c t i v i t y i n l i v e r has been shown t o be more s e n s i t i v e t o p y r o p h o s p h a t e i n h i b i t i o n t h a n the g l u c a g o n - s t i m u l a t e d a c t i v i t y ( 4 3 , 1 4 4 ) . The k i n e t i c n a t u r e of the p y r o p h o s p h a t e i n h i b i t i o n of s k e l e t a l m u s c l e a d e n y l c y c l a s e was d e t e r m i n e d . The e f f e c t of two c o n c e n t r a t i o n s of p y r o p h o s p h a t e (0.2 mM and 1.0 mM) on ATP s a t u r a t i o n i n the p r e s e n c e of f l u o r i d e i s - 83 -F i g u r e 13: E f f e c t o f p y r o p h o s p h a t e on s k e l e t a l m u s c l e a d e n y l c y c l a s e . A c t i v i t y i n a membrane p r e p a r a t i o n ( 350 pg p r o t e i n ) was measured by a s s a y A i n the p r e s e n c e of 6 mM magnesium and v a r y i n g c o n c e n t r a t i o n s of p y r o p h o s p h a t e w i t h no f u r t h e r a d d i t i o n s ( 0 ~ 0 ^ » w i t h 0 . 0 1 mM e p i n e p h r i n e ( A ~ A ) » o r w i t h 12 mM f l u o r i d e ( Q - Q ) . - 84 -F i g u r e 13 - 85 -F i g u r e 14: E f f e c t of pyrophosphate on ATP s a t u r a t i o n of s k e l e t a l muscle adenyl c y c l a s e . Enzyme a c t i v i t y was measured by assay B with a membrane p r e p a r a t i o n (290 pg p r o t e i n ) incubated with 9 mM magnesium, 12 mM f l u o r i d e and two f i x e d c o n c e n t r a t i o n s of pyrophosphate. C o n c e n t r a t i o n s of pyro-phosphate (mM) are i n d i c a t e d by the numbers on each c u r v e . - 98 -- 87 -shown i n F i g . 14. From the d o u b l e r e c i p r o c a l p l o t s i t i s a p p a r e n t t h a t the i n h i b i t i o n by p y r o p h o s p h a t e i s c o m p e t i t i v e w i t h r e s p e c t to ATP w i t h a of 0.45 mM. E f f e c t of n u c l e o s i d e t r i p h o s p h a t e s on s k e l e t a l m u s c l e a d e n y l e y e l a s e — N u c l e o s i d e t r i p h o s p h a t e s o t h e r t h a n ATP c o u l d p o s s i b l y s e r v e as s u b s t r a t e s f o r a d e n y l c y c l a s e . The a b i l i t y of o t h e r n u c l e o s i d e t r i p h o s p h a t e s t o b i n d t o a d e n y l c y c l a s e i n s k e l e t a l m u s c l e membranes was examined by o b s e r v i n g the e f f e c t s of c o n c e n t r a t i o n s of GTP, ITP, UTP and CTP e q u i -m o l a r w i t h ATP c o n c e n t r a t i o n on b a s a l enzyme a c t i v i t y , UTP and CTP had no e f f e c t , b u t s u r p r i s i n g l y b o t h ITP and GTP i n c r e a s e d a d e n y l c y c l a s e a c t i v i t y . The e f f e c t of 0.1 mM ITP and 0.1 mM GTP on b a s a l , e p i n e p h r i n e - s t i m u 1 a t e d , and f l u o r i d e - s t i m u l a t e d enzyme a c t i v i t y i s shown i n T a b l e X. B a s a l a c t i v i t y was i n c r e a s e d f r o m 30 t o 50%, by GTP and I T P , however e p i n e p h r i n e - s t i m u l a t e d a c t i v i t y was i n c r e a s e d by 2.5-f o l d t o an o v e r a l l s t i m u l a t i o n of 5 to 6 - f o l d . In c o n t r a s t , GTP and ITP had no e f f e c t on f l u o r i d e - s t i m u l a t e d a c t i v i t y . The a b i l i t y of a v a r i e t y of n u c l e o t i d e s to enhance e p i n e p h r i n e -s t i m u l a t e d a d e n y l c y c l a s e a c t i v i t y i s shown i n T a b l e X I . CTP, UTP and v a r i o u s a d e n i n e n u c l e o t i d e s and ATP a n a l o g s a p p e a r e d t o have no a b i l i t y t o i n c r e a s e the s t i m u l a t i o n by e p i n e p h r i n e . ITP was as e f f e c t i v e as GTP at c o n c e n t r a t i o n s of 0.1 mM but no t a t 1 uM c o n c e n t r a t i o n s . GDP was a l s o e f f e c t i v e a t 1 pM c o n c e n t r a t i o n s , but 1 pM GMP had no e f f e c t . The e f f e c t of v a r i o u s c o n c e n t r a t i o n s of GTP on b a s a l , e p i n e p h r i n e - s t i m u l a t e d ) and f l u o r i d e - s t i m u l a t e d a d e n y l c y c l a s e and of GDP on e p i n e p h r i n e - 88 -TABLE X E f f e c t of GTP and ITP on s k e l e t a l muscle adenyl c y c l a s e . Adenyl c y c l a s e a c t i v i t y of a s k e l e t a l muscle plasma membrane p r e p a r a t i o n (205 pg p r o t e i n ) was determined by assay A i n the presence of 9 mM magnesium with the a d d i t i o n s i n d i -c a t e d . Epinephrine and f l u o r i d e were present i n c o n c e n t r a t i o n s of 0.01 mM and 12 mM r e s p e c t i v e l y . A d d i t i o n s Adenyl c y c l a s e a c t i v i t y B a s a l E p i F " pmoles/min/mg None 36.3 107.8 568.9 0.1 mM ITP 53 .9 261.1 592.1 0.1 mM GTP 43.0 270.4 543 .0 - 89 -TABLE X I E f f e c t o f n u c l e o t i d e s on e p i n e p h r i n e - s t i m u 1 a t e d a d e n y l c y c l A s k e l e t a l m u s c l e membrane p r e p a r a t i o n (77 pg) was a s s a y e d f o r e p i n e p h r i n e - s t i m u l a t e d a d e n y l c y c l a s e a c t i v i t y w i t h t h e a d d i t i o n s i n d i c a t e d by a s s a y A and w i t h 9 mM m a g n e s i u m . The c o n c e n t r a t i o n o f e p i n e p h r i n e was 0.01 mM. A d d i t i o n s A d e n y l c y c l a s e a c t i v i t y praole s/min/mg None 148.9 0.1 mM 0(, 6 -me t h y l e n e - A T P 110.2 0.1 mM B,X - m e t h y l e n e - A T P 116.6 0.1 mM ADP 112.7 0.1 mM AMP 98.5 0.1 mM GTP 301.7 0.001 mM GTP 24 2. 7 0.001 mM GDP 315.2 0.001 mM GMP 137.0 0.1 mM ITP 33 8 .0 0.001 mM ITP 130.5 0.1 mM CTP 115.9 0.1 mM UTP 118.1 - 90 -s t i m u l a t e d enzyme a c t i v i t y i s shown i n F i g . 15. GTP had o n l y a s l i g h t e f f e c t on b a s a l a c t i v i t y and no s i g n i f i c a n t e f f e c t on f l u o r i d e - s t i m u l a t e d a c t i v i t y . B o th GDP and GTP i n c r e a s e d e p i n e p h r i n e - s t i m u l a t e d a c t i v i t y by t w o - f o l d ( f r o m 2 . 5 - f o l d s t i m u l a t i o n t o 5 - f o l d ) . Half-maximal s t i m u l a t i o n by g u a n y l n u c l e o t i d e s o c c u r r e d at 0.5 t o 1 uM c o n c e n t r a t i o n s . S i n c e t h e s e e f f e c t s of GDP were o b s e r v e d i n r e a c t i o n s c o n t a i n i n g t h e A T P - r e g e n e r a t i n g s y s t e m , the p o s s i b i l i t y must be c o n s i d e r e d t h a t GDP i s e f f e c t i v e o n l y a f t e r c o n v e r s i o n t o GTP. However, s i n c e GDP i s even more e f f e c t i v e t h a n GTP and s i n c e the Km of GDP f o r p y r u v a t e k i n a s e i s a t h o u s a n d t i m e s g r e a t e r t h a n t h e c o n c e n t r a t i o n of GDP r e q u i r e d f o r h a l f - m a x i m a l enhancement of e p i n e p h r i n e s t i m u l a t i o n , i t i s l i k e l y t h a t GDP a c t s d i r e c t l y on the enzyme. S i m i l a r e f f e c t s o f GTP and GDP on b a s a l and g l u c a g o n - s t i m u l a t e d a d e n y l c y c l a s e i n l i v e r p l a s m a membranes were r e p o r t e d by R o d b e l l et_ a_l_ ( 1 4 5 ) . GDP was e f f e c t i v e i n the a b s e n c e of any A T P - r e g e n e r a t i n g s y s t e m ( 1 4 5 ) . However, i n c o n t r a s t t o the r e s u l t s i n F i g . 15, GTP or the B , X - m e t h y l e n e p h o s p h o n a t e a n a l o g of GTP r e s u l t e d i n a 30 t o 50% i n h i b i t i o n of f l u o r i d e - s t i m u l a t e d h e p a t i c enzyme a c t i v i t y ( 1 4 5 ) . The r e a s o n f o r t h i s d i s c r e p a n c y between r e s u l t s i s not c l e a r . The p r e c e d i n g e f f e c t s of g u a n y l n u c l e o t i d e s on e p i n e -p h r i n e - s t i m u l a t e d a d e n y l c y c l a s e were o b t a i n e d w i t h c o n c e n -t r a t i o n s o f e p i n e p h r i n e p r o d u c i n g a maximal s t i m u l a t i o n of enzyme a c t i v i t y ( s e e F i g . 1 0 ) . GTP s t i m u l a t e d r e a c t i o n v e l o c i t y w i t h e p i n e p h r i n e c o n c e n t r a t i o n s r a n g i n g f r o m 10~ 7 t o 10" 4 M, w i t h no e f f e c t on the a p p a r e n t a f f i n i t y of the enzyme - 91 -F i g u r e 1 5 : E f f e c t o f GTP a n d GDP o n s k e l e t a l m u s c l e a d e n y l c y c l a s e . E n z y m e a c t i v i t y i n a m e m b r a n e p r e p a r a t i o n ( 1 4 7 u g p r o t e i n ) was d e t e r m i n e d b y a s s a y A i n t h e p r e s e n c e o f 9 mM m a g n e s i u m a n d v a r y i n g c o n c e n t r a t i o n s o f GTP ( } o r GDP ( ) w i t h n o f u r t h e r a d d i t i o n s « 3 ~ O) > w i t h 0 . 0 1 mM e p i n e p h r i n e ( A ~ A ) > o r w i t h 1 2 mM f l u o r i d e < • - Q ) . - 92 -- 93 -f o r e p i n e p h r i n e . 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 of the b i n d i n g of r_ 3Hl-ep i n e p h r i n e t o s k e l e t a l m u s c l e p l a s m a membranes a l s o i n d i c a t e t h a t GTP (0.1 mM) has no e f f e c t on hormone b i n d i n g . - 94 -DISCUSSION A d e n y l c y c l a s e i s t h o u g h t to be l o c a t e d p r i m a r i l y , but n o t e x c l u s i v e l y , i n the c e l l u l a r p l a s m a membrane of most mammalian t i s s u e s . The l o c a t i o n of the enzyme i n the p l a s m a membrane i s an i m p o r t a n t f e a t u r e of the s e c o n d messenger c o n c e p t . A d e n y l c y c l a s e has been shown t o be p r e s e n t i n p u r i f i e d p l a s m a membrane p r e p a r a t i o n s f r o m f a t c e l l s ( 1 4 6 , 1 4 7 ) , l i v e r ( 1 4 8 ) , t h y r o i d t i s s u e (149,150) and e r y t h r o c y t e s ( 1 5 1 , 1 5 2 ) . A t t e m p t s to p u r i f y p l a s m a membranes from c a r d i a c t i s s u e were u n s u c c e s s f u l . However, the l a r g e p r o p o r t i o n of a c t i v i t y p r e s e n t i n the washed p a r t i c l e s and the p a r t i a l p u r i f i c a t i o n by s h o r t - t e r m L i B r e x t r a c t i o n s ( T a b l e I I ) s u g g e s t t h a t the m a j o r i t y of m y o c a r d i a l a d e n y l c y c l a s e r e s i d e s i n the p l a s m a membrane. In s k e l e t a l m u s c l e , a c o n s i d e r a b l e p o r t i o n of a d e n y l c y c l a s e a c t i v i t y was p r e s e n t i n a p l a s m a membrane f r a c t i o n ( T a b l e IV, p a r t B) i n c o n t r a s t t o e a r l i e r r e p o r t s t h a t the enzyme was p r e s e n t i n m i t o c h o n d r i a l and m i c r o s o m a l f r a c t i o n s ( 3 1 , 3 2 ) . T h i s d i s c r e p a n c y w i t h the r e s u l t s p r e s e n t e d i n T a b l e IV i s l i k e l y due t o t h e l o n g e r p e r i o d s of h omogeni-z a t i o n (60 t o 120 s e c o n d s ) employed i n the e a r l i e r s t u d i e s . A number o f methods e x i s t f o r the p r e p a r a t i o n of s k e l e t a l m u s c l e p l a s m a membranes ( 1 2 9 , 1 3 0 , 1 3 2 , 1 3 3 , 1 5 3 , 1 5 4 ) . The p r o c e d u r e a d o p t e d f o r t h e s e i n v e s t i g a t i o n s was a m o d i f i c a t i o n of the s a l t - e x t r a c t i o n t e c h n i q u e of Kono and C o l o w i c k (130) a f t e r an i n i t i a l h o m o g e n i z a t i o n i n 50 mM c a l c i u m c h l o r i d e as d e s c r i b e d by M c C o l l e s t e r ( 1 2 9 ) . R e l a t i v e l y m i l d c o n d i t i o n s were r e q u i r e d d u r i n g the p r o c e d u r e s b e c a u s e of the l a b i l i t y - 9 5 -of t h e enzyme. T h i s method a l l o w s t h e i s o l a t i o n o f p l a s m a membranes i n a r e l a t i v e l y s h o r t t i m e i n t e r v a l w i t h a h i g h y i e l d o f a d e n y l c y c l a s e a c t i v i t y . A number of c r i t e r i a w ere e x a m i n e d t o e s t a b l i s h t h a t t h e membranes i s o l a t e d by t h i s p r o c e d u r e d i d i n d e e d c o n s i s t p r i m a r i l y o f p l a s m a mem-b r a n e s . Phase c o n t r a s t m i c r o s c o p y ( F i g . 5) r e v e a l e d t h e e x i s t e n c e o f e m p t y , t u b u l a r , s a c - l i k e c e l l s e g m e n t s s i m i l a r t o t h o s e o b s e r v e d p r e v i o u s l y ( 1 2 9 , 1 3 0 , 1 5 3 , 1 5 4 ) . The l i p i d c o m p o s i t i o n of the membrane f r a c t i o n ( T a b l e V) was v e r y s i m i l a r t o t h a t r e p o r t e d by F i e h n e t a l ( 1 3 6 ) f o r s k e l e t a l m u s c l e p l a s m a membranes i s o l a t e d by t h e method d e s c r i b e d by P e t e r ( 1 3 2 ) . I n p a r t i c u l a r , t h e h i g h m o l a r r a t i o ( 0 . 5 -0.6) of c h o l e s t e r o l t o p h o s p h o l i p i d i s c h a r a c t e r i s t i c o f p l a s m a membranes ( 1 3 5 ) . O t h e r c e l l u l a r membrane s t r u c t u r e s s u c h as m i t o c h o n d r i a o r m i c r o s o m e s have much l e s s c h o l e s t e r o l and c h a r a c t e r i s t i c a l l y have c h o l e s t e r o l t o p h o s p h o l i p i d m o l a r r a t i o s o f 0.1 t o 0.2 ( 1 5 5 ) . The a b s e n c e o f c y t o c h r o m e c o x i d a s e , a c i d m a l t a s e , a c i d p h o s p h a t a s e , and p y r o p h o s p h a t a s e i n d i c a t e d l i t t l e o r no c o n -t a m i n a t i o n o f t h e p l a s m a membrane p r e p a r a t i o n by m i t o c h o n d r i a or l y s o s o m e s . The p r e s e n c e of m a g n e s i u m - A T P a s e , s o d i u m , p o t a s s i u m - a c t i v a t e d , m a g n e s i u m - A T P a s e , and c a l c i u m - A T P a s e o b s e r v e d by S e v e r s o n , Drummond and S u l a k h e ( 1 3 1 ) i n t h i s membrane p r e p a r a t i o n i s a l s o c o n s i s t e n t w i t h t h e i r l o c a t i o n i n o t h e r s k e l e t a l m u s c l e p l a s m a membrane p r e p a r a t i o n s ( 1 3 2 -1 3 4 ) . The s o d i u m , p o t a s s i u m - a c t i v a t e d , m agnesium-ATPase i s - 96 -c o n s i d e r e d to be a s s o c i a t e d with a c t i v e c a t i o n t r a n s p o r t across c e l l membranes, and the presence of t h i s enzyme a c t i v i t y i n f r o g s k e l e t a l muscle membranes has been used to d i s t i n g u i s h plasma membranes from sar c o p l a s m i c r e t i c u l u m fragments which con t a i n e d only a magnesium-ATPase (156). The calcium-ATPase i n t h i s plasma membrane p r e p a r a t i o n d i d not r e q u i r e magnesium f o r i t s a c t i v i t y and had s i m i l a r p r o p e r t i e s when compared with calcium-ATPase from hamster s k e l e t a l muscle plasma membranes (157) and from c a r d i a c muscle sarcolemmal p r e p a r a t i o n s (158, 159). The s k e l e t a l muscle membrane p r e p a r a t i o n d e s c r i b e d here a l s o possessed the a b i l i t y to bind calcium i n an ATP-dependent manner which could be f u r t h e r s t i m u l a t e d by the a d d i t i o n of phosphate (131). Calcium has a l s o been r e p o r t e d to bind to plasma membranes from b u l l f r o g s k e l e t a l muscle (160) and l i v e r (161) and c o r r e l a t i o n s have been observed between calcium b i n d i n g , calcium t r a n s p o r t and calcium-ATPase a c t i v i t y i n e r y t h r o c y t e ghosts (162,163). Microsomal con-ta m i n a t i o n i s not l i k e l y to account f o r e i t h e r the c a l c i u m -ATPase a c t i v i t y or the ATP-dependent c a l c i u m b i n d i n g present i n the s k e l e t a l muscle membrane p r e p a r a t i o n d e s c r i b e d i n t h i s i n v e s t i g a t i o n . Heavy microsomes that were i n i t i a l l y present in the f i r s t 2000 x g p e l l e t were e f f e c t i v e l y removed by the s e r i e s of washes and low speed c e n t r i f u g a t i o n steps employed du r i n g the p r e p a r a t i o n . In a d d i t i o n , Severson, Drummond and Sulakhe (131) have r e p o r t e d that e x t r a c t i o n of s a r c o p l a s m i c r e t i c u l u m fragments with 0.4 M l i t h i u m bromide g r e a t l y reduced t h e i r a b i l i t y to sequester c a l c i u m , as had been observed - 97 -p r e v i o u s l y by Repke and K a t z w i t h c a r d i a c s a r c o p l a s m i c r e t i c u l u m ( 1 6 4 ) . These c o n s i d e r a t i o n s and the c r i t e r i a p r e s e n t e d e a r l i e r s u g g e s t t h a t the membrane f r a c t i o n does c o n s i s t of p l a s m a membranes ( s a r c o l e m m a ) i n a h i g h d e g r e e of p u r i t y . The k i n e t i c p r o p e r t i e s of a d e n y l c y c l a s e f r o m h e a r t and s k e l e t a l m u s c l e a r e q u a l i t a t i v e l y s i m i l a r and w i l l be d i s c u s s e d t o g e t h e r . A d e n y l c y c l a s e i s s t i m u l a t e d by c o n c e n t r a t i o n s of m e t a l i o n s g r e a t l y i n e x c e s s of the ATP c o n c e n t r a t i o n , s u g g e s t i n g t h a t m e t a l i o n s can b i n d t o some s i t e on the enzyme i n d e p e n d e n t of t h e i r p r o b a b l e b i n d i n g t o ATP at t h e c a t a l y t i c s i t e . The c o n s e q u e n c e of s u c h m e t a l i o n b i n d i n g may be t o s t a b i l i z e a p a r t i c u l a r c o n f o r m a t i o n of the enzyme r e s u l t i n g i n e n h a n c e d r e a c t i v i t y of the c a t a l y t i c s i t e and an i n c r e a s e d r e a c t i o n v e l o c i t y . The e f f e c t of magnesium on m y o c a r d i a l a d e n y l c y c l a s e was i n i t i a l l y r e p o r t e d by Drummond and Duncan ( 2 8 ) . The a b i l i t y of magnesium t o s t i m u l a t e the c a r d i a c enzyme by i n t e r -a c t i n g a t some s i t e i n d e p e n d e n t of the s u b s t r a t e w i t e was c o n f i r m e d by Drummond, S e v e r s o n and Duncan (108) and i n F i g . 2. S i m i l a r e f f e c t s of magnesium have been o b s e r v e d i n l i v e r ( 4 3 , 1 0 8 , 1 4 8 , 1 6 5 ) , b r a i n ( 1 0 8 ) , and s k e l e t a l m u s c l e ( F i g . 7 ) . In a d d i t i o n , i n c r e a s i n g the r a t i o of magnesium t o ATP i n c r e a s e d the r e a c t i o n v e l o c i t y of a d e n y l c y c l a s e i n b a c t e r i a (142) and i n a d i p o c y t e s ( 5 3 ) . The s t i m u l a t i o n of a d e n y l c y c l a s e i n f a t c e l l s by magnesium was u n i q u e i n t h a t the enzyme c o u l d n o t be s a t u r a t e d w i t h the m e t a l i o n and t h a t h i g h c o n c e n -t r a t i o n s of magnesium c o u l d s u b s t i t u t e f o r ACTH and f l u o r i d e - 98 -s t i m u l a t i o n (107). Manganese was more e f f e c t i v e t h a n magnesium i n a c t i v a t i n g m y o c a r d i a l a d e n y l c y c l a s e , w h i l e c o b a l t was l e s s e f f e c t i v e ( F i g . 2). An i d e n t i c a l p a t t e r n of magnesium and manganese a c t i v a t i o n was o b s e r v e d by Menon and Sm i t h w i t h s a l m o n t e s t e s a d e n y l c y c l a s e (37), and a s i m i l a r example of m e t a l i o n a c t i v a t i o n was o b s e r v e d w i t h the a d i p o s e t i s s u e enzyme (107). Burke o b s e r v e d t h a t s t i m u l a t i o n by 5 mM man-ganese was g r e a t e r t h a n w i t h 5 mM magnesium f o r t h y r o i d a d e n y l c y c l a s e (166) and i t was r e p o r t e d t h a t the o p t i m a l magnesium c o n c e n t r a t i o n f o r the f r o g e r y t h r o c y t e enzyme was 3 t o 5 mM, whereas manganese was o p t i m a l a t 1 mM c o n c e n t r a t i o n (152). Manganese p r o d u c e d a much g r e a t e r s t i m u l a t i o n of a d e n y l c y c l a s e of monkey s p e r m a t o z o a t h a n d i d magnesium (167). Manganese was a l s o more e f f e c t i v e t h a n magnesium i n s t i m u l a t i n g a d e n y l c y c l a s e of N o c a r d i a e r y t h r o p o l i s (39); a t low c o n c e n -t r a t i o n s , the s t i m u l a t i o n of the enzyme f r o m E s c h e r i c h i a c o l i and B r e v i b a c t e r i u m l i q u e f a c i e n s by manganese was g r e a t e r t h a n by magnesium, however a t h i g h e r c o n c e n t r a t i o n s , manganese e x h i b i t e d s e v e r e i n h i b i t i o n (39). The d i f f e r e n c e between m e t a l i o n s w i t h r e s p e c t to t h e i r a b i l i t y t o a c t i v a t e s k e l e t a l m u s c l e a d e n y l c y c l a s e ( F i g . 7) was n o t as p r o n o u n c e d as w i t h the c a r d i a c enzyme. B o t h c o b a l t and manganese a c t i v a t e d the s k e l e t a l m u s c l e enzyme at s l i g h t l y l ower c o n c e n t r a t i o n s t h a n magnesium, b u t the e x t e n t of a c t i v a t i o n by a l l t h r e e i o n s was q u i t e s i m i l a r . The K m of ATP f o r m y o c a r d i a l a d e n y l c y c l a s e was r e p o r t e d to be 0.08 mM i n the p r e s e n c e of magnesium ( 2 8 ) . T h i s was c o n f i r m e d by Drummond, S e v e r s o n and Duncan (108) when sub-s t r a t e s a t u r a t i o n was examined o v e r a g r e a t e r range of ATP - 99 -c o n c e n t r a t i o n s . A s i m i l a r K of 0.1 mM was o b t a i n e d f o r ATP m w i t h the c a r d i a c enzyme i n the p r e s e n c e of manganese ( F i g . 3 ) . A K m of a p p r o x i m a t e l y 0.3 mM ATP was o b s e r v e d w i t h the s k e l e t a l m u s c l e enzyme ( F i g . 8 ) . A much h i g h e r K m of about 1 mM f o r ATP was f o u n d by Hofman f o r a d e n y l c y c l a s e i n s k e l e t a l m u s c l e p a r t i c l e s ( 3 2 ) . The r e a s o n f o r t h i s d i s c r e p a n c y i s n o t a p p a r e n t . V a l u e s f o r the K m of ATP u s u a l l y r a n g e d f r o m 0.1 t o 0.5 mM i n s t u d i e s on a d e n y l c y c l a s e i n f a t c e l l s ( 1 0 7 , 1 2 4 ) , t o a d b l a d d e r ( 2 9 ) , and f r o m E s c h e r i c h i a c o l i (40) and N o c a r d i a e r y t h r o p o l i s ( 3 9 ) . K m v a l u e s of a p p r o x i m a t e l y 1.0 mM ATP were o b s e r v e d w i t h enzyme p r e p a r a t i o n s f r o m B r e v i -b ac te r ium 1 i q u e f ac i e n s (142) and S t r e p t o c o c c u s s a l i v a r i u s ( 3 8 ) . Hepp, E d e l and W i e l a n d (43) r e p o r t e d t h a t the K m of mouse l i v e r a d e n y l c y c l a s e f o r ATP was 4 mM and the enzyme fr o m r e n a l c o r t e x was n o t s a t u r a t e d w i t h c o n c e n t r a t i o n s of ATP as l a r g e as 5 mM ( 1 6 8 ) . As c o n c e n t r a t i o n s of ATP a p p r o a c h or e x c e e d the m e t a l i o n c o n c e n t r a t i o n , an i n h i b i t i o n of a d e n y l c y c l a s e i n c a r d i a c (108 and F i g . 3) and s k e l e t a l m u s c l e t i s s u e ( F i g . 8) was o b s e r v e d . S i m i l a r e f f e c t s of i n c r e a s i n g ATP c o n c e n t r a t i o n s have been r e p o r t e d f o r a d e n y l c y c l a s e from t u r k e y and r a t e r t h r o c y t e s ( 4 4 , 4 5 ) , b r a i n ( 1 0 8 ) , a d i p o s e t i s s u e ( 1 0 7 ) , l i v e r (148,169) and t h y r o i d t i s s u e ( 1 5 0 ) . These f i n d i n g s have been t a k e n as f u r t h e r e v i d e n c e t h a t the true s u b s t r a t e f o r a d e n y l c y c l a s e may be an A T P - m e t a l i o n c o m p l e x . The i n h i b i t i o n by ATP was n o t r e v e r s e d by f l u o r i d e or by the v a r i o u s hormones, b u t was overcome by i n c r e a s i n g the c o n c e n t r a t i o n of m e t a l i o n s . The i n h i b i t i o n by ATP c o u l d be due t o c o m p e t i t i o n w i t h the m e t a l - 100 -i o n - A T P s u b s t r a t e at the c a t a l y t i c s i t e , or by c o m p e t i t i o n w i t h the enzyme f o r f r e e magnesium. ATP i n h i b i t i o n can be overcome by the a d d i t i o n of manganese i n c o n c e n t r a t i o n s l e s s t h a n t h a t r e q u i r e d to f o r m a s t o i c h i o m e t r i c o n e - t o - o n e complex w i t h ATP ( F i g . 4 ) , s u g g e s t i n g t h a t m e t a l i o n s a r e p r i m a r i l y i n t e r a c t i n g w i t h the enzyme to overcome the i n h i b i t i o n . The same r e s u l t was o b t a i n e d by Drummond, S e v e r s o n and Duncan (108) w i t h a d d i t i o n s of magnesium i n the p r e s e n c e of f l u o r i d e . M e t a l a c t i v a t i o n o f a d e n y l c y c l a s e , t h e r e f o r e , may o c c u r by two m e c h a n i s m s . The f i r s t of t h e s e i s by an i n t e r a c t i o n w i t h the s u b s t r a t e , ATP. T h i s m e t a l i o n e f f e c t o c c u r s w i t h most enzyme r e a c t i o n s i n v o l v i n g n u c l e o s i d e d i p h o s p h a t e s and n u c l e o -s i d e t r i p h o s p h a t e s ( 1 7 0 ) . The s e c o n d mechanism i n v o l v e s an i n t e r a c t i o n of the m e t a l i o n at some o t h e r s i t e on the enzyme i n some s t r u c t u r a l r o l e , p e r h a p s by s t a b i l i z i n g a c a t a 1 y t i c a 1 l y a c t i v e p r o t e i n c o n f o r m a t i o n . M e t a l i o n s a c t i v a t e a d e n y l c y c l a s e a t a l l c o n c e n t r a t i o n s of ATP, w i t h the r e s u l t t h a t maximal v e l o c i t y i s i n c r e a s e d by m e t a l i o n s w i t h o u t an e f f e c t on the a f f i n i t y of the m e t a l i o n - A T P complex at t h e c a t a l y t i c s i t e of t h e enzyme i n h e a r t (28,108 and F i g . 3 ) , s k e l e t a l m u s c l e ( F i g . 8) and f a t c e l l s ( 1 0 7 ) . Thus a d e n y l c y c l a s e i n c a r d i a c and s k e l e t a l m u s c l e t i s s u e c o u l d e x i s t i n two c o n f o r m a t i o n a l f o r m s c o r r e s p o n d i n g t o the f r e e enzyme and m e t a l i o n - b o u n d enzyme, w i t h b o t h c o n f o r m a t i o n a l forms h a v i n g e q u a l a f f i n i t y f o r the A T P - m e t a l i o n c o m p l ex, but w i t h the m e t a l - b o u n d enzyme h a v i n g g r e a t e r c a t a l y t i c r e a c t i v i t y . I t i s a p p a r e n t t h a t c hanges i n the r a t i o of m e t a l i o n s t o ATP w i t h i n the c e l l c o u l d - 101 -e x e r t a p r o f o u n d i n f l u e n c e on a d e n y l c y c l a s e and so c o n t -r i b u t e t o the p h y s i o l o g i c a l r e g u l a t i o n of enzyme a c t i v i t y . M e t a l i o n a c t i v a t i o n i n v o l v i n g b o t h s u b s t r a t e and enzyme i s a l s o i n v o l v e d i n the r e g u l a t i o n of p y r u v a t e c a r b o x y l a s e (171) as w e l l as s e v e r a l o t h e r enzyme s y s t e m s ( 1 7 0 ) . The s t i m u l a t i o n of a d e n y l c y c l a s e i n most mammalian t i s s u e s by f l u o r i d e i o n r e m a i n s one of the most p u z z l i n g f e a t u r e s of the p r o p e r t i e s of the enzyme. F l u o r i d e i o n s a r e i n h i b i t o r y t o many m e t a l - a c t i v a t e d enzyme s y s t e m s s u c h as the g l y c o l y t i c enzymes, e n o l a s e and p h o s p h o g l u c o m u t a s e , and v a r i o u s p h o s p h a t a s e s ( 1 7 2 ) . F l u o r i d e was f i r s t o b s e r v e d t o s t i m u l a t e a d e n y l c y c l a s e i n b r o k e n c e l l p r e p a r a t i o n s f r o m v a r i o u s dog t i s s u e s ( 7 , 1 8 ) . The e f f e c t o f f l u o r i d e on a d e n y l c y c l a s e a p p e a r e d t o be s e p a r a t e f r o m i t s i n h i b i t o r y e f f e c t on ATPase a c t i v i t y and r e s u l t e d i n maximal c a t a l y t i c a c t i v i t y s i n c e hormones d i d not r e s u l t i n f u r t h e r enzyme s t i m u l a t i o n i n t h e p r e s e n c e of f l u o r i d e . A number of s t u d i e s have i n d i c a t e d t h a t the a d e n y l c y c l a s e s t i m u l a t i o n by f l u o r i d e i s not due t o ATPase i n h i b i t i o n , w i t h some of the e v i d e n c e summarized by Weiss ( 1 7 3 ) . C o n c e n t r a t i o n s of f l u o r i d e t h a t r e s u l t e d i n m aximal s t i m u l a t i o n of a d e n y l c y c l a s e i n h i b i t e d ATPase a c t i v i t y o n l y s l i g h t l y . In a d d i t i o n , o t h e r ATPase i n h i b i t o r s or the use of s a t u r a t i n g l e v e l s of ATP had no e f f e c t on a d e n y l c y c l a s e a c t i v i t y or on the s t i m u l a t o r y e f f e c t of f l u o r i d e . Numerous s t u d i e s , i n c l u d i n g t h i s i n v e s t i g a t i o n , have shown t h a t f l u o r i d e s t i m u l a t e d a d e n y l c y c l a s e under c o n d i t i o n s i n w h i c h ATP l e v e l s were m a i n t a i n e d by A T P - r e g e n e r a t i n g s y s t e m s . F l u o r i d e a l s o - 102 -s t i m u l a t e d the s y n t h e s i s of c y c l i c AMP from the 5 1-adeny1-imido diphosphate (AMP-PNP) analog of ATP which i s not degraded by ATPases (148). Thus, the s t i m u l a t i o n by f l u o r i d e would appear to i n v o l v e a d i r e c t e f f e c t upon adenyl c y c l a s e . The e f f e c t of f l u o r i d e on b a c t e r i a l adenyl c y c l a s e depends on the s t r a i n ; f l u o r i d e a c t i v a t e d the enzyme from Strep tococcus s a l i v a r ius (38), i n h i b i t e d the E s c h e r i c h i a c o l i enzyme (143) and had no e f f e c t on the enzyme from Nocardia e r y t h r o p o l i s ( 3 9 ) . A c u r i o u s f e a t u r e of the a c t i o n of f l u o r i d e has been the o b s e r v a t i o n that adenyl c y c l a s e i n i n t a c t c e l l s or s l i c e p r e p a r a t i o n s was not s t i m u l a t e d by f l u o r i d e . The a d d i t i o n of f l u o r i d e to i n t a c t turkey e r y t h r o c y t e s (44), i n t a c t spermatazoa (167) or to s l i c e p r e p a r a t i o n s from t h y r o i d t i s s u e (174) and f e t a l r a t c a l v a r i a (52) had no e f f e c t on c y c l i c AMP l e v e l s , whereas homogenates of e r y t h r o c y t e s , t h y r o i d and c a l v a r i a d i d respond to f l u o r i d e with an i n c r e a s e i n adenyl c y c l a s e a c t i v i t y (25,44,52). The a d d i t i o n of f l u o r i d e to p e r f u s e d l i v e r s a c t u a l l y decreased t i s s u e l e v e l s of c y c l i c AMP (175). W i l l i a m s et a l have r e p o r t e d a s t i m u l a t i o n of adenyl c y c l a s e by f l u o r i d e i n i s o l a t e d a d i p o c y t e s (53) but another i n v e s t i g a t i o n found that f l u o r i d e had no e f f e c t on adenyl c y c l a s e i n i n t a c t a d i p o c y t e s and s t i m u l a t e d enzyme a c t i v i t y only i n homogenates (14). It has been suggested that the i n a b i l i t y of f l u o r i d e to s t i m u l a t e enzyme a c t i v i t y i n i n t a c t c e l l s and s l i c e p r e p a r a t i o n s i s because the a c t i v e c a t a l y t i c s i t e of adenyl c y c l a s e i s l o c a t e d on the inner s u r f a c e of the c e l l membrane and that these i n t a c t c e l l p r e p a r a t i o n s are not permeable to f l u o r i d e . This s u g g e s t i o n , however, cannot be r e c o n c i l e d with the a b i l i t y of f l u o r i d e to i n h i b i t g l y c o l y s i s i n i n t a c t e r y t h r o c y t e s (176) and i n i n t a c t - 1 0 3 -s p e r m a t o z o a ( 1 7 7 ) , p r e s u m a b l y by i n h i b i t i o n of e n o l a s e and p h o s p h o g l u c o m u t a s e . T h e r e f o r e , t h e r e a s o n t h a t i n t a c t c e l l u l a r p r e p a r a t i o n s do n o t r e s p o n d to f l u o r i d e w i t h i n c r e a s e d a d e n y l c y c l a s e a c t i v i t y r e m a i n s t o be f o u n d . A l t h o u g h f l u o r i d e a c t i v a t i o n i s n o t s p e c i f i c w i t h r e s p e c t t o the t i s s u e s o u r c e of a d e n y l c y c l a s e i n c o n t r a s t t o the e f f e c t s of hormones, the e f f e c t of the f l u o r i d e i o n i s v e r y s e l e c t i v e s i n c e no o t h e r a n i o n o r o r g a n i c f l u o r i d e compound c o u l d s u b -s t i t u t e f o r f l u o r i d e i o n i n a c t i v a t i n g m y o c a r d i a l a d e n y l c y c l a s e ( 2 8 ) . F l u o r i d e p r o f o u n d l y s t i m u l a t e d s k e l e t a l m u s c l e a d e n y l c y c l a s e ( F i g . 9 ) . The e f f e c t of f l u o r i d e must be i n s t a n t a n e o u s and n o t i n v o l v e a t i m e - d e p e n d e n t a c t i v a t i o n p r o c e s s s i n c e r e a c t i o n r a t e s a r e l i n e a r b o t h i n the absence and i n the p r e s e n c e of f l u o r i d e ( F i g . 6 ) . T e m p e r a t u r e had a much g r e a t e r e f f e c t on f l u o r i d e - s t i m u l a t e d a c t i v i t y t h a n on b a s a l a c t i v i t y , a l t h o u g h t h e c o n c e n t r a t i o n of f l u o r i d e f o r maximal s t i m u l a t i o n r e m a i n e d the same ( F i g , 9 ) . T h i s e f f e c t of t e m p e r a t u r e was r e f l e c t e d by an i n c r e a s e i n the e n e r g y of a c t i v a t i o n i n the p r e s e n c e of f l u o r i d e ( F i g . 9 ) . T h i s i s l i k e l y due to changes i n the p r o t e i n c o n f o r m a t i o n o r changes i n the r a t e - 1 i m i t i n g e v e n t i n the c a t a l y t i c r e a c t i o n . A g r e a t e r e f f e c t of t e m p e r a t u r e on f l u o r i d e -s t i m u l a t e d a c t i v i t y i n c o m p a r i s o n t o b a s a l or h o r m o n e - s t i m u l a t e d a c t i v i t y has a l s o been o b s e r v e d i n f a t c e l l s (26,107) and i n c e r e b r a l c o r t e x ( 1 3 8 ) . In agreement w i t h o t h e r i n v e s t i g a t o r s ( 1 3 8 - 1 4 1 ) , the s t i m u l a t i o n of s k e l e t a l m u s c l e a d e n y l c y c l a s e by f l u o r i d e was p a r t i a l l y i r r e v e r s i b l e a f t e r d i a l y s i s to r e -move the f l u o r i d e ( T a b l e V I I I ) . T h i s i s b e s t e x p l a i n e d by s u g g e s t i n g t h a t f l u o r i d e p r o m o t e s some c a t a l y t i c a 1 l y - a c t i v e - 104 -c o n f o r m a t i o n a l change i n a d e n y l c y c l a s e , p e r h a p s by d i s s o c i a t i o n of some i n h i b i t o r y s u b s t a n c e ( 1 3 9 , 1 7 8 ) , w h i c h i s s t a b l e and r e v e r s e d o n l y s l o w l y a f t e r r e m o v a l of the f l u o r i d e . I t i s of i n t e r e s t t h a t i n r a t b r a i n , f l u o r i d e s t i m u l a t i o n was e v i d e n t o n l y a f t e r a p p r o x i m a t e l y n i n e days p o s t - p a r t u m ( 1 7 8 ) . I t was s u g g e s t e d t h a t f l u o r i d e was a b l e t o r e v e r s e some i n h i b i t o r y i n f l u e n c e w h i c h was i n i t i a l l y n o t p r e s e n t a t b i r t h . Of p o s s i b l e f u r t h e r r e l e v a n c e t o the mechanism of a c t i o n of f l u o r i d e i s t h e o b s e r v a t i o n t h a t p y r o p h o s p h a t e c a u s e d a much g r e a t e r i n h i b i t i o n of f l u o r i d e - s t i m u l a t e d enzyme a c t i v i t y t h a n w i t h e i t h e r b a s a l or e p i n e p h r i n e - s t i m u l a t e d s k e l e t a l m u s c l e a d e n y l c y c l a s e a c t i v i t y ( F i g . 1 3 ) . The i n h i b i t i o n by p y r o p h o s p h a t e i n the p r e s e n c e of f l u o r i d e was c o m p e t i t i v e w i t h r e s p e c t t o ATP ( F i g . 1 4 ) . T h i s s u g g e s t s t h a t the r e l e a s e o f p r o d u c t s i n the a d e n y l c y c l a s e r e a c t i o n i s o r d e r e d , w i t h c y c l i c AMP b e i n g r e l e a s e d f r o m the enzyme b e f o r e p y r o p h o s p h a t e . The e f f e c t of hormones on m y o c a r d i a l a d e n y l c y c l a s e was modest b u t v e r y r e p r o d u c i b l e . The s t i m u l a t i o n of the c a r d i a c enzyme by e p i n e p h r i n e was a l w a y s l e s s i n the p r e s e n c e of man-ganese t h a n i n the p r e s e n c e of magnesium. Hormonal a c t i v a t i o n i n t h y r o i d (166) and i n f a t c e l l s (107) was a l s o l e s s i n the p r e s e n c e of manganese i n c o m p a r i s o n t o h o r m o n a l a c t i v a t i o n of a d e n y l c y c l a s e i n the p r e s e n c e of magnesium. The p r e s e n c e of manganese c o m p l e t e l y i n h i b i t e d the s t i m u l a t i o n of a d e n y l c y c l a s e i n r e n a l c o r t e x by p a r a t h y r o i d hormone (168) as w e l l as the s t i m u l a t i o n of the t o a d b l a d d e r enzyme by a n t i d i u r e t i c hormone ( 1 7 9 ) . The r e a s o n f o r t h i s s e l e c t i v e m e t a l i o n e f f e c t i s not known. C a t e c h o l a m i n e s a c t i v a t e d m y o c a r d i a l a d e n y l c y c l a s e w i t h an - 105 -o r d e r of p o t e n c y i d e n t i c a l t o t h a t r e p o r t e d w i t h dog h e a r t p a r t i c l e s ( 4 1 ) . The b l o c k a d e of the e p i n e p h r i n e s t i m u l a t i o n by p r o p r a n o l o l and the s i m i l a r i t i e s between the p o t e n c y of c a t e c h o l a m i n e s to s t i m u l a t e a d e n y l c y c l a s e and t o i n c r e a s e c a r d i a c c o n t r a c t i l i t y (70) a r e c o n s i s t e n t w i t h the h y p o t h e s i s t h a t a d e n y l c y c l a s e i s a t l e a s t a p a r t of a b e t a - a d r e n e r g i c r e c e p t o r . A d e n y l c y c l a s e i n s k e l e t a l m u s c l e was a l s o s t i m u l a t e d by c a t e c h o l a m i n e s ( F i g . 10) and the o r d e r of p o t e n c y was the same as t h a t o b s e r v e d f o r the s t i m u l a t i o n of g l y c o g e n o l y s i s i n r a t d i a p h r a g m ( 1 8 0 ) . The i n h i b i t i o n of e p i n e p h r i n e s t i m u l -a t i o n of s k e l e t a l m u s c l e a d e n y l c y c l a s e by p r o p r a n o l o l i s c o n s i s t e n t w i t h an a c t i o n on b e t a - a d r e n e r g i c r e c e p t o r s . The a b i l i t y of e p i n e p h r i n e to s t i m u l a t e s k e l e t a l m u s c l e a d e n y l c y c l a s e a c t i v i t y was e n h a n c e d by v e r y low c o n c e n t r a t i o n s of GTP and GDP ( F i g . 1 5 ) , i n a manner a n a l o g o u s t o t h a t o b s e r v e d f o r g l u c a g o n s t i m u l a t i o n o f a d e n y l c y c l a s e i n l i v e r p l a s m a membranes ( 1 4 8 ) . The s t i m u l a t i o n by e p i n e p h r i n e of a d e n y l c y c l a s e i n g u i n e a p i g h e a r t L i B r - e x t r a c t e d p a r t i c l e s was a l s o e n h a n c e d by 1.8 f o l d w i t h the a d d i t i o n of 0.1 mM GTP. A d e n y l c y c l a s e i n s k e l e t a l m u s c l e was u n a f f e c t e d by v a r i o u s o t h e r h ormones. I n s u l i n had no e f f e c t on e i t h e r b a s a l or e p i n e -p h r i n e - s t i m u l a t e d a d e n y l c y c l a s e a c t i v i t y . These r e s u l t s a g r e e w i t h the f i n d i n g s of C r a i g , R a i l and L a r n e r (181) t h a t the a c t i v a t i o n of muscle g l y c o g e n s y n t h e t a s e by i n s u l i n was not a s s o c i a t e d w i t h changes i n the t i s s u e l e v e l s of c y c l i c AMP. D e s p i t e i n i t i a l r e p o r t s t h a t a d e n y l c y c l a s e a c t i v i t y i n a d i p o s e t i s s u e (182) and l i v e r (169) was d e c r e a s e d by i n s u l i n , r e c e n t - 106 -i n v e s t i g a t i o n s have f o u n d t h a t i n s u l i n does n o t d e c r e a s e a d e n y l c y c l a s e a c t i v i t y i n f a t c e l l membranes (27,183) or i n l i v e r p l a s m a membranes ( 1 4 8 ) . The k i n e t i c n a t u r e of the s t i m u l a t i o n of a d e n y l c y c l a s e i n c a r d i a c and s k e l e t a l m u s c l e t i s s u e by e p i n e p h r i n e i s v e r y s i m i l a r t o the k i n e t i c n a t u r e of f l u o r i d e s t i m u l a t i o n . F l u o r i d e and e p i n e p h r i n e i n c r e a s e d the r e a c t i o n v e l o c i t y a t a l l c o n c e n -t r a t i o n s of ATP ( 2 8 , 108 and F i g . 3, F i g . 1 2 ) , t h u s r e s u l t i n g i n i n c r e a s e d maximal v e l o c i t y w i t h o u t an a l t e r a t i o n i n the a f f i n i t y f o r the A T P - m e t a l i o n s u b s t r a t e . Hofman a l s o r e p o r t e d t h a t the K m f o r ATP i n s k e l e t a l m u s c l e p a r t i c u l a t e p r e p a r a t i o n s was n o t changed s u b s t a n t i a l l y by e p i n e p h r i n e or f l u o r i d e ( 3 2 ) . S i m i l a r e f f e c t s on maximal v e l o c i t y have been o b t a i n e d w i t h f l u o r i d e , ACTH, and e p i n e p h r i n e w i t h the f a t c e l l enzyme ( 2 7 , 107,124); f l u o r i d e and g l u c a g o n w i t h the enzyme i n l i v e r p l a s m a membranes ( 1 4 8 ) ; f l u o r i d e and t h y r o i d - s t i m u l a t i n g hormone w i t h t h y r o i d a d e n y l c y c l a s e ( 1 5 0 ) ; and w i t h l u t e i n i z i n g hormone and f l u o r i d e w i t h a d e n y l c y c l a s e i n c o r p u s l u t e u m ( 1 8 4 ) . F l u o r i d e a l s o i n c r e a s e d maximal v e l o c i t y w i t h o u t c h a n g i n g s u b s t r a t e a f f i n i t y w i t h a d e n y l c y c l a s e i n b r a i n ( 1 0 8 ) , p i n e a l g l a n d (173) and i n two of the a d e n y l c y c l a s e f r a c t i o n s f r o m S t r e p t o c o c c u s s a l i v a r i u s ( 3 8 ) . F l u o r i d e i n c r e a s e d maximal v e l o c i t y and d e c r e a s e d the s u b s t r a t e a f f i n i t y i n the t h i r d enzyme f r a c t i o n f r o m t h i s b a c t e r i a l s o u r c e ( 3 8 ) . P y r u v a t e s t i m u l a t e d a d e n y l c y c l a s e i n B r e v i b a c t e r i u m 1 i q u e f ac i e n s by i n c r e a s i n g maximal v e l o c i t y w i t h no change i n the K m f o r ATP ( 1 4 2 ) . A n t i d i u r e t i c hormone a l s o s t i m u l a t e d r e a c t i o n v e l o c i t y w i t h o u t a l t e r i n g the - 107 -K f o r ATP w i t h t o a d b l a d d e r enzyme ( 2 9 ) . An e x c e p t i o n m t o t h e s e f i n d i n g s o f p r i m a r i l y maximal v e l o c i t y e f f e c t s was r e p o r t e d by Rosen and Rosen w i t h the f r o g e r y t h r o c y t e enzyme ( 1 5 2 ) . The K f o r ATP was 1.25 mM i n the p r e s e n c e of i s o p r o p y l -m n o r e p i n e p h r i n e b ut was d e c r e a s e d t o 0.116 mM i n the p r e s e n c e of f l u o r i d e , w i t h b o t h a g e n t s p r o d u c i n g the same maximal v e l o c i t y . F l u o r i d e and e p i n e p h r i n e a l s o i n c r e a s e d r e a c t i o n v e l o c i t y at a l l c o n c e n t r a t i o n s o f m e t a l i o n s w i t h the c a r d i a c enzyme (28,108 and F i g . 2) and w i t h the s k e l e t a l m u s c l e enzyme ( F i g . 1 1 ) . M a x i m a l v e l o c i t y was i n c r e a s e d by f l u o r i d e and e p i n e -p h r i n e w i t h no s i g n i f i c a n t change i n the K f o r m e t a l i o n s . A s i m i l a r e f f e c t was n o t e d w i t h f l u o r i d e and e p i n e p h r i n e on b r a i n a d e n y l c y c l a s e ( 1 0 8 ) , w i t h g l u c a g o n and f l u o r i d e on the h e p a t i c enzyme (43,108,148) and w i t h f l u o r i d e and t h y r o i d -s t i m u l a t i n g hormone on t h y r o i d a d e n y l c y c l a s e ( 1 5 0 ) . In c o n t r a s t to t h e s e f i n d i n g s , B i r n b a u m e r , P o h l and R o d b e l l have r e p o r t e d t h a t t h e a f f i n i t y f o r magnesium was i n c r e a s e d by ACTH and f l u o r i d e ( 1 0 7 ) . T h i s d i s c r e p a n c y may r e s u l t f r o m the f a c t t h a t the f a t c e l l enzyme c o u l d n o t be s a t u r a t e d w i t h magnesium, whereas a d e n y l c y c l a s e f r o m h e a r t (28,108 and F i g . 2 ) , s k e l e t a l m u s c l e ( F i g . 7 ) , l i v e r ( 4 3 , 1 0 8 , 1 6 5 ) , b r a i n (108) and t h y r o i d (150) i s s a t u r a t e d w i t h magnesium. In g e n e r a l however, hormones and f l u o r i d e p r i m a r i l y r e s u l t i n i n c r e a s e d maximal v e l o c i t y of the a d e n y l c y c l a s e r e a c t i o n , w i t h no e f f e c t s on a f f i n i t y f o r m e t a l i o n s or s u b s t r a t e . E p i n e p h r i n e and f l u o r i d e i n h e a r t and s k e l e t a l m u s c l e may s t a b i l i z e or i n d u c e p a r t i c u l a r c o n f o r m a t i o n a l - 108 -s t a t e s o f a d e n y l c y c l a s e as was d i s c u s s e d p r e v i o u s l y f o r m e t a l i o n a c t i v a t i o n . T hese c o n f o r m a t i o n a l s t a t e s o f t h e enzyme w o u l d have t h e same a f f i n i t y f o r m e t a l i o n s and ATP, b u t w o u l d p o s s e s s a g r e a t e r c a t a l y t i c r e a c t i v i t y , r e s u l t i n g i n i n c r e a s e d m a x i m a l v e l o c i t y . T h u s , t h e r e g u l a t i o n of m y o c a r d i a l and s k e l e t a l m u s c l e a d e n y l c y c l a s e may be d e s -c r i b e d as a "V" a l l o s t e r i c s y s t e m , u s i n g the t e r m i n o l o g y o f Monod, Wyman and Changeux ( 1 8 5 ) . A c l a s s i c a l e x a m p l e o f a mammalian "V" a l l o s t e r i c enzyme i s f r u c t o s e 1 , 6 - d i p h o s -p h a t a s e f o r w h i c h AMP i s an a l l o s t e r i c i n h i b i t o r ( 1 8 6 ) . The same m e c h a n i s m of a l l o s t e r i c r e g u l a t i o n has a l s o b e e n a p p l i e d t o t h e a c t i v a t i o n of p r o t e i n k i n a s e i n s k e l e t a l m u s c l e by c y c l i c AMP ( 9 5 ) . S t u d i e s on t h e a c t i v a t i o n o f a d e n y l c y c l a s e by hormones and o t h e r a g e n t s a r e c o m p l i c a t e d by t h e p a r t i c u l a t e n a t u r e o f t h e enzyme. V a r i o u s m a t e r i a l s c o u l d i n f l u e n c e a d e n y l c y c l a s e a c t i v i t y i n d i r e c t l y by i n t e r a c t i n g w i t h t h e membrane and t h u s p o s s i b l y a f f e c t i n g t h e a c c e s s i b i l i t y o f s u b s t r a t e a t t h e c a t a l y t i c s i t e . I n s p i t e o f t h e s e c o n s t r a i n t s h o w e v e r , t h e s t u d y of t h e p r o p e r t i e s of t h e membrane-bound enzyme may p r o v i d e more r e l e v a n t i n f o r m a t i o n c o n c e r n i n g p h y s i o l o g i c a l r e g u l a t o r y m e c h a n i s m s t h a n t h e s t u d y o f h i g h l y p u r i f i e d , s o l u b i l i z e d p r e p a r a t i o n s . I t i s l i k e l y t h a t t h e r e g u l a t i o n of a d e n y l c y c l a s e i n membranes w i l l be c l o s e l y i n t e g r a t e d w i t h o t h e r membrane e n z y m a t i c p r o c e s s e s and membrane t r a n s p o r t s y s t e m s s i n c e t h e p l a s m a membrane i s s u c h an i m p o r t a n t f u n c t i o n a l u n i t of the c e l l . - 109 -The p r o p e r t i e s of adenyl c y c l a s e w i l l now be d i s c u s s e d i n terms of the r o l e of the enzyme i n hormonal a c t i o n . Hormones may be c o n s i d e r e d as the chemical i n t e r m e d i a t e s i n v o l v e d i n i n t e r c e l l u l a r communication i n m u l t i c e l l u l a r organisms (187). Since hormones are a p a r t of an inform-a t i o n a l t r a n s f e r u n i t , the mechanism of hormone a c t i o n i n v o l v i n g adenyl c y c l a s e has been d i s c u s s e d i n c y b e r n e t i c terms by Hechter e_t a_l_ (188) and more r e c e n t l y by Rodbell and h i s co-workers (189,190). The components of the i n f o r m a t i o n a l t r a n s f e r u n i t c o n s i s t of a d i s c r i m i n a t o r which r e c o g n i z e s the input s i g n a l (hormone), an a m p l i f i e r u n i t to a m p l i f y the input s i g n a l by enzymatica 1ly i n c r e a s i n g the i n t r a c e l l u l a r t i s s u e l e v e l s of c y c l i c AMP, and a t r a n s d u c e r which t r a n s f e r s the s i g n a l to the a m p l i f i e r f o l l o w i n g s i g n a l r e c o g n i t i o n by the d i s c r i m i n a t o r . These components of the i n f o r m a t i o n a l t r a n s f e r u n i t are shown s c h e m a t i c a l l y i n F i g . 16. The i n t e r a c t i o n of hormones with the d i s c r i m i n a t o r i s i n v o l v e d i n the s p e c i f i c i t y of t i s s u e responses to v a r i o u s hormones. Many t i s s u e s have the c a p a c i t y to respond to more than one hormone. Glucagon and e p inephrine a c t i v a t e adenyl c y c l a s e i n c a r d i a c t i s s u e (68,69) and the enzyme i n f a t c e l l s responds to a number of hormones (27,53-56). In examples of m u l t i p l e hormonal i n t e r a c t i o n s with adenyl c y c l a s e , the hormones a l l seem to act on the same c a t a l y t i c ( a m p l i f i e r ) u n i t of adenyl c y c l a s e , s i n c e combinations of maximal c o n c e n t r a t i o n s of the hormones are not a d d i t i v e . Each hormone i n t e r a c t s with a separate d i s c r i m i n a t o r , however, s i n c e p r o p r a n o l o l i n h i b i t e d adenyl c y c l a s e s t i m u l a t i o n by e p inephrine but not by glucagon - 110 -F i g u r e 16: Schematic r e p r e s e n t a t i o n of hormone a c t i o n on adenyl c y c l a s e as an i n f o r m a t i o n a l t r a n s f e r u n i t . DISCRIMINATOR TRANSDUCER input Hormone • Hormone bin d i n g •> Calc ium? Guanyl n u c l e o t i d e s F i g u r e 16 AMPLIFIER Adenyl c y c l a s e ATP 1» eye l i e ou tpu t AMP K P magnesium? f l u o r i d e ? - 112 -i n heart (68,69). S i m i l a r l y , b e t a - a d r e n e r g i c b l o c k i n g agents d i d not a f f e c t adipose t i s s u e adenyl c y c l a s e s t i m u l a t i o n by ACTH or glucagon (54,55) but d i d block s t i m u l a t i o n by epi n e -p h r i n e (27,55). Pretreatment of adipose t i s s u e with t r y p s i n completely a b o l i s h e d the s t i m u l a t o r y e f f e c t of glucagon on adenyl c y c l a s e with no e f f e c t on epi n e p h r i n e a c t i v a t i o n and only a p a r t i a l i n h i b i t i o n of the s t i m u l a t i o n by ACTH ( 5 6 ) . Calcium was r e q u i r e d f o r the s t i m u l a t i o n of f a t c e l l adenyl c y c l a s e by ACTH; EGTA blocked the enzyme a c t i v a t i o n by ACTH with no e f f e c t on the a c t i v a t i o n by ep i n e p h r i n e or glucagon (54-56). In a d d i t i o n , c e r t a i n ACTH analogs i n h i b i t e d only the s t i m u l a t o r y a c t i o n of ACTH (55). Braun and Hechter have shown that the ACTH d i s c r i m i n a t o r i n f a t c e l l s i s a separate molecular e n t i t y which can be d i f f e r e n t i a t e d by an absolute requirement f o r g l u c o c o r t i c o i d s (191). Adenyl c y c l a s e i n f a t c e l l membranes from adrenalectomized or hypophysectomized r a t s was s t i m u l a t e d by e p i n e p h r i n e , glucagon and f l u o r i d e but not by ACTH. It i s apparent that heart and adipose t i s s u e have d i s c r i m i n a t o r s which are separate and d i s t i n c t f o r each hormone. The b i n d i n g of l a b e l l e d glucagon and epi n e -p h r i n e to l i v e r plasma membranes and l a b e l l e d ACTH to adrenal membranes have been i n v e s t i g a t e d (192-194) i n d i c a t i n g that hormone b i n d i n g to the d i s c r i m i n a t o r can be examined by c l a s s i c a l b i n d i n g t e c h n i q u e s . The a m p l i f i e r u n i t c o n t a i n s the s i t e of the c a t a l y t i c r e a c t i o n of adenyl c y c l a s e . The input s i g n a l i s a m p l i f i e d by the enzymatic c o n v e r s i o n of ATP to c y c l i c AMP. Because of - 113 -the n o n - s e l e c t i v e n a t u r e of the s t i m u l a t i o n o f a d e n y l c y c l a s e i n v a r i o u s t i s s u e s by m e t a l i o n s and f l u o r i d e , i t i s p o s s i b l e t h a t t h e s e a g e n t s may a c t d i r e c t l y on the c a t a l y t i c u n i t of the a m p l i f i e r . A number of s t u d i e s have s u g g e s t e d t h a t f l u o r i d e does not a c t a t the d i s c r i m i n a t o r s i t e s i n c e the f l u o r i d e - s t i m u l a t e d enzyme a c t i v i t y can be d i s s o c i a t e d f r o m h o r m o n a l s t i m u l a t i o n . F l u o r i d e a p p e a r s t o promote maximal c a t a l y t i c r e a c t i v i t y i n most t i s s u e s . S t i m u l a t i o n of a d e n y l c y c l a s e by e p i n e p h r i n e c a n n o t be a c h i e v e d i n the p r e s e n c e of m a x i m a l l y - s t i m u l a t i n g c o n c e n t r a t i o n s of f l u o r i d e ( T a b l e I X ) ; however s t i m u l a t i o n by maximal c o n c e n t r a t i o n s of e p i n e p h r i n e was a d d i t i v e i n the p r e s e n c e of s u b - m a x i m a l c o n c e n t r a t i o n s of f l u o r i d e , s u g g e s t i n g t h a t f l u o r i d e and e p i n e p h r i n e a c t by d i f f e r e n t mechanisms b u t on the same a d e n y l c y c l a s e enzyme. The a c t i v a t i o n of a d e n y l c y c l a s e i n the p i n e a l g l a n d by f l u o r i d e i s n o t b l o c k e d by b e t a - a d r e n e r g i c b l o c k i n g a g e n t s ( 1 7 3 ) . A d i f f e r e n t i a l e f f e c t of t e m p e r a t u r e between f l u o r i d e -s t i m u l a t e d and A C T H - s t i m u l a t e d a d e n y l c y c l a s e i n f a t c e l l s has been n o t e d ( 1 0 7 ) . A d i s t i n c t s e p a r a t i o n between f l u o r i d e and hormone s t i m u l a t i o n of a d e n y l c y c l a s e can a l s o be a c h i e v e d by v a r i o u s d e t e r g e n t s and o t h e r a g e n t s . T r e a t m e n t of l i v e r p l a s m a membranes w i t h d i g i t o n i n and phospho 1 i p a s e A e l i m i n a t e d s t i m u l a t i o n by g l u c a g o n w i t h no e f f e c t on f 1 u o r i d e - s t i m u l a t e d enzyme a c t i v i t y ( 1 4 4 ) . The a d d i t i o n of membrane l i p i d s p a r t i a l l y r e s t o r e d the a b i l i t y of g l u c a g o n t o s t i m u l a t e a d e n y l c y c l a s e ( 1 9 5 ) . D i g i t o n i n t r e a t m e n t of f a t c e l l g h o s t s i n h i b i t e d the a c t i v a t i o n of a d e n y l c y c l a s e by ACTH, s e c r e t i n , e p i n e p h r i n e - 114 -and glucagon but not by f l u o r i d e ( 1 4 4 ) . A v a r i e t y of agents ( p h e n o t h i a z i n e s , d e t e r g e n t s , b a s i c p r o t e i n s , ionophores such as v a l i n o m y c i n , and l e c i t h i n a s e C) a b o l i s h e d the s e n s i t i v i t y of t h y r o i d adenyl c y c l a s e to t h y r o i d - s t i m u l a t i n g hormone without a l t e r i n g the response to f l u o r i d e ( 1 5 0 ) . S o l u b i l i z e d p r e p a r a t i o n s of adenyl c y c l a s e from cat heart are s t i m u l a t e d by f l u o r i d e but not by hormones ( 8 3 ) . F u r t h e r evidence concerning the s e p a r a t i o n of hormonal i n t e r a c t i o n at the d i s c r i m i n a t o r s i t e and the f l u o r i d e - s t i m u l a t e d enzyme a c t i v i t y was p r o v i d e d by s t u d i e s i n which the development of hormonal s e n s i t i v i t y by the adenyl c y c l a s e of the tadpole e r y t h r o c y t e d u r i n g metamorphosis occurred much l a t e r than the s e n s i t i v i t y to f l u o r i d e ( 1 9 6 ) . Hemolyzates of tadpole e r y t h r o c y t e s are s t i m u l a t e d by f l u o r i d e but not by catecholamines; s e n s i t i v i t y to catecholamines occurred only a f t e r the onset of t a i l shor t e n i n g . The transducer i s i n v o l v e d with the t r a n s l a t i o n of the e f f e c t s of hormonal b i n d i n g to the d i s c r i m i n a t o r i n t o the sequence of events l e a d i n g to the a c t i v a t i o n of adenyl c y c l a s e i n the a m p l i f i e r u n i t . Calcium was r e q u i r e d f o r ACTH s t i m u l -a t i o n of adenyl c y c l a s e i n f a t c e l l s ( 5 4 - 5 6 , 1 9 7 ) and i n the adrenal ( 1 9 7 ) . This e f f e c t of c a l c i u m was s e l e c t i v e f o r ACTH s t i m u l a t i o n , s i n c e the removal of ca l c i u m by EGTA had l i t t l e e f f e c t on b a s a l a c t i v i t y or on the s t i m u l a t i o n of enzyme a c t i v i t y by other hormones. However, the b i n d i n g of ACTH to adrenal membranes was not i n h i b i t e d i n the absence of calcium ( 1 9 8 ) . T h e r e f o r e , c a l c i u m may be r e q u i r e d at the transducer s i t e to allow the b i n d i n g of ACTH at the d i s c r i m i n a t o r s i t e to r e s u l t - 115 -i n t h e a c t i v a t i o n of a d e n y l c y c l a s e a t t h e a m p l i f i e r s i t e . A. s i m i l a r r o l e f o r g u a n y l n u c l e o t i d e s may be i n v o l v e d i n g l u c a g o n -s t i m u l a t i o n of a d e n y l c y c l a s e i n l i v e r membranes ( 1 4 5 ) and t h e e p i n e p h r i n e s t i m u l a t i o n o f a d e n y l c y c l a s e i n s k e l e t a l m u s c l e p l a s m a membranes ( T a b l e s X, X I ; F i g . 1 5 ) . The a d d i t i o n o f low c o n c e n t r a t i o n s o f GTP o r GDP s e l e c t i v e l y i n c r e a s e d t h e s e n s i t i v i t y o f s k e l e t a l m u s c l e a d e n y l c y c l a s e t o e p i n e p h r i n e , w i t h l i t t l e o r no e f f e c t on b a s a l o r f l u o r i d e - s t i m u l a t e d a c t i v i t y ( F i g . 1 5 ) . T h i s e f f e c t o f g u a n y l n u c l e o t i d e s w o u l d be c o n s i s t e n t w i t h an a c t i o n a t t h e t r a n s d u c e r u n i t t o i m p r o v e t h e t r a n s f e r of i n f o r m a t i o n b e t w e e n t h e r e c o g n i t i o n o f t h e s i g n a l a t t h e d i s c r i m i n a t o r and t h e c a t a l y t i c a c t i v i t y a t t h e a m p l i f i e r . A s p e c i f i c i n v o l v e m e n t o f g u a n y l n u c l e o t i d e s a t t h e t r a n s d u c e r u n i t w o u l d a l s o e x p l a i n t h e l a c k o f e f f e c t of g u a n y l n u c l e o t i d e s on b a s a l and f 1 u o r i d e - s t i m u 1 a t e d enzyme a c t i v i t y a t t h e a m p l i f i e r s i t e . P a r a d o x i c a l l y , g u a n y l n u c l e o t i d e s d e c r e a s e d t h e b i n d i n g and b i n d i n g a f f i n i t y o f l a b e l l e d g l u c a g o n and i n c r e a s e d t h e r a t e o f d i s s o c i a t i o n o f bound l a b e l l e d g l u c a g o n w i t h l i v e r p l a s m a membranes ( 1 9 9 ) . E x p e r i m e n t s a r e c u r r e n t l y i n p r o g r e s s t o d e t e r m i n e t h e e f f e c t of g u a n y l n u c l e o t i d e s on t h e b i n d i n g o f l a b e l l e d e p i n e p h r i n e t o s k e l e t a l m u s c l e p l a s m a membranes, a l t h o u g h p r e l i m i n a r y r e s u l t s s u g g e s t t h a t GTP does n o t a f f e c t e p i n e p h r i n e b i n d i n g a t t h e d i s c r i m i n a t o r s i t e . As m e n t i o n e d p r e v i o u s l y , h o r m o n a l s t i m u l a t i o n o f a d e n y l c y c l a s e i n v a r i o u s t i s s u e s i s more s e n s i t i v e t o d e t e r g e n t t r e a t m e n t t h a n t he b a s a l and f l u o r i d e -s t i m u l a t e d a c t i v i t y ( 1 4 4 , 1 5 0 ) . H o r m o n a l r e s p o n s i v e n e s s o f s o l u b i l i z e d c a r d i a c a d e n y l c y c l a s e p r e p a r a t i o n s c a n be r e s t o r e d - 116 -by the a d d i t i o n of p h o s p h o l i p i d s (84,85). I t would be of i n t e r e s t to determine i f the p h o s p h o l i p i d s are r e q u i r e d f o r the b i n d i n g of the hormones to the d i s c r i m i n a t o r or f o r the t r a n s f e r of i n f o r m a t i o n by the transducer u n i t . D i g i t o n i n and phospholipase A treatment of l i v e r plasma membranes did decrease the b i n d i n g of l a b e l l e d glucagon; b i n d i n g was p a r t i a l l y r e s t o r e d by the a d d i t i o n of membrane l i p i d s (195). It i s obvious that the membrane-bound adenyl c y c l a s e system i s very complex. The p r e c e d i n g d i s c u s s i o n has d e a l t with a c o n c e p t u a l approach to the q u e s t i o n of the mechanism of a c t i o n of hormones on t a r g e t t i s s u e s i n v o l v i n g adenyl c y c l a s e . However, the components of the i n f o r m a t i o n a l t r a n s f e r u n i t r e f e r r e d to as the d i s c r i m i n a t o r , t r a n s d u c e r , and a m p l i f i e r can be envisaged as being macromolecular p r o t e i n subunits of the membrane. The t r a n s f e r of i n f o r m a t i o n could i n v o l v e membrane conformation changes induced by subunit i n t e r a c t i o n s c o n s i s t e n t with c u r r e n t models r e l a t i n g membrane s t r u c t u r e and f u n c t i o n . This concept of hormone a c t i o n i s based p r i m a r i l y on i n d i r e c t methods, and so f u r t h e r i n v e s t i g a t i o n s w i l l have to deal with the i n d i v i d u a l membrane components. B i n d i n g s t u d i e s with l a b e l l e d hormones to the d i s c r i m i n a t o r u n i t w i l l y i e l d u s e f u l i n f o r m a t i o n . The b i n d i n g of epine-p h r i n e to l i v e r plasma membranes (193) and of ACTH to adrenal membranes ( 2 0 0 ) .has been shown to i n v o l v e both high and low-a f f i n i t y s i t e s , with the g r e a t e s t number of s i t e s being of the l o w - a f f i n i t y type. S o l u b i l i z a t i o n and p u r i f i c a t i o n of the c a t a l y t i c a c t i v i t y w i l l allow the d e t e r m i n a t i o n of the r e a c t i o n mechanism and the p r e c i s e mode of a c t i o n of f l u o r i d e . The adenyl c y c l a s e enzyme r e a c t i o n i s r e v e r s i b l e (201,202) - 117 -and so h i g h l y p u r i f i e d p r e p a r a t i o n s w i l l a l s o a l l o w the e x a m i n a t i o n of t h e mechanism of the r e v e r s e r e a c t i o n . P u r i f i c a t i o n of the t r a n s d u c e r component w i l l p e r m i t the e v a l u a t i o n of i t s r o l e i n the p r o c e s s of i n f o r m a t i o n t r a n s f e r . The i s o l a t i o n of the v a r i o u s c omponents, the d e t e r m i n a t i o n of t h e i r p h y s i c a l and c h e m i c a l p r o p e r t i e s and the i n v e s t i g a t i o n of the complex i n t e g r a t i o n of the f u n c t i o n a l and s t r u c t u r a l components i n v o l v e d i n h o r m o n a l a c t i o n and a d e n y l c y c l a s e r e g u l a t i o n w i l l p r o v i d e c h a l l e n g i n g p r o b l e m s f o r f u t u r e r e s e a r c h . - 118 -BIBLIOGRAPHY 1. S u t h e r l a n d , E.W., Recent Progr. Hormone Res., 5_, 441 (1950). 2. S u t h e r l a n d , E.W. and C o r i , C.F., J . B i o l . Chem., 188, 531 (1951). 3. R a i l , T.W., Su t h e r l a n d , E.W. and W o s i l a i t , W.D., J . B i o l . Chem., 218, 483 (1956) . 4. R a i l , T.W., S u t h e r l a n d , E.W. and B e r t h e t , J . , J . B i o l . Chem. , _224, 463 (1957) . 5. S u t h e r l a n d , E.W. and R a i l , T.W., J . Am. Chem. S o c , 79 , 3608 (1957). 6. S u t h e r l a n d , E.W. and R a i l , T.W., J . B i o l . Chem., 232, 1077 (1958). 7. R a i l , T.W. and S u t h e r l a n d , E.W., J . B i o l . Chem., 23 2, 1065 (1958). 8. S u t h e r l a n d , E.W. and R a i l , T.W., Pharmacol. Rev., 1_2, 265 (1960) . 9. Su t h e r l a n d , E.W., 0ye, I . and Butcher, R.W., Recent Progr. Hormone Res., _2_1 , 623 (1965). 10. Robison, G.A., Butcher, R.W. and Su t h e r l a n d , E.W., Ann. Rev. Biochem., 3_7, 149 (1968). 11. S u t h e r l a n d , E.W., Robison, G.A. and Butcher, R.W., C i r c u l a t i o n , 37, 279 (1968). 12. S u t h e r l a n d , E.W.,and Robison, G.A., D i a b e t e s , 18, 797 (1969). 13. J o s t , J.-P. and Rickenberg, H.V., Ann, Rev. Biochem,, 40 741 (1971). 14. Robison, G.A., Butcher, R.W. and Su t h e r l a n d , E.W., C y c l i c AMP, Academic P r e s s , New York (1971). 15. Costa, E. and Greengard, P. ( E d i t o r s ) , Role of C y c l i c AMP i n C e l l F u n c t i o n , Advances i n Bioch e m i c a l Ps yc hop harms: o lo gy , V o l . I l l , Raven P r e s s , New York (1970). 16. C o n d l i f f e , P. and R o d b e l l , M, ( E d i t o r s ) , Colloquium on the  Role of Adenyl Cyclase and C y c l i c AMP i n B i o l o g i c a l P r o c e s s e s , Fogarty I n t e r n a t i o n a l Center Proceedings No. 4~j United States Government P r i n t i n g O f f i c e , Washington (1971). - 1 1 9 -1 7 . R o b i s o n , G.A., Nahas , G.G. and T r i n e r , L . ( E d i t o r s ) , C y c l i c AMP and C e l l F u n c t i o n , A n n a l s of t h e New Yo r k Academy of S c i e n c e s , V o l . 1 8 5 , New Yo r k Academy of S c i e n c e s , New Y o r k , ( 1 9 7 1 ) . 1 8 . S u t h e r l a n d , E.W., R a i l , T.W. and Menon, T., J. B i o l . Chem. , 2 3 7 , 1 2 2 0 ( 1 9 6 2 ) . 1 9 . R a i l , T.W. and S u t h e r l a n d , E.W., J. B i o l . Chem., 2 3 7 , 1 2 2 8 ( 1 9 6 2 ) . 2 0 . B u t c h e r , R.W. and S u t h e r l a n d , E.W., J. B i o l . Chem., 2 3 7 1 2 4 4 ( 1 9 6 2 ) . 2 1 . A z h a r j S. and K r i s h n a M u r t i , C.R., B i o c h e m . B i o p h y s . R e s . Comm. , 4 3 _ , 5 8 ( 1 9 7 1 ) . 2 2 . Solomon, D. and M a s c a r e n h a s , J.P., L i f e S c i e n c e s , 1 0 , p a r t I I , 8 7 9 ( 1 9 7 1 ) . 2 3 . W e i s s , B. and C o s t a , E . , B i o c h e m . P h a r m a c o l . , 1 7 , 2 1 0 7 ( 1 9 6 8 ) . 2 4 . Dousa, T. and R y c h l x k , I., L i f e S c i e n c e s , 7 , p a r t I I , 1 0 3 9 ( 1 9 6 8 ) . 2 5 . B u r k e , G. , E n d o c r i n o l o g y , _ 8 6 _ , 3 4 6 ( 1 9 7 0 ) . 2 6 . S k a l a , J . , Hahn, P. and B r a u n , T., L i f e S c i e n c e s , 9 , p a r t I, 1 2 0 1 ( 1 9 7 0 ) . 2 7 . Vaughan, M. and Murad , F., B i o c h e m i s t r y , J 5 , 3 0 9 2 ( 1 9 6 9 ) . 2 8 . Drummond, G.I. and Duncan, L . , J. B i o l . Chem., 2 4 5 , 9 7 6 ( 1 9 7 0 ) . 2 9 . H y n i e , S. and S h a r p , G.W.G., B i o c h i m . B i o p h y s . A c t a , 2 3 0 , 4 0 ( 1 9 7 1 ) . 3 0 . R o s e n , O.M. and Rosen, S.M., B i o c h e m . B i o p h y s . R e s . Comm., 3 1 , 8 2 ( 1 9 6 8 ) . 3 1 . R a b i n o w i t z , M., D e s a l l e s , L . , M e i s l e r , J. and L o r a n d , L ., B i o c h i m . B i o p h y s . A c t a , 9 7 2 9 ( 1 9 6 5 ) . 3 2 . Hofman, L . F . , Ph.D. T h e s i s , U n i v e r s i t y of W a s h i n g t o n ( 1 9 7 0 ) . 3 3 . H e c h t e r , 0 . , B a r , H.P., M a t s u b a , M. and S o i f e r , D., L i f e S c i e n c e s , _ 8 , p a r t I, 9 3 5 ( 1 9 6 9 ) . 3 4 . S a t r e , M. , Chambaz, E.M. and V i g n a i s , P.V., FEBS L e t t e r s , 1 2 , 2 0 7 ( 1 9 7 1 ) . - 120 -35 . S c h o r r , I . and Ney, R.L., J . C l i n . I n v e s t . , 50.> 1295 (1971) . 36. T a u n t o n , O.D., R o t h , J . and P a s t a n , I . , J . B i o l . Chem., 244, 247 (1969) . 37. Menon, K.M.J, and S m i t h , M. , B i o c h e m i s t r y , 1_0 , 1186 (1971) . 38. K h a n d e l w a l , R. L. and H a m i l t o n , I.R., J . B i o l . Chem. 246 3297 (1971) . 39 . I d e , M., A r c h . B i o c h e m . B i o p h y s . , 144, 262 (1971) . 40 . Tao, M. and Huberman, A., A r c h . B i o c h e m . B i o p h y s . , 141 236 (1970) . 4 1 . Murad, F., C h i , Y.-M., R a i l , T.W. and S u t h e r l a n d , E.W., J . B i o l . Chem., 237, 1233 (1962) . 42. B i t e n s k y , M.W., R u s s e l l , V. and R o b e r t s o n , W., Bi o c h e m . B i o p h y s . R e s . Comm., 3JL_> 706 (1968) . 43 . Hepp, K.D., E d e l , R. and W i e l a n d , 0 . , E u r . J . B i o c h e m . , 17, 171 (1970) . 44 . 0ye , I, and S u t h e r l a n d , E.W., B i o c h i m . B i o p h y s . A c t a , 127 347 (1966) . 45 . S h e p p a r d , H. and B u r g h a r d t , C.R., M o l . P h a r m a c o l . , 6, 425 (1970) . ~~ 46. P a s t a n , I . and K a t z e n , R., B i o c h e m . B i o p h y s . R es. Comm., 29, 792 (1967) . 47. C h a s e , L.R. and A u r b a c h , G.D., S c i e n c e , 159 , 545 (1968) . 48 . M a r c u s , R. and A u r b a c h , G.D., E n d o c r i n o l o g y , j3_5 , 801 (1969) . 49. Dousa, T., H e c h t e r , 0 . , S c h w a r t z , I . L . and W a l t e r , R., P r o c . N a t . A c a d . S c i . ( U S A ) , 6_8, 1693 ( 1971). 50. B a r , H.P., H e c h t e r , 0 . , S c h w a r t z , I . L . and W a l t e r , R., P r o c . N a t . A c a d . S c i . ( U S A ) , 6j7_, 7 ( 1970). 51 . C h a s e , L.R., Fed a k , S.A. and A u r b a c h , G.D., E n d o c r i n o l o g y , 84 , 761 (1969) . 52. C h a s e , L.R. and A u r b a c h , G.D., J . B i o l . Chem., 245, 1520 (1970) . 53. W i l l i a m s , R.H., Wals h , S.A., Hepp, D.K. and E n s i n c k , J.W. M e t a b o l i s m , 17, 653 (1968) . - 121 -54. B a r , H.P. and Hechter, 0., Proc. Nat. Acad. S c i . (USA), 63_, 350 (1969). 55. Birnbaumer, L. and R o d b e l l , M. , J. B i o l . Chem., 244 , 3477 (1969). 56. R o d b e l l , M. , Birnbaumer, L. and Pohl , S.L., J . B i o l . Chem., 245, 718 (1970). 57. Schorr, I . , Rathnam, P., Saxena, B.B. and Ney, R.L., J. B i o l . Chem., 246 , 5806 (1971). 58. Mayer, S.E., Fed. P r o c , 29^ , 1367 (1970). 59. E p s t e i n , S.E., S k e l t o n , C.L., Levey, G.S. and Entman, M. , Annals I n t . Med., ]_2, 561 (1970). 60. E p s t e i n , S.E., Levey, G.S. and S k e l t o n , C.L., C i r c u l a t i o n , 4_3 , 437 (1971). 61. Cheung, W.Y. and W i l l i a m s o n , J.R., Nature, 207 , 979 ( 1965). 62. Robison, G.A., Butcher, R.W., 0ye , I . , Morgan, H.E. and Sut h e r l a n d , E.W., Mol. Pharmacol., 1_, 168 (1965). 63. Hammermeister, K.E., Yun i s , A.A. and Krebs, E.G., J. B i o l . Chem., 240, 986 (1965) . 64. Drummond, G.I., Duncan, L. and Hertzman, E., J. B i o l . Chem., 241, 5899 (1966). 65. Namm, D.H. and Mayer, S.E., Mol. Pharmacol., 4_, 61 (1968). 66. LaRaia, P.J. and Reddy, W.J., Biochim. Biophys. A c t a , 17 7, 189 (1969). 67. Lee, T.P., Kuo, J.F. and Greengard, P., Biochem. Biophys. Res. Comm., 4_5 , 991 (1971). 68. Murad, F. and Vaughan, M. , Biochem. Pharmacol., 18, 1053 (1969). 69. Levey, G.S. and E p s t e i n , S.E., C i r c . Res., 2_4 , 151 (1969). 70. Mayer, S.E. and Moran, N.C., J. Pharmacol. Exp. Therap., 129, 271 (1960). 71. Mayer, S.E., Namm, D.H. and R i c e , L., C i r c . Res., 26, 225 (1970). — 72. Levey, G.S. and E p s t e i n , S.E., Biochem. Biophys. Res. Comm., 33, 990 (1968). - 122 -73. K l e i n , I. and Levey, G.S., J . C l i n . I n v e s t . , 5_0, 1 0 1 2 (1971) . 74. K l e i n , I . and Levey, G.S., Metabolism, 20., 890 (1971). 75. Levey, G.S., Palmer, R.F., L a s s e t e r , K.C. and McCarthy, J . , J . C l i n . E n d o c r i n . Metab., _33 . 371 (1971). 76. LaRaia, P.J. and Sonnenblick, E.H., C i r c . Res., ^ _8, 377 (1971) . 77. Sobel, B.E., Dempsey, P.J. and Cooper, T., Biochem. Biophys. Res. Comm., 33_, 758 (1968). 78. Levey, G.S., S k e l t o n , C.L. and E p s t e i n , S.E., E n d o c r i n o l o g y , 85, 1004 (1969). 79. Levey, G.S., Am. J . Med., 50_, 413 (1971). 80. Sobel, B.E., Henry, P.D., Robison, A., B l o o r , C. and Ross, J . , C i r c . Res., 24_, 507 (1969). 81. G e r t l e r , M.M., S a l u s t e , E. and Spencer, F., Proc. Soc. Exp. B i o l . Med., 135, 817 (1970). 82. Levey, G.S., P r i n d l e , K.H. and E p s t e i n , S.E., J . Mol. C e l l . C a r d i o l . , _1 , 403 (1970) . 83. Levey, G.S., Biochem. Biophys. Res. Comm., 3_8, 86 (1970). 84. Levey, G.S., Biochem. Biophys. Res. Comm., 43^, 108 (1971). 85. Levey, G.S., J . B i o l . Chem., 246, 7405 (1971). 86. Brody, T.M. and M c N e i l l , J.H., Fed. P r o c , _29_, 1375 (1970). 87. Raper, C., C i r c . Res., Supp . I l l , 147 (1967). 88. Posner, J.B., S t e r n , R. and Krebs, E.G., J . B i o l . Chem., 240, 982 (1965). 89. Lyon, J.B. and Mayer, S.E., Biochem. Biophys. Res. Comm., 34, 459 (1969). 90. Drummond, G.I. Harwood, J.P. and Powell, C.A., J . B i o l . Chem. , 244 , 4235 (1969) . 91. Krebs, E.G., Graves, D.J. and F i s c h e r , E.H., J . B i o l . Chem., 234, 2867 (1959). 92. Krebs, E.G., Love, D.S., B r a t v o l d , G.E., T r a y s e r , K.A., Meyer, W.L. and F i s c h e r , E.H., B i o c h e m i s t r y , 3, 1022 (1964). - 123 -93. DeLange, R.J., Kemp, R.G., R i l e y , W.D., Cooper, R. A. and Krebs, E.G., J. B i o l . Chem., 243, 2200 (1968). 94. Walsh, D.A., P e r k i n s , J.P. and Krebs, E.G., J. B i o l . Chem., 243 , 3763 (1968). 95. Reimann, E.M., Walsh, D.A. and Krebs, E.G., J. B i o l . Chem., 246 , 1986 (1971). 96. Reimann, E.M. Brostrom, CO., C o r b i n , J.D., King, C.A. and Krebs, E.G., Biochem. Biophys. Res. Comm., 4_2, 187 (1971). 97. S c u l l , J.T. and Mayer, S.E., J. B i o l . Chem., 246, 5716 (1971). 98. Friedman, D.L. and Larner , J . , B i o c h e m i s t r y , 2, 669 (1963). 99. C r a i g , J.W. and L a r n e r , J. , Nature, 202, 971 (1964). 100. W i l l i a m s , B.J. and Mayer, S.E., Mol. Pharmacol., 2, 454 (1966) . 101. Rose 11-Perez, M. and La r n e r , J. , B i o c h e m i s t r y , 3_, 81 (1964). 102. H u i j i n g , F. and L a r n e r , J ., Biochem. Biophys. Res. Comm., 23 , 259 (1966). 103. Schlender, K.K., Wei, S.H. and V i 1 l a r - P a l a s i , C., Biochim. Biophys. A c t a , 191, 272 (1969). 104. S o d e r l i n g , T.R., Hiekenbottorn, J.P., Reimann, E.M., Hunkeler, F.L., Walsh, D.A. and Krebs, E.G., J. B i o l . Chem. 24 5, 6317 (1970). 105. S e r a y d a r i a n , K. and Mommaerts, W.F.H.M., J. C e l l . B i o l . 26, 641 (1965). 106. K l a i n e r , L.M., C h i , Y.-M., F r e i d b e r g , S.L., R a i l , T.W. and Sut h e r l a n d , E.W., J . B i o l . Chem., 237 , 1239 (1962). 107. Birnbaumer, L. , P o h l , S.L. and R o d b e l l , M., J. B i o l . Chem., 244 , 3468 (1969). 108. Drummond, G.I., Severson, D.L. and Duncan, L., J. B i o l . Chem., 246, 4166 (1971). 109. Bllcher, T. and P f l e i d e r e r , G., i n S.P. Colowick and N.O. Kaplan, ( E d i t o r s ) , Methods i n Enzymology V o l . 1, pg. 435, Academic P r e s s , New York (1955). - 1 2 4 -110. K r i s h n a , G., Weiss, B. and Brodie , B.B., J. Pharmacol. Exp. Therap., 163, 379 (1968). 111. Bray, G.A., A n a l . Biochem. _1, 279 (1960). 112. F i s k e , C.H. and SubbaRow, Y., J. B i o l . Chem., _66, 375 (1925) . 113. N o r d l i e , R.C. and A r i o n , W.J. i n S.P. Colowick and N.O. Kaplan ( E d i t o r s ) , Methods i n Enzymology, V o l . 9, pg. 621, Academic Press , New York (19 66) . 114. Drummond, G.I., Eng. D.Y. and Mcintosh, C.A., B r a i n Res., 28, 153 (1971). 115. Hers, H.G. and vanHoof, F. i n S.P. Colowick and N.O. Kaplan ( E d i t o r s ) , Methods i n Enzymology, V o l . 8, pg. 531, Academic P r e s s , New York (1966) . 116. C o o p e r s t e i n , S.J. and Lazarow, A., J . B i o l . Chem., 189, 665 (1951). 117. Ellraan, G.L., Courtney, K.D., Andres, V. and Featherstone , R.M., Biochem. Pharmacol., 7_, 88 (1961). 118. Zak, B., Dickenman, R.C., White, F.C., B u r n e t t , H. and Cherney, P.J., Am. J. C l i n . Path., 24_, 1307 (1954). 119. B a r t l e t t , G.R., J. B i o l . Chem., 234, 466 (1959). 120. Warren, L. , J. B i o l . Chem., 234, 1971 (1959). 121. Svennerholm, L., A c t a . Chem. Scand., 1_2, 547 (1958). 122. Yamamura, S.S., Wade, M.A. and S i k e s , J.H., A n a l . Chem., 34, 1308 (1962). 123. Lowry, O.H., Rosebrough, N.J., F a r r , A.L. and R a n d a l l , R.J., J. B i o l . Chem., 193 , 265 (1951). 124. BHr, H.P. and Hechter, 0., A n a l . Biochem., Z9_, 476 (1969). 125. Emmelot, P. and Bos, C.J., Biochim. Biophys. A c t a . , 249 285 (1971). 126. Entman, M.L., Levey, G.S. and E p s t e i n , S.E., Biochem. Biophys. Res. Comm., 3_5 , 728 ( 1969). 127. D h a l l a , N.S., Sulakhe, P.V., Khandelwal, R.L. and Hamilton, I.R., L i f e S c i e n c e s , p a r t I, 625 (1970). 128. P o r t i u s , H.J. and Repke, K.R.H., A c t a . B i o l . Med. German., 19, 879 (1967). - 125 -1 2 9 . M c C o l l e s t e r , D.L., Biochim. Biophys. A c t a , 57_, 4 2 7 ( 1 9 6 2 ) . 1 3 0 . Kono, T. and Colowick, S.P., Arch Biochem. Biophys., 9 3 _ , 5 2 0 ( 1 9 6 1 ) . 1 3 1 . Severson, D.L., Drummond, G.I. and Sulakhe, P.V., J . B i o l . Chem., accepted f o r p u b l i c a t i o n , May, 1 9 7 2 . 1 3 2 . P e t e r , J.B., Biochem. Biophys. Res. Comm., 4 _ 0 , 1 3 6 2 ( 1 9 7 0 ) . 1 3 3 . Sulakhe, P.V., Fed e l e s o v a , M., McNamara, D.B. and D h a l l a , N.S., Biochem. Biophys. Res. Comm., 4 _ 2 , 7 9 3 ( 1 9 7 1 ) . 1 3 4 . Ferdman, D.L., Himmelreich, N.G. and Dyadyusha, G.P., Biochim. Biophys. A c t a , 2 1 9 , 3 7 2 ( 1 9 7 0 ) . 135. Coleman, R. and Fin e a n , J.B., Biochim. Biophys. A c t a , 125, 197 (1966). 136. F i e h n , W., P e t e r , J.B., Mead, J.F. and Gan-Elepano, M., J . B i o l . Chem., 246, 5617 (1971). 137. Boyer, P.D., Lardy, H.A. and P h i l l i p s , P.H., J . B i o l . Chem., 149, 529 (1943). 138. P e r k i n s , J.P. and Moore, M.M., J . B i o l . Chem., 246, 62 (1971) . 139. Schramm, M. and Nairn, E., J . B i o l . Chem., 245, 3225 (1970). 140. Pastan, I . , P r i c e r , W. and Bla n c h e t t e - M a c k i e , J . , Metab-o l i s m , 1_9 , 809 (1970) . 141. K e l l y , L.A. and K o r i t z , S.B., Biochim. Biophys. A c t a , 237, 141 (1971). 142. H i r a t a , M. and H a y a i s h i , 0 . , Biochim. Biophys. A c t a , 149, 1 (1967). 143. Tao, M. and Lipmann, F., Proc. Nat. Acad. S c i . (USA), 63, 86 (1969). — 144. Birnbaumer, L. , P o h l , S.L. and R o d b e l l , M. , J . B i o l . Chem., 246, 1857 (1971). 145. R o d b e l l , M., Birnbaumer, L., P o h l , S.L. and Krans, M.J., J . B i o l . Chem., 246, 1877 (1971). - 1 2 6 -1 4 6 . R o d b e l l , M. , J . B i o l . Chem., 2 4 2 , 5 7 4 4 ( 1 9 6 7 ) . 1 4 7 . M c K e e l , D.W. and J a r e t t , L . , J . C e l l B i o l . , 4 4 - , 4 1 7 ( 1 9 7 0 ) . 1 4 8 . P o h l , S.L., B i r n b a u m e r , L . and R o d b e l l , M., J . B i o l . Chem. , _ 2 4 6 _ , 1 8 4 9 ( 1 9 7 1 ) . 1 4 9 . Y a m a s h i t a , K. and F i e l d , J.B., B i o c h e m . B i o p h y s . R e s . Comm. , _ 4 0 , 1 7 1 ( 1 9 7 0 ) . 1 5 0 . W o l f f , J . and J o n e s , A.B., J . B i o l . Chem., 2 4 6 , 3 9 3 9 ( 1 9 7 1 ) . 1 5 1 . D a v o r e n , P.R. and S u t h e r l a n d , E.W., J . B i o l . Chem., 2 3 8 , 3 0 1 6 ( 1 9 6 3 ) . 1 5 2 . Rosen, O.M. and Rosen, S.M., A r c h , B i o c h e m . B i o p h y s . , 1 3 1 , 4 4 9 ( 1 9 6 9 ) . 1 5 3 . R o s e n t h a l , S.L., E d e l m a n , P.M. and S c h w a r t z , I . L . , B i o c h i m . B i o p h y s . A c t a , 1 0 9 , 5 1 2 ( 1 9 6 5 ) . 1 5 4 . W e s t o r t , C. and H u l t i n , H.O., A n a l . B i o c h e m . , J J 6 , 3 1 4 ( 1 9 6 6 ) . 1 5 5 . C o l b e a u , A., N a c h b a u r , J . and V i g n a i s , P.M., B i o c h i m . B i o p h y s . A c t a , 2 4 9 , 4 6 2 ( 1 9 7 1 ) . 1 5 6 . Boegraan, R . J . , Manery, J . F . and P i n t e r i c , L . , B i o c h i m . B i o p h y s . A c t a , 2 0 3 , 5 0 6 ( 1 9 7 0 ) . 1 5 7 . McNamara, D.B., S u l a k h e , P.V. and D h a l l a , N.S., B i o c h e m , J . , 1 _ 2 5 , 5 2 5 ( 1 9 7 1 ) . 1 5 8 . S u l a k h e , P.V. and D h a l l a , N.S., L i f e S c i e n c e s , 1 0 , p a r t I , 1 8 5 ( 1 9 7 1 ) . — 1 5 9 . D i e t z e , G. and Hepp, K.D., B i o c h e m . B i o p h y s . R e s . Comm., 4 4 , 1 0 4 1 ( 1 9 7 1 ) . 1 6 0 . K o k e t s u , K., K i t a m u r a , R. and T a n a k a , R., Am. J . P h y s i o l . , 2 0 7 , 5 0 9 ( 1 9 6 4 ) . 1 6 1 . S h l a t z , L . and M a r i n e t t i , G.V., B i o c h e m . B i o p h y s . Res. Coram., 4 5 , 5 1 ( 1 9 7 1 ) . 1 6 2 . S c h a t z m a n n , H.J. and V i n c e n z i , F . F . , J . P h y s i o l . ( L o n d o n ) , 2 0 1 , 3 6 9 ( 1 9 6 9 ) . 1 6 3 . Cha, Y.N., S h i n , B.C. and L e e , K.S., J . Gen. P h y s i o l . , 5 7 , 2 0 2 ( 1 9 7 1 ) . - 127 -164. Repke, D.I. and Katz, A.M., Biochim. Biophys. A c t a , 172, 348 (1969). 165. Gorman, R.E. and B i t e n s k y , M.W., E n d o c r i n o l o g y , 87 , 1075 (1970). 166. Burke, G., Biochim. Biophys. A c t a , 220, 30 (1970). 167. C a s i l l a s , E.R. and Hoskins, D .D . , Arch. Biochem. Biophys., 1_4_7, 148 (1971). 168. Marcus, R. and Aurbach, G.D., Biochim. Biophys. A c t a , 242, 410 (1971). 169. Ray, T.K., Tomasi, V. and M a r i n e t t i , G.V., Biochim. Biophys. A c t a , 211, 20 (1970). 170. Mildvan, A.S. i n P.D. Bayer ( E d i t o r ) , The Enzymes, T h i r d e d i t i o n , pg. 445, Academic P r e s s , New York (19 70) . 171. Keech, B. and B a r r i t t , G.J., J . B i o l . Chem., 24 2, 1983 (1967). 172. Hewitt, E . J . and N i c h o l a s , D.J.D. i n R.M. Hochster and J.H. Quastel ( E d i t o r s ) , M e t a b o l i c I n h i b i t o r s , V o l . I I , pg. 311, Academic P r e s s , New York (1963) . 173. Weiss, B., J . Pharmacol. Exp. Therap., 166, 330 (1969). 174. Kaneko, T., Zor, U. and F i e l d , J.B., S c i e n c e , 163, 1062 (1969). 175. Exton, J.H., Robison, G.A., S u t h e r l a n d , E.W. and Park, C.R., J . B i o l . Chem., 246, 6166 (1971). 176. F e i g , S.A., Shohet, S.B. and Nathan, D.G., J . C l i n . I n v e s t . , 50, 1731 (1971). 177. Lardy, H.A. and P h i l l i p s , P.H., J . B i o l . Chem., 148, 343 (1943). 178. Schmidt, M.J., Palmer, E.C., Dett b a r n , W-D., Robison, G.A., Dev. P s y c h o b i o l . , _3> 53 (1970). 179. Hynie, S. and Sharp, G.W.G., J . E n d o c r i n . , _50 , 231 (1971). 180. E l l i s , S., D a v i s , A.H. and Anderson, H.L., J . Pharmacol. Exp. Therap., 115, 120 (1955). 181. C r a i g , J.W., R a i l , T.W. and Larn e r , J . , Biochim. Biophys. Acta , 1_7_7 , 213 (1969) . 182. Jungas, R.L., Proc. Nat. Acad. S c i . (USA), _56 , 757 (1966). 183. C r y e r , P.E., J a r e t t , L. and K i p n i s , D.M., Biochim. Biophys. Acta , 177 , 586 (1969) . - 128 -184. Marsh, J.M., J . B i o l . Chem., 245 , 1596 (1970). 185. Monod, J . , Wyraan , J . and Changeux, J . -P., J . Mol. B i o l . , 12, 88 (1965). 186. Taketa, K. and P o g e l l , B.M., J . B i o l . Chem., 240, 651 (1965) . 187. Huxley, J.S., B i o l . Rev., 1_0, 427 (1935). 188. Hechter, 0., Yoshinaga, K., H a l k e r s t o n , I.D.K., Cohn, C. and Dodd, P. i n 0. Walaas ( E d i t o r ) , M o l ecular B a s i s of  Some Aspects of Mental A c t i v i t y , p. 291, Academic P r e s s , New York (1966). 189. Birnbaumer, L., P o h l , S., Krans, H.M.J, and R o d b e l l , M. i n P. Greengard and E. Costa ( E d i t o r s ) , Advances i n B i o c h e m i c a l Psychopharmacology, V o l . 3, p. 185, Raven P r e s s , New York (1970). 190. R o d b e l l , M., Birnbaumer, L., and P o h l , S.L., i n M. Rodbell and P. C o n d l i f f e ( E d i t o r s ) , Fogarty I n t e r n a t i o n a l Center Proceedings No. 4, United States Government P r i n t i n g O f f i c e , Washington (1971). 191. Braun, T. and Hechter, 0., Proc. Nat. Acad. S c i . (USA), 66 , 995 (1970). 192. R o d b e l l , M. , Krans, H.M.J., P o h l , S.L. and Birnbaumer, L., J . B i o l . Chem., 246, 1861 (1971). 193. Dunnick, J.K. and M a r i n e t t i , G.V., Biochim. Biophys. A c t a , 249, 122 (1971). 194. L e f k o w i t z , R.J., Roth, J . , P r i c e r , W. and Pastan, I . , Proc. Nat. Acad. S c i . (USA), 6_5, 745 (1970). 195. P o h l , S.L., Krans, H.M.J., K o z y r e f f , V., Birnbaumer, L. and R o d b e l l , M., J . B i o l . Chem., 246, 4447 (1971). 196. Rosen, O.M. and Er l i c h m a n , J . , Arch. Biochem. Biophys. 133 , 171 ( 1969). 197. BHr, H. P. and Hechter, 0., Biochem. Biophys. Res. Comm., 3_5 , 681 (1969). 198. L e f k o w i t z , R.J., Roth, J . and Pastan, I . , Nature, 228 864 (1970). 199. R o d b e l l , M., Krans, H.M.J., Poh l , S.L. and Birnbaumer, L., J . B i o l . Chem., 246, 1872 (1971). 200. L e f k o w i t z , R.J., Roth, J . and Pastan, I. i n G.A. Robison, G.G. Nahas and L. T r i n e r ( E d i t o r s ) , Annals of the New York Academy of S c i e n c e s , V o l . 185, pg. 195 (1971). - 129 -201. H a y a i s h i , 0„, Greengard, P. and Colowick, S.P., J . B i o l . Chem., 246, 5840 (1971). 202. T a k a i , K., Kurashina, Y., Suzuki, C., Okamoto, H., Ue k i , A. and H a y a i s h i , 0., J . B i o l . Chem., 246, 5843 (1971). 6 PUBLICATIONS: Severson, D.L. and S u t t e r , M.C., "Smooth Muscle C o n t r a c t i o n : E f f e c t s of Zero-Calcium and Cation S u b s t i t u t i o n s . " ( A b s t r a c t ) , Proc. Can. Fed. B i o l . Soc. 12, 36 (1969). Drummond, G.I. and Severson, D.L., "Adenyl Cyclase: K i n e t i c P r o p e r t i e s and Nature of F l u o r i d e and Hormone S t i m u l a t i o n . " ( A b s t r a c t ) , I n t . Congr. C y c l i c AMP, M i l a n (1971). Drummond, George I . and Severson, David L., " B i o l o g i c a l A ctions of C y c l i c AMP Analogs" i n Annual Reports i n M e d i c i n a l Chemistry (1970), Chapter 21, pp. 215-226, Academic Press, New York (1971). Drummond, George I . , Severson, David L. and Duncan, Loverne. "Adenyl Cyclase: K i n e t i c P r o p e r t i e s and Nature of F l u o r i d e and Hormone S t i m u l a t i o n . " J . B i o l . Chem. 246, 4166-4173 (1971). Severson, David L., Drummond, George I . and Sulakhe, Prakash V. "Adenyl Cyclase i n S k e l e t a l Muscle: K i n e t i c P r o p e r t i e s and Hormonal S t i m u l a t i o n . " Accepted f o r p u b l i c a t i o n , J . B i o l . Chem., May (1972). Drummond, George I . , Severson, David L. and Sulakhe, Prakash V., "Nature of Hormone and F l u o r i d e S t i m u l a t i o n of Adenyl Cyclase i n Muscle" i n The Proceedings of the Symposium on I n s u l i n A c t i o n , Academic P r e s s , New York, (1972). 

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