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Cardiac adenylate metabolism : possible relationship to autoreguation of coronary blood flow Nakatsu, Kanji 1971

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CARDIAC ADENYLATE METABOLISM: POSSIBLE RELATIONSHIP TO AUTOREGULATION OF CORONARY BLOOD FLOW by KANJI NAKATSU B . S c , U n i v e r s i t y of A l b e r t a , 1964. M.Sc, U n i v e r s i t y of 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 the Department of Pharmacology We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1971. In present ing th i s thes is in p a r t i a l f u l f i lmen t of the requirements for an advanced degree at the Un iver s i t y of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f r e e l y ava i l ab le for reference and study. I f u r ther agree that permission for extens ive copying of t h i s thes i s fo r s cho la r l y purposes may be granted by the Head of my Department or by h i s representat ives . It i s understood that copying or pub l i ca t i on o f th i s thes i s f o r f i nanc i a l gain sha l l not be allowed without my wr i t ten permiss ion. Department of t A# f c P . The Un ivers i ty o f B r i t i s h Columbia Vancouver 8. Canada y - •>/ ( i ) ABSTRACT The metabolism of 5'-AMP by 5 1 - n u c l e o t i d a s e , adenylate deaminase and adenylate kinase was examined i n heart homogenates of r a t , r a b b i t , dog, pigeon and t u r t l e . The study was conducted i n c o n s i d e r a t i o n of the p o s s i b i l i t y that adenosine, a c a t a b o l i c product of 5'-AMP, may c o n t r o l vasotone f o r the a u t o r e g u l a t i o n of coronary blood f l o w . The r e l a t i v e a c t i v i t i e s of homogenates of hearts from v a r i o u s species to form adenosine by the a c t i o n of 5'-nucleotidase g e n e r a l l y supported such a r o l e f o r t h i s n u c l e o s i d e . Those sp e c i e s a n t i c i p a t e d to have the l a r g e s t p o t e n t i a l requirements f o r coronary v a s o d i l a t i o n , i . e . those whose oxygen consumption i s known to in c r e a s e s i g n i f i c a n t l y d u r i n g p h y s i c a l e x e r t i o n , had the highest l e v e l s of c a r d i a c 5'-nucleo-t i d a s e . An exception to t h i s was the pigeon which had no d e t e c t a b l e c a r d i a c 5'-nucleotidase; the order of l e v e l s of t h i s enzyme i n h e a r t s of the other s p e c i e s t e s t e d was: r a t > dog > r a b b i t > t u r t l e . The t u r t l e v e n t r i c l e , by v i r t u e of i t s h i g h content of adenylate deaminase and low content of 5'-nucleotidase appeared to c a t a b o l i z e 5'-AMP l a r g e l y by deamination to IMP. Homogenates of pigeon v e n t r i c l e contained the gr e a t e s t a c t i v i t y of adenylate k i n a s e , i n d i c a t i n g that the heart of t h i s s p ecies i s equipped f o r p r e s e r v a t i o n of ATP by r e s y n t h e s i s from ADP. Enzyme h i s t o c h e m i s t r y revealed that most 5'-nucleotidase of mammalian hear t s was l o c a l i z e d i n the e n d o t h e l i a l c e l l s of c a p i l l a r i e s . Therefore, i f adenosine i s i n v o l v e d i n r e g u l a t i o n of coronary p e r f u s i o n , i t s source may be c a p i l l a r y e n d o t h e l i a l c e l l s r a t h e r than c a r d i a c muscle c e l l s . 5'-Nucleotidase was p a r t i a l l y p u r i f i e d from an acetone powder of r a t h e a r t . I t was a c t i v e over a broad range of pH w i t h an optimum a t ( i i ) pH 8.5. The enzyme was s t i m u l a t e d up t o 5 - f o l d by Mg (K = 1.9 x 10 M); a I | | [ _ £^  Mn and N i a l s o s t i m u l a t e d a c t i v i t y . The K f o r 5'-AMP was 2.1 x 10 M m I | _5 i n t h e absence o f Mg and 2.3 x 10 M i n t h e p r e s e n c e o f 16 mM MgC^. C e r t a i n o f i t s p r o p e r t i e s i n d i c a t e d t h a t t h e p r o d u c t i o n o f a d e n o s i n e might be f a v o u r e d under c o n d i t i o n s i n w h i c h c o r o n a r y v a s o d i l a t i o n would be r e q u i r e d and v i c e - v e r s a . F o r example, t h e enzyme was i n h i b i t e d by ATP, whose l e v e l s a r e g r e a t e s t i n w e l l o x y g e n a t e d h e a r t s i n w h i c h energy c h a r g e i s h i g h . Not a l l p r o p e r t i e s o f 5 ' - n u c l e o t i d a s e were c o n s i s t e n t w i t h enhanced a d e n o s i n e f o r m a t i o n a t red u c e d energy c h a r g e . B o t h ADP and o r t h o -p h o s p h a t e , t h e l e v e l s o f w h i c h i n c r e a s e when energy c h a r g e d e c r e a s e s , i n h i b i t e d t h e enzyme; i n f a c t ADP was a more p o w e r f u l i n h i b i t o r t h a n ATP. I n a d d i t i o n , t h e enzyme was n o t s p e c i f i c f o r 5'-AMP but h y d r o l y z e d a v a r i e t y o f n u c l e o s i d e 5'-monophosphates; and t h e h y d r o l y s i s o f 5'-AMP was c o m p e t i t i v e l y i n h i b i t e d by UMP. I | I n t h e absence o f Mg , i n h i b i t i o n by ADP was o f t h e mixed ( c o m p e t i -t i v e - n o n - c o m p e t i t i v e ) t y p e . I n t h e p r e s e n c e o f 16 mM MgC^, i n h i b i t i o n was n o n - c o m p e t i t i v e . On t h e b a s i s o f t h e s e d a t a and D i x o n p l o t s o f i n h i b i t i o n as a f u n c t i o n o f ADP c o n c e n t r a t i o n , i t i s s u g g e s t e d t h a t two c o n f o r m a t i o n s o f t h e enzyme a r e p o s s i b l e ; one w h i c h i s c o m p e t i t i v e l y i n h i b i t e d by ADP. The s i m p l e n o n - c o m p e t i t i v e i n h i b i t i o n by ADP, o b s e r v e d I | i n t h e p r e s e n c e o f 16 mM MgC^, i s a t t r i b u t e d t o Mg - i n d u c e d p r e f e r e n c e f o r t h e l a t t e r c o n f o r m a t i o n . ( i i i ) TABLE OF CONTENTS Page INTRODUCTION 1 METHODS AND MATERIALS A. G e n e r a l 17 B. Survey o f C a r d i a c Enzymes w h i c h U t i l i z e 5'-AMP and A d e n o s i n e 17 C. P e r f u s i o n o f R a t , R a b b i t and T u r t l e H e a r t s .21 D. H i s t o c h e m i s t r y 22 E. A s s a y o f P a r t i a l l y P u r i f i e d 5 ' - N u c l e o t i d a s e 22 F. M a t e r i a l s ' 24 RESULTS A. P r e l i m i n a r y 25 B. C a r d i a c Enzymes w h i c h U t i l i z e 5'-AMP and A d e n o s i n e 26 C. E f f e c t o f A d e n o s i n e on Co r o n a r y F l o w 30 D. H i s t o c h e m i c a l L o c a l i z a t i o n o f C a r d i a c 5 ' - N u c l e o t i d a s e 31 E. P a r t i a l P u r i f i c a t i o n o f 5 ' - N u c l e o t i d a s e 36 F. P r o p e r t i e s o f 5 ' - N u c l e o t i d a s e 39 DISCUSSION 52 N a t u r e o f ADP I n h i b i t i o n 63 LITERATURE CITED 67 (iv) LIST OF TABLES Page TABLE I. Survey of Ventricular Adenosine Deaminase 29(a) TABLE I I . Summary of P u r i f i c a t i o n 38(a) TABLE I I I . Effect of Ca** on 5'-Nucleotidase A3(a) (v) LIST OF FIGURES S t a n d a r d c u r v e f o r a s s a y o f i n o r g a n i c p h o s p h a t e . L i n e a r i t y o f 5 ' - n u c l e o t i d a s e a s s a y w i t h t i m e and p r o t e i n . Survey o f v e n t r i c u l a r 5 ' - n u c l e o t i d a s e . Survey o f v e n t r i c u l a r a d e n y l a t e deaminase. Survey o f v e n t r i c u l a r a d e n y l a t e k i n a s e . E f f e c t o f a d e n o s i n e on c o r o n a r y f l o w . H i s t o c h e m i c a l l o c a l i z a t i o n o f 5 ' - n u c l e o t i d a s e i n v e n t r i c l e s e c t i o n s . P l a t e •A. Rat v e n t r i c l e ; s u b s t r a t e 5'-AMP. P l a t e B. Rat v e n t r i c l e ; s u b s t r a t e 5'-AMP P l a t e C. D o g - v e n t r i c l e ; s u b s t r a t e 5'-AMP. P l a t e ' D. Human p a p i l l a r y m u s c l e ; s u b s t r a t e 5'-AMP. P l a t e E. Human p a p i l l a r y m u s c l e ; c o n t r o l u s i n g 3'-AMP. P l a t e F. Human p a p i l l a r y m u s c l e ; h e m a t o x y l i n and e o s i n . P l a t e G. Gu i n e a p i g v e n t r i c l e ; s u b s t r a t e 5'-AMP. P l a t e H. Gu i n e a p i g v e n t r i c l e ; c o n t r o l . P l a t e I . Mouse v e n t r i c l e ; s u b s t r a t e 5'-AMP. P l a t e J . G u i n e a p i g v e n t r i c l e ; s u b s t r a t e 5'-AMP. P l a t e K. P i g e o n v e n t r i c l e ; s u b s t r a t e 5'-AMP. P l a t e L. T u r t l e v e n t r i c l e ; s u b s t r a t e 5'-AMP. Figure 8. Summary of 5'-nucleotidase p u r i f i c a t i o n . Figure 9, A c t i v i t y of 5'-nucleotidase as a function of pH. Figure 10. Effect of Mg, ,-Mn , and Ni on 5'-nucleotidase. Figure 11. Substrate s p e c i f i c i t y . Figure 12. Effect of 5'-AMP concentration. Figure 13. I n h i b i t i o n by ATP and ADP at high concentrations of 5'-AMP, Figure 14. ADP i n the absence of Mg Figure 15. I n h i b i t i o n by ADP i n the,t;presence of MgC^. Figure 16. Effect of ADP concentration on i n h i b i t i o n . Figure 17. I n h i b i t i o n by UMP and orthophosphate i n the presence of MgC^. Figure 18. Effect of MgC^ concentration on i n h i b i t i o n by ADP and ATP. ( v i i ) A b b r e v i a t i o n s and T r i v i a l Names Customary b i o l o g i c a l a b b r e v i a t i o n s and t r i v i a l names have been used t h r o u g h o u t . t h e t e x t o f t h i s t h e s i s . The o f f i c i a l names o f t h e s e a r e e x p l a i n e d below. A d e n o s i n e Deaminase: A d e n o s i n e A m i n o h y d r o l a s e , E.C. 3.5.4.4. A d e n y l a t e Deaminase: AMP A m i n o h y d r o l a s e , E.C. 3.5.4.6. A d e n y l a t e K i n a s e : ATP:AMP P h o s p h o t r a n s f e r a s e , E.C. 2.7.4.3-5 ' - N u c l e o t i d a s e : 5 ' - R i b o n u c l e o t i d e P h o s p h o h y d r o l a s e , E.C. 3.1.3.5. 5'-AMP, a d e n o s i n e 5'-monophosphate; 3'-AMP, a d e n o s i n e 3'-monophosphate; ADP, a d e n o s i n e 5 ' - d i p h o s p h a t e ; ATP, a d e n o s i n e 5 ' - t r i p h o s p h a t e ; CMP, c y t i d i n e 5'-monophosphate; CDP, c y t l d i n e 5 ' - d i p h o s p h a t e ; CTP, c y t l d i n e 5 ' - t r i p h o s p h a t e ; GMP, g u a n o s i n e 5'-monophosphate; GDP, g u a n o s i n e 5 ' - d i p h o s p h a t e ; GTP, g u a n o s i n e 5 ' - t r i p h o s p h a t e ; P_^ , o r t h o p h o s p h a t e ; UMP, u r i d i n e 5'-monophosphate; 3GP, 3 - g l y c e r o p h o s p h a t e ; UDP, u r i d i n e 5 ' - d i p h o s p h a t e ; UTP, u r i d i n e 5 ' - t r i p h o s p h a t e . A s m a l l " d " as t h e f i r s t l e t t e r o f an a b b r e v i a t i o n f o r a n u c l e o t i d e i n d i c a t e s t h e 2'-deoxy- compound. T r i s , tr±s,(hydroxymethyl)amino-methane; EDTA, e t h y l e n e d i a m i n e t e t r a a c e t l c a c i d . ( v i i i ) \ • ' • \ ACKNOWLEDGEMENTS I would l i k e to express my gratitude to Dr. George I. Drummond who, by word and example, gave guidance, encouragement and instruction during the course of this work. I also thank a l l those who contributed to my education of the past three years. I wish to express my appreciation to the H.R. MacMillan Family and the Medical Research Council of Canada for f i n a n c i a l support. For My Friends INTRODUCTION The d e l i v e r y o f oxygen to c a r d i a c m u s c l e i s a symphony f o r w h i c h t h e m u s i c i s s t i l l u n w r i t t e n . The mammalian h e a r t i s p o o r l y e q u i p p e d f o r a n o x i c f u n c t i o n ; 180 seconds o f a n o x i a r e d u c e s t h e r a t e by 60% and f o r c e o f c o n t r a c t i o n by 70% ( W i l l i a m s o n , 1 9 6 6 ) . Due t o t h e l i m i t e d amount of energy a v a i l a b l e from c a r d i a c g l y c o l y s i s , an i n c r e a s e i n c a r d i a c energy u t i l i z a t i o n must be matched by p r o c e s s e s w h i c h r e s u l t i n i n c r e a s e d oxygen c o n s u m p t i o n ( O p i e , 1 9 6 8 ) . The h e a r t , u n l i k e o t h e r organs suc h as b r a i n , k i d n e y and s k e l e t a l m u s c l e , e x t r a c t s a l a r g e p o r t i o n ( o v e r 75%) o f t h e oxygen p r o v i d e d by i t s a r t e r i a l s u p p l y (Van C i t t e r s , 1 9 6 5 ) . S i n c e t h e r e i s l i t t l e oxygen a v a i l a b l e f o r f u r t h e r e x t r a c t i o n , any i n c r e a s e d demand f o r oxygen must be f u l f i l l e d by an i n c r e a s e i n c o r o n a r y b l o o d f l o w . B l o o d f l o w i n t h e c o r o n a r y c i r c u l a t i o n i s a f f e c t e d by p e r f u s i o n p r e s s u r e , v a s c u l a r r e s i s t a n c e and c o n t r a c t i o n s o f t h e h e a r t . I t i s p h a s i c b ecause c o n t r a c t i o n o f t h e myocardium compresses b l o o d v e s s e l s and r e s t r i c t s f l o w , w h i l e f l o w t h r o u g h t h e r e l a x e d h e a r t i s r e l a t i v e l y u n r e s t r i c t e d . I f c o r o n a r y f l o w i s examined under b a s a l , u n s t r e s s e d c o n d i t i o n s , i t i s about 4 t i m e s g r e a t e r d u r i n g d i a s t o l e t h a n d u r i n g s y s t o l e (Van C i t t e r s , 1965). I n r e s p o n s e t o a demand f o r g r e a t e r c a r d i a c o u t p u t , b o t h s t r o k e volume and h e a r t r a t e may i n c r e a s e . The i n c r e a s e d c a r d i a c o u t p u t due t o e x e r c i s e i s a c c o m p l i s h e d p r i m a r i l y by i n c r e a s e d h e a r t r a t e (Rushmer, Van C i t t e r s and F r a n k l i n , 1963). An i n c r e a s e i n r a t e , however, r e s u l t s i n a d e c r e a s e i n t h e p r o p o r t i o n of t i m e s p e n t i n d i a s t o l e (Van C i t t e r s , 1965). T h i s t e n d s to r e d u c e r a t h e r t h a n enhance c o r o n a r y b l o o d f l o w i n p e r i o d s o f i n c r e a s e d work because f l o w i s g r e a t e s t d u r i n g d i a s t o l e . W h i l e d i a s t o l i c as w e l l as s y s t o l i c b l o o d pressure.may i n c r e a s e w i t h i n c r e a s e d c a r d i a c o u t p u t , changes i n t h e f o r m e r a r e more i m p o r t a n t w i t h r e s p e c t t o p e r f u s i o n o f t h e c a r d i a c v a s c u l a t u r e . S i n c e t h e r e i s l i t t l e o r no change i n d i a s t o l i c p r e s s u r e 2. d u r i n g , e x e r c i s e ( B e v e g a r d , Holmgren and J o n s s o n , 1 9 6 0 ) , i t i s u n l i k e l y t h a t a change i n p e r f u s i o n p r e s s u r e c a n a c c o u n t f o r l a r g e , e x e r t i o n -i n d u c e d i n c r e a s e s i n c o r o n a r y b l o o d f l o w such as t h a t o b s e r v e d i n dogs s u b j e c t e d t o m i l d t r e a d - m i l l e x e r c i s e (Gregg, 1 9 6 3 ) . I t i s a p p a r e n t t h a t n e i t h e r i n c r e a s e d p e r f u s i o n p r e s s u r e n o r i n c r e a s e d d u r a t i o n o f d i a s t o l e c a n a c c o u n t f o r enhancement o f b l o o d f l o w and oxygen d e l i v e r y t o t h e s t r e s s e d h e a r t . I t f o l l o w s , t h e r e f o r e , t h a t a l t e r a t i o n s i n c o r o n a r y r e s i s t a n c e must p l a y a major r o l e i n t h e mai n t e n a n c e o f adequate o x y g e n a t i o n o f t h e myo-c a r d i u m . The a b i l i t y o f t h e h e a r t t o r e g u l a t e v a s c u l a r r e s i s t a n c e was de m o n s t r a t e d some y e a r s ago by E c k e l e _ t a _ l . ; (1949) i n a n e s t h e t i z e d , o p e n - c h e s t dogs. ' When c o r o n a r y a r t e r i e s were c a n n u l a t e d and p e r f u s e d a t c o n s t a n t p r e s s u r e , r e s i s t a n c e was c o n s t a n t . However, i f t h e p e r f u s i o n p r e s s u r e was i n c r e a s e d , r e s i s t a n c e i n c r e a s e d r a p i d l y ; t h u s a r e l a t i v e l y s t e a d y f l o w was m a i n t a i n e d d e s p i t e p r e s s u r e .changes. A s a t i s f a c t o r y e x p l a n a t i o n o f t h i s a u t o r e g u l a t i o n o f c o r o n a r y b l o o d f l o w i s s t i l l b e i n g s o u g h t ; s e v e r a l h y p o t h e s e s a r e d i s c u s s e d below. - M y o g e n i c , n e u r a l and m e t a b o l i c c o n t r o l a r e t h e t h r e e main e x p l a n a t i o n s w h i c h have been o f f e r e d f o r t h e r e g u l a t i o n o f c o r o n a r y r e s i s t a n c e . W h i l e n e u r a l c o n t r o l i s thought t o i n v o l v e a c e t y l c h o l i n e , a d r e n a l i n e and/or n o r a -d r e n a l i n e , m e t a b o l i c c o n t r o l t h e o r i e s s u g g e s t t h e r e l e a s e of;; v a s o a c t i v e m e t a b o l i t e s f r o m h y p o x i c h e a r t s . Myogenic c o n t r o l , as f i r s t s u g g e s t e d by B a y l i s s (1902) and r e v i e w e d r e c e n t l y by Berne ( 1 9 6 4 ) , i s based on t h e o b s e r v a t i o n t h a t m u s c l e c o n t r a c t s i n r e s p o n s e t o s t r e t c h . I t i s h y p o t h e -s i z e d t h a t c o r o n a r y b l o o d f l o w c o u l d be m a i n t a i n e d a t a c o n s t a n t r a t e by the c o n t r a c t i o n o f smooth m u s c l e o f r e s i s t a n c e v e s s e l s i n r e s p o n s e t o t h e s t r e t c h s t i m u l u s o f i n c r e a s e d p r e s s u r e and v i c e - v e r s a . F o r example, i f a r t e r i a l p r e s s u r e were i n c r e a s e d , t h e w a l l s o f r e s i s t a n c e v e s s e l s would be s t r e t c h e d and would respond by c o n t r a c t i o n t o t h e i r o r i g i n a l s i z e . T h i s would remove t h e s t i m u l u s f o r f u r t h e r c o n t r a c t i o n and c o u l d a c c o u n t f o r t h e m a i n t e n a n c e o f r e s i s t a n c e v e s s e l d i a m e t e r a t a c o n s t a n t v a l u e . Such m a i n t e n a n c e of c o n d u i t s i z e i s n o t s u f f i c i e n t f o r m a i n t e n a n c e o f a c o n s t a n t r a t e of p e r f u s i o n because f l o w would i n c r e a s e due t o t h e i n c r e a s e d a r t e r i a l p r e s s u r e . However, i f t h e s t i m u l u s f o r c o n t r a c t i o n were t e n s i o n o f smooth m u s c l e f i b r e s r a t h e r t h a n l e n g t h , t h i s c r i t i c i s m i s s a t i s f i e d b ecause r e s i s t a n c e v e s s e l s c o u l d c o n s t r i c t u n t i l w a l l t e n s i o n s r e t u r n e d t o c o n t r o l v a l u e s . A c c o r d i n g t o t h e L a p l a c e r e l a t i o n s h i p , i n t e r n a l p r e s s u r e i s p r o p o r t i o n a l t o v e s s e l w a l l t e n s i o n and i n v e r s e l y p r o p o r t i o n a l t o r a d i u s (P = T/r).. An i n c r e a s e i n a r t e r i a l p r e s s u r e m ight w e l l r e s u l t i n a d e c r e a s e i n v e s s e l s i z e t o l e s s t h a n c o n t r o l o r u n t i l t e n s i o n was r e t u r n e d t o t h e o r i g i n a l v a l u e . A c c o r d i n g t o t h i s myogenic h y p o t h e s i s , c o r o n a r y b l o o d f l o w m i g h t be m a i n t a i n e d a t a c o n s t a n t r a t e o v e r a w i d e range of a r t e r i a l p r e s s u r e . A s h o r t c o m i n g of t h i s myogenic t h e o r y t o e x p l a i n . r e g u l a t i o n o f c o r o n a r y r e s i s t a n c e i s t h a t i t c a n o n l y a c c o u n t f o r t h e m a i n t e n a n c e of a c o n s t a n t f l o w r a t e . Myogenic r e g u l a t i o n , as d e s c r i b e d , c o u l d n o t p e r m i t i n c r e a s e d oxygen d e l i v e r y t o the h e a r t d u r i n g e x e r c i s e , when c a r d i a c demands a r e h i g h but d i a s t o l i c p r e s s u r e i s unchanged (Bevegard e t a l . , 1 9 6 0 ) . C e r t a i n n e u r a l . o r n e u r o h u m o r a l s u b s t a n c e s have been s u g g e s t e d as m e d i a t o r s i n t h e r e g u l a t i o n o f c o r o n a r y b l o o d f l o w . B o t h p a r a s y m p a t h e t i c and s y m p a t h e t i c v a s o d i l a t i o n o f c o r o n a r y v e s s e l s have been d e m o n s t r a t e d . F o r example, i n o p e n - c h e s t dogs, v a g a l s t i m u l a t i o n r e s u l t e d i n i n c r e a s e d c o r o n a r y p e r f u s i o n d e s p i t e d e c r e a s e d p e r f u s i o n p r e s s u r e s ; t h e mean a o r t i c p r e s s u r e d e c r e a s e d 13% ( F e i g l , 1 9 6 9 ) . T h i s v a s o d i l a t i o n was n o t o b s e r v e d a f t e r t r e a t m e n t w i t h a t r o p i n e , a c o m p e t i t i v e i n h i b i t o r o f a c e t y l c h o l i n e 4. at muscarinic receptors. Fe i g l (1969) could not explain t h i s i n terms of alterations i n oxygen tension or metabolism, because vagal stimulation has a negative inotropic effect. Therefore, he concluded that the vaso-d i l a t i o n observed was t r u l y a parasympathetic phenomenon. Bussmann and Lochner (1966) demonstrated sympathetic coronary vasodilation i n isolated guinea pig hearts, using adrenaline and isoproterenol. In Langendorf preparations (see Wedd, 1931), infusion of these catecholamines increased both rate and coronary flow. The increased rate of perfusion could not be attributed to hypoxia secondary to increased metabolic ratej because venous pO^ was actually greater than control.' If the vasodilation had been a result of hypoxia, venous pC^ should have been reduced rather than increased. These workers concluded that a direct S-adrenergic receptor effect was involved because the enhancement of coronary flow by isoproterenol and adrenaline was inhibited by the 3-receptor blocking agents, propranolol and pronethalol. This i n h i b i t i o n was s p e c i f i c for g-adrenergic receptors; the increase i n coronary flow induced by other vasodilator substances (dipyridamol, ATP, adenosine and nitroglycerine) was not blocked. These experiments serve to i l l u s t r a t e the potential of catecholamines and acetylcholine i n the control of blood flow through the myocardium. Although neural regulation of*the coronary c i r c u l a t i o n may be active i n normal hearts i n s i t u , t his i s not l i k e l y . t h e case i n denervated and isolated hearts. For example, i n isolated hearts, neural and neuro-humoral substances are precluded because nerves and blood vessels which might convey vasoactive substances are severed. In these cases (Markwalder and S t a r l i n g , 1913; Berne, Blackmon and Gardner, 1957) 5. m e t a b o l i c r e g u l a t i o n may be i n v o l v e d ; i n whole a n i m a l s , perhaps b o t h m e t a b o l i c and n e u r a l f a c t o r s c o n t r i b u t e t o r e g u l a t i o n o f c o r o n a r y p e r f u s i o n . M e t a b o l i c c o n t r o l o f c o r o n a r y b l o o d f l o w was i m p l i c a t e d many y e a r s ago by Ma r k w a l d e r and S t a r l i n g ( 1 9 1 3 ) . F l o w t h r o u g h t h e c o r o n a r y c i r c u -l a t i o n was i n c r e a s e d 5 - f o l d by a s p h y x i a i n t h e dog h e a r t - l u n g p r e p a r a t i o n . H i l t o n and E i c h h o l t z (1925) c o n c l u d e d t h a t oxygen was t h e r e g u l a t o r o f v a s o t o n e because an inverse'< r e l a t i o n s h i p was found between c o r o n a r y b l o o d f l o w and oxygen s a t u r a t i o n i n t h e dog h e a r t - l u n g p r e p a r a t i o n . B o t h l a c t a t e and CC^ were e x c l u d e d as v a s o r e g u l a t o r s because b o t h a l t e r e d f l o w o n l y s l i g h t l y , even when u n p h y s i o l o g i c a l l y l a r g e q u a n t i t i e s were a d m i n i s t e r e d . Other m e t a b o l i t e s were e l i m i n a t e d because no v a s o d i l a t o r s u b s t a n c e s c o u l d be d e m o n s t r a t e d i n b l o o d w h i c h had been r e c i r c u l a t e d f o r one hour. T h i s c o n c l u s i o n was r e a c h e d because t h e r e was no d e c r e a s e i n c o r o n a r y b l o o d f l o w when r e c i r c u l a t e d b l o o d was r e p l a c e d w i t h f r e s h b l o o d , as would be e x p e c t e d i f v a s o d i l a t o r compounds had a c c u m u l a t e d d u r i n g t h e p e r i o d o f r e c i r c u l a t i o n . T h i s would n o t e x c l u d e t h e p o s s i -b i l i t y t h a t v a s o a c t i v e s u b s t a n c e s were r e l e a s e d b u t degraded r a p i d l y i n b l o o d . Perhaps no r e l e a s e o f v a s o a c t i v e m e t a b o l i t e s s h o u l d have o c c u r r e d under t h e c o n d i t i o n s u s e d , because w e l l o x y genated b l o o d was d e l i v e r e d t o t h e h e a r t . The r e s u l t s o f t h e s e e a r l y w o r k e r s a r e n o t c o n s i s t e n t w i t h t h o s e o f Berne, Blackmon and Gardner ( 1 9 5 7 ) , who c o n d u c t e d s i m i l a r e x p e r i m e n t s on op e n - c h e s t dogs. The l a t t e r found t h a t an i n v e r s e c o r r e l a t i o n between f l o w and oxygen c o n t e n t c o u l d o n l y be d e m o n s t r a t e d i f c o r o n a r y s i n u s oxygen l e v e l s f e l l b elow 5.5 volumes %. They c o n c l u d e d t h a t c o r o n a r y f l o w was n o t dependent on a r t e r i a l b l o o d ' oxygen c o n t e n t p e r se b u t was a f u n c t i o n o f t h e degree of h y p o x i a . 6. In addition to pO^j lactate and pCO^j other metabolites have been invoked to explain coronary vasodilation i n response to hypoxia. Histamine, a known vasodilator, has been examined because i t appeared i n venous blood of dog gastrocnemius after contraction (Anrep and Barsoum, 1935). I t was also reported i n cardiac venous blood of the dog heart-lung preparation after hypoxia (Anrep, Barsoum and Talaat, 1936). However, since these observations could not be repeated (Code, Evans and Gregory, 1938) and histamine i s released from lung by hypoxia (Hauge, 1968; Hauge and Melmon,, 1968);; this has not been an a t t r a c t i v e p o s s i b i l i t y . . Potassium causes an increase i n coronary flow at low concentrations but decreases i t at higher concentrations (Katz and Lindner, 1938). Potassium i s given l i t t l e consideration as a mediator of coronary autoregulation, because the maximum increases i n flow rate induced by this cation are much smaller than those induced by asphyxia or adrenaline. In.addition, increases i n sinus potassium concentrations do not correlate well with the increases i n coronary blood flow (Driscol and Berne, 1957). The substance which appears to claim the most experimental support as mediator of coronary vasodilation i s adenosine.' Drury and Szent-GyeJrgyi (1929) f i r s t demonstrated both general and coronary vasodilatory effects of adenosine and adenylic acid. These workers showed that adenosine.(10 mg) increased coronary blood flow 5-fold i n both atropine treated arid non^atropinized dog heart-lung preparations. That this effect was due to a reduction of resistance alone was assured by maintenance of perfusion pressure at 82 mm Hg and maintaining a constant rate by e l e c t r i c a l pacing. Adenosine acts as a coronary vasodilator not only i n isolated preparations; the vasodilator properties of adenosine 7. and i t s n u c l e o t i d e s were d e m o n s t r a t e d i n dog c o r o n a r y v a s c u l a t u r e i n s i t u by Wedd and D r u r y (1934) . I n t h e s e a n i m a l s t h e i n t r a v e n o u s a d m i n i s t r a t i o n of a d e n o s i n e (7.5 mg) i n c r e a s e d t h e r a t e o f c o r o n a r y b l o o d f l o w t o 3 t i m e s c o n t r o l . Such v a s o d i l a t i o n l a s t e d 20 m i n u t e s when e i t h e r a d e n o s i n e o r 5'-AMP was i n j e c t e d and 100 m i n u t e s when 3'-AMP was i n j e c t e d . The h e a r t i t s e l f was e s t a b l i s h e d as t h e s i t e o f a c t i o n o f a d e n o s i n e by t h e e x p e r i m e n t s o f Wedd (1931). He d e m o n s t r a t e d t h a t a d e n o s i n e was 20 t i m e s more p o t e n t t h a n sodium n i t r i t e as a c o r o n a r y v a s o d i l a t o r i n r a b b i t h e a r t s p e r f u s e d by t h e Lang e n d o r f method. I n t h i s p r e p a r a t i o n , c o r o n a r y p e r f u s i o n i s e f f e c t e d t h r o u g h an a o r t i c c a n n u l a and t h e p e r f u s a t e must pass t h r o u g h t h e c o r o n a r y c i r c u l a t i o n , b e c a u s e t h e o n l y o t h e r escape r o u t e i s s e a l e d by t h e a o r t i c v a l v e . S i n c e t h e h e a r t was p e r f u s e d w i t h R i n g e r ' s s o l u t i o n and i s o l a t e d w i t h no pulmonary c o n n e c t i o n s , t h e a c t i o n o f a d e n o s i n e c o u l d n o t have been due t o t h e r e l e a s e o f some f a c t o r from t h e b l o o d o r l u n g s , b u t must have been a d i r e c t a c t i o n on t h e h e a r t ; The p o s s i b i l i t y t h a t d e r i v a t i v e s o f a d e n i n e might be i n v o l v e d i n t h e p h y s i o l o g i c a l r e g u l a t i o n o f b l o o d f l o w was r a i s e d by R i g l e r ( 1 9 3 2 ) . He s u g g e s t e d t h a t r e l e a s e o f a d e n i n e n u c l e o t i d e s c o u l d r e g u l a t e b l o o d f l o w i n m u s c l e t o match i t s r e q u i r e m e n t s . C l a r k e t a l . (1952) d e m o n s t r a t e d t h a t t h i s r e g u l a t i o n c o u l d o c c u r o n l y w i t h d e r i v a t i v e s o f a d e n i n e . They e s t a b l i s h e d t h a t a d e n o s i n e and i t s 2 - s u b s t i t u t e d a n a l o g u e s ( 2 - a c e t a m i d o - , 2-amino-, 2 - m e t h y l ^ , 2-hydroxy-, and 2 - c h l o r o - ) were a c t i v e as v a s o d e p r e s s o r s i n t h e whole c a t , whereas g u a n o s i n e , i n o s i n e and x a n t h o s i n e were i n a c t i v e . I n p u r s u i t o f t h e s e c o n c e p t s , t h e v a s o -d i l a t o r y r e s p o n s e o f t h e c o r o n a r y v a s c u l a t u r e o f o p e n - c h e s t dogs were shown t o be s e l e c t i v e f o r t h e 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 (Wolf and Berne, 1956). When ATP, ADP, AMP or adenosine was infused into the coronary a r t e r i e s , a large increase i n coronary flow followed. The dog cardiac vessels, l i k e the general systemic supply of cats, were not responsive to non-adenine derivatives such as hypoxanthine, inosine, IMP, IDP, guanine, guanosine, GDP, GTP, cytidine, CMP, CDP, CTP, u r a c i l , uridine UMP and UDP. Both ITP and UTP were exceptions i n that they caused vasodilation. Hence i f Rigler's suggestion i s confined to the heart, the release of adenine derivatives would be most l i k e l y to match blood flow with i t s metabolic demands. Berne, Blackmon and•Gardner (1957) demonstrated the importance of the metabolic state of the myocardium as a determinant of coronary blood flow. They perfused the coronary vasculature open-chest dogs at high pressures so that a high coronary sinus pC^ was obtainable. This technique combined with controlled deoxygenation of blood using N^, allowed them to investigate the effects of blood oxygen content on coronary flow over a wide range of sinus pO^. As long as coronary sinus blood oxygen content was greater than 5.5,.ivolumes %, perfusion rate was not affected, by changes i n a r t e r i a l or venous blood oxygen content. When sinus blood oxygen content was less than 5.5 volumes %, the perfusion rate was inversely proportional to the oxygen content of coronary venous'blood. Thus alterations i n blood oxygen content alone were not s u f f i c i e n t to a l t e r vasotone; coronary resistance was not a simple function of blood oxygen content but was a function of myocardial oxygen sufficiency. The discrepancy between these data and those of Hilton and Eichholtz (1925) can be reconciled. These early workers were probably limited to measurements injonly the oxygen dependent zone because the special high perfusion pressures necessary for demonstration of oxygen independence were not used. Dissociation of a direct l i n k 9. between blood oxygen content and vascular tone, and establishment of the importance of the metabolic state led to attempts to demonstrate the release of vasodilator adenine derivatives from the heart. I n i t i a l attempts to extract adenosine or i t s nucleotides from perfusates of metabolically d e b i l i t a t e d cat hearts were unsuccessful (Jacob and Berne, 1960). However, other data of considerable importance was obtained from these experiments. Further evidence of a direct l i n k between coronary blood flow and metabolic state were e l i c i t e d from experiments using Langendorf preparations of cat hearts. When anoxia was simulated by uncoupling oxidative phosphorylation with d i n i t r o -phenol, coronary flow increased by 25 to 50%. Although adenine deriva-tives were not found i n the perfusate, indirect evidence for the release of adenosine was obtained because the quantity of degradation products of adenosine (inosine and hypoxanthine) found i n the effluent was increased up to 48-fold. These workers also showed that adenosine, i n the coronary vasculature, was extremely short-lived and that adenosine 14 crossed c e l l membranes readily. When adenosine-8-C was infused, 50% of the r a d i o a c t i v i t y was trapped by the heart as adenine nucleotides. The other 50% was degraded to inosine and hypoxanthine. Hence the impaired metabolic state induced by dinitrophenol was thought to result i n the formation of adenosine, which could then diffuse out of the c e l l s . Recovery of adenosine per se could not be expected because of i t s rapid degradation. The suggestion that inosine and hypoxanthine, found i n coronary perfusate, originated from adenosine after i t s d i f f u s i o n from cardiac c e l l s was supported by the findings of Gerlach, Deuticke and Driesbach (1963). The major metabolic pathway for 5'-AMP degradation i n rat heart 10. was demonstrated to involve dephosphorylation to adenosine followed by deamination to inosine. This i s i n contrast to skeletal muscle i n which the major route of 5'-AMP catabolism i s deamination to IMP (Imai, Riley and Berne, 1964). Incorporating the knowledge that adenosine: ( i ) i s a potent coronary vasodilator; ( i i ) penetrates membranes readily; ( i i i ) i s produced i n preference to IMP i n heart; (iv) i s deaminated rapidly ;in the coronary c i r c u l a t i o n ; and (v) i t s degradation products are found i n the perfusate of dinitrophenol-treated and hypoxic dog-hearts, an elegant theory for autoregulation of coronary blood flow has been proposed by Berne (1963). He has suggested that i n response to increased oxygen u t i l i z a t i o n or decreased oxygen delivery, the oxygen tension of the myocardial c e l l would decrease; .this decrease would lead to a breakdown of adenine nucleotides to produce adenosine. Adenosine could then diffuse through the i n t e r s t i t i a l f l u i d to r e s i s -tance vessels to induce coronary d i l a t i o n and increase the blood and oxygen supply to the myocardium; th i s would act as a feedback mechanism to reduce the production of adenosine as oxygen tension returned to an adequate l e v e l . This has become known as the "adenosine hypothesis". In support of this hypothesis, the formation of adenosine has been demonstrated i n severely anoxic rat hearts (Gerlach et al_. -, 1963). Respiring rat hearts were placed i n a "moist" anoxic environment at 37° and analysed for nucleoside and nucleotide content after various incubation times. Before i n i t i a t i o n of anoxia, neither adenosine nor i t s degradation products could be detected. After 5 minutes of anoxia, small but s i g n i f i c a n t amounts of adenosine, inosine and hypoxanthine were found. After 60 minutes, considerable quantities-of these substances 11. were recovered while the ATP and ADP content of the hearts decreased greatly. This experiment has been repeated and i d e n t i c a l r e s u l t s were obtained using rabbit hearts (Imai, Rile y and Berne, 1963). Moreover, these workers, extracted traces of hypoxanthine, inosine and adenosine from hypoxic perfused hearts. To t h i s point, no d i r e c t evidence had been presented f o r the e x t r a c e l l u l a r presence of adenosine, although i n d i r e c t evidence was provided by the experiments of Klibler and Bretschneider (1964). These studies showed that the coronary vaso-d i l a t o r dipyridamol could i n h i b i t the removal of adenosine from blood. Since dipyridamol also potentiated the e f f e c t s of exogenously administered adenosine i n hearts, they suggested that i t s mechanism of action was by preserving adenosine. It would follow then that the v a s o d i l a t i o n induced by the administration of dipyridamol alone (Kadatz, 1959) was due to preserving adenosine of endogenous o r i g i n . If t h i s were the case, adenosine per se must have been present i n the e x t r a c e l l u l a r f l u i d of blood. The f i r s t d i r e c t demonstration of adenosine i n the e x t r a c e l l u l a r f l u i d of hearts was reported by Riehman and Wyborny (1964). When Langendorf preparations of rabbit hearts were treated with bishydroxy-coumarin f or 20 minutes to e f f e c t uncoupling of oxidation from phosphory-l a t i o n , 'adenosine was recovered from the r e c i r c u l a t e d perfusate. However, no adenosine was detected i n the perfusate of the same preparation a f t e r hypoxia f o r 20 minutes. In the presence of 8-azaguanine, an adenosine deaminase i n h i b i t o r , adenosine could be detected i n perfusates of both uncoupled and anoxic hearts. These observations were confirmed i n guinea pig and cat hearts (Katori and Berne, 1966). Adenosine was recovered from perfusates of Langendorf preparations of these hearts a f t e r a s i n g l e pass of the perfusate through the vasculature, which 12. compares w e l l w i t h t h e r e c i r c u l a t i o n t e c h n i q u e of Richman and Wyborny (1964). The p r e s e n c e of a d e n o s i n e has s i n c e been d e t e c t e d i n the c o r o n a r y s i n u s b l o o d o f o p e n - c h e s t dogs under much more p h y s i o l o g i c a l c o n d i t i o n s ( R u b i o , Berne and K a t o r i , 1 9 6 9 ) . "One o f t h e g r e a t e s t p r o b l e m s , d e g r a d a t i o n o f a d e n o s i n e by a d e n o s i n e deaminase, was overcome w i t h t h e a i d o f a s p e c i a l s i n u s c a n n u l a . As b l o o d p a s s e d t h r o u g h t h e c a n n u l a t o t h e c o l l e c t i o n t u b e , i t was d i l u t e d and c o o l e d w i t h one t o two volumes o f i c e - c o l d s a l i n e ; b o t h d i l u t i o n a n d - c o o l i n g would e f f e c t i v e l y r e d u c e a d e n o s i n e deaminase a c t i v i t y . C e l l s were removed f r o m t h i s d i l u t e d b l o o d by c e n t r i f u g a t i o n and plasma p r o t e i n s were h e a t d e n a t u r e d . The r e s u l t i n g f l u i d was c o n c e n t r a t e d and a n a l y s e d f o r n u c l e o s i d e c o n t e n t . By u s i n g t h i s t e c h n i q u e , a d e n o s i n e was d e t e c t e d i n t h e c o r o n a r y s i n u s b l o o d a f t e r p e r i o d s of a n o x i a as s h o r t as 30 t o 60 s e c o n d s . By e x t r a -p o l a t i o n , t h e c o n c e n t r a t i o n of a d e n o s i n e i n t h e e x t r a c e l l u l a r f l u i d o f s u c h a n o x i c h e a r t s was c a l c u l a t e d t o be 0.75 yM. This.was g r e a t e r t h a n t h e a r t e r i a l c o n c e n t r a t i o n r e q u i r e d f o r maximal v a s o d i l a t i o n i n . t h i s p r e p a r a t i o n (0.56 yM)'. I n a d d i t i o n , a d e n o s i n e has been e x t r a c t e d f r o m t h e p e r i c a r d i a l space o f n o r m a l l y o x y g e n a t e d as w e l l as h y p o x i c dog-h e a r t s (Rubio and B e r n e , 1969). T h i s i s p a r t i c u l a r l y s i g n i f i c a n t b e cause f o r a d e n o s i n e t o be a t r u e a u t o r e g u l a t o r , t h e r e s h o u l d be a mechanism f o r r e d u c i n g as w e l l as i n c r e a s i n g c o r o n a r y b l o o d f l o w . T h i s f i n d i n g a l l o w s f o r a d e c r e a s e i n a d e n o s i n e c o n c e n t r a t i o n w h i c h c o u l d e f f e c t v a s o c o n s t r i c t i o n . As f u r t h e r s u p p o r t f o r t h e a d e n o s i n e h y p o t h e s i s , a d e n o s i n e r e c o v e r y f r o m t h e p e r i c a r d i a l f l u i d was i n c r e a s e d 70% by p a r t i a l a s p h y x i a . I n n o r m a l l y oxygenated d o g - h e a r t s t h e e x t r a c e l l u l a r a d e n o s i n e c o n c e n t r a t i o n was e s t i m a t e d a t 1.09 yM o r 55% g r e a t e r t h a n t h e p r e v i o u s d e t e r m i n a t i o n f o r a n o x i c h e a r t s (Rubio e t a l . , 1 969). V a l u e s o b t a i n e d by 13. t h e l a t t e r p r o c e d u r e might l e a d t o t h e c o n c l u s i o n t h a t normal h e a r t s produce s u f f i c i e n t a d e n o s i n e t o m a i n t a i n maximal v a s o d i l a t i o n a t a l l t i m e s . O l s s o n (1970) d e t e c t e d a d e n o s i n e i n n o r m a l l y oxygenated myo-c a r d i u m o f dog, u s i n g a q u i c k - f r e e z e t e c h n i q u e combined w i t h a s e n s i t i v e a s s a y w h i c h e x p l o i t e d t h e s p e c i f i c i t y o f a d e n o s i n e deaminase. M o d i f i e d r o n g e u r s w h i c h had been c h i l l e d i n l i q u i d n i t r o g e n were used t o t a k e samples o f v e n t r i c l e and f r e e z e them s i m u l t a n e o u s l y . E x t r a c t s o f t h e s e samples were made and t h e a d e n o s i n e c o n t e n t was d e t e r m i n e d . A d e n o s i n e was e n z y m a t i c a l l y c o n v e r t e d t o u r i c a c i d and t h e change i n ab s o r b a n c e was m o n i t o r e d w i t h a d u a l w a v e l e n g t h , s p l i t - b e a m r e c o r d i n g s p e c t r o p h o t o m e t e r . When t h e c o r o n a r y s u p p l y t o t h e v e n t r i c l e was o c c l u d e d f o r 15 seconds,, a d e n o s i n e c o n c e n t r a t i o n i n c r e a s e d about 6 - f o l d . D e s p i t e t h i s w e l l documented s u p p o r t , t h e a d e n o s i n e h y p o t h e s i s has n o t been u n i v e r s a l l y a c c e p t e d . F o r example, A f o n s o (1969) q u e s t i o n e d t h e h y p o t h e s i s because l i d o f l a z i n e , a c o r o n a r y v a s o d i l a t o r and a d e n o s i n e s p a r i n g a g e n t , p o t e n t i a t e d v a s o d i l a t i o n i n d u c e d by exogenous a d e n o s i n e but not by h y p o x i a i n dog h e a r t s i n s i t u . He s u g g e s t e d t h a t i f a d e n o s i n e were r e l e a s e d i n a p p r o p r i a t e amounts d u r i n g h y p o x i a , v a s o d i l a t i o n i n d u c e d by t h i s c o n d i t i o n s h o u l d have been p o t e n t i a t e d by l i d o f l a z i n e . T h i s o b j e c t i o n s h o u l d be tempered because t h e e x a c t mechanism and s i t e o f a c t i o n , o f l i d o f l a z i n e as c o r o n a r y v a s o d i l a t o r have n o t been e s t a b l i s h e d . K l l b l e r , Spieckermann and B r e t s c h n e i d e r (1970) have d e m o n s t r a t e d t h a t , t h e c o r o n a r y v a s o d i l a t o r , d i p y r i d a m o l i n h i b i t e d b o t h u p t a k e and r e l e a s e o f a d e n o s i n e by t h e c a n i n e myocardium. They s u g g e s t e d t h a t i f r e l e a s e o f a d e n o s i n e were n e c e s s a r y f o r r e g u l a t i o n o f c o r o n a r y f l o w , d i p y r i d a m o l s h o u l d i n d u c e . v a s o c o n s t r i c t i o n by i n h i b i t i o n o f a d e n o s i n e r e l e a s e ,by t h e s e c e l l s . - Hence t h e y c o n c l u d e d t h a t s u c h d a t a was n o t c o n s i s t e n t 14. with the adenosine hypothesis. However, vasotone i s not l i k e l y a function of adenosine release per se but may be related to extra c e l l u l a r adenosine concentration. The quantity of adenosine i n the extr a c e l l u l a r pool would be expected to be affected by movement of adenosine into and away from i t . Dipyridamol i n h i b i t s movement i n both directions (Kdbler et: a l . , 1970). If i n h i b i t i o n of adenosine penetration into c e l l s were inhibited to a greater degree than flow out, an increase i n i n t e r s t i t i a l adenosine concentration would be possible i n the face of an inhibited rate of release. This p o s s i b i l i t y i s supported by the observation that dipyridamol has only minimal vasodilatory properties i n hearts perfused with Tyrode's solution (Kadatz and Schroter, 1962) i n which one of the major sources of adenosine i n a c t i v a t i o n , the red blood c e l l s (Ktibler and Bretschneider, 1964) i s absent. In summary, the adenosine hypothesis has considerable data to support i t , but there are inconsistencies within t h i s data, and the questions posed by Afonso (1969) and Ktibler et a l . (1970) have helped to maintain channels of doubt. Although i t has not been unequivocally established whether adenosine mediates the regulation of coronary flow,,the formation of adenosine i n the mammalian heart i s well documented (Gerlach et_ a l . , 1963; Imai et a l . , 1964; Richman and Wyborny, 1965; Olsson, 1970). Adenosine i s produced by the dephosphorylation of 5'-AMP by 5'-nucleotidase according to the reaction 5'-nucleotidase 5'-AMP •.—» adenosine + P It i s evident that a f u l l understanding of the properties of this enzyme i n cardiac tissue, especially with respect to i t s l o c a l i z a t i o n , k i n e t i c s and regulation, could y i e l d important information regarding the possible 15. r o l e of adenosine i n coronary autoregulation. 5'-Nucleotidase i s not confined to cardiac t i s s u e , i t i s almost u n i v e r s a l l y d i s t r i b u t e d i n the l i v i n g world; 5'-nucleotidases have been studied from b a c t e r i a , yeast, plants and numerous tissues from higher animals (for review see Drummond and Yamamoto, 1971). The c h a r a c t e r i s t i c s of such enzymes are almost as varied as t h e i r sources. For example, the 5'-nucleotidase from bovine p i t u i t a r y i s soluble (Lisowski, 1966) while that from E h r l i c h Ascites-tumour c e l l s i s mem-brane bound (Murray and F r i e d r i c h s , 1969). The r e l a t i v e rates of hydrolysis of 5'-nucleotides by the enzyme from d i f f e r e n t sources are quite d i f f e r e n t (Drummond and Yamamoto, 1971). In add i t i o n , both a soluble ( F r i t z s o n , 1969) and a membrane bound (Song and Bodansky, 1967) 5'-nucleotidase have been extracted from rat l i v e r . The properties of these enzymes are s u b s t a n t i a l l y d i f f e r e n t . The soluble enzyme had a K (5.'-AMP) of 6.8 mM, an absolute requirement f o r Mg and a pH optimum of 6.2. In contrast, the membrane bound enzyme had a (5'-AMP) of 0.022 mM.and pH optima at 7.5 and 9.3; Mg enhanced a c t i v i t y but was not e s s e n t i a l . From these data, i t i s apparent that properties of the enzyme should not be extrapolated from one source to another and that caution should be exercised i n comparison and i n t e r -p r etation. Therefore, the involvement of 5'-nucleotidase i n coronary autoregulation must be discussed with s p e c i f i c reference to the enzyme from heart. Fortunately, 5'-nucleotidase i s present i n cardiac t i s s u e from several species i n quantities quite adequate for examination (Baer, Drummond and Duncan, 1966). By the use of histpchemical and' electron microscopic techniques, the enzyme from rat heart has been demonstrated 16. i n t h e T-system, i n t e r c e l l u l a r s p a ces and e n d o t h e l i a l c e l l s ( R o s t g a a r d and Behnke, 1959). Baer e t a l . , (1966) r e p o r t e d t h a t 83% of t h e r a t h e a r t enzyme was membrane bound w h i l e 17% was s o l u b l e ; t h e membrane bound enzyme c o u l d be s o l u b i l i z e d w i t h sodium d e o x y c h o l a t e . • The (5'-AMP) of t h e 5 ' - n u c l e o t i d a s e f r o m membrane, a f t e r s o l u b i l i z a t i o n , was 1.8 x 10 ~* M; pH optimum was 9.5 and i t was c o m p e t i t i v e l y i n h i b i t e d by ATP. Edwards and M a g u i r e (1970) r e p o r t e d t h a t t h e K m (5'-AMP) was 1.65 x 10 ^ M, pH optimum 7.6 and ATP i n h i b i t e d i n a mixed c o m p e t i t i v e -n o n - c o m p e t i t i v e manner; t h e s e d a t a were a l s o d e r i v e d f r o m r a t h e a r t enzyme. I f c a r d i a c 5 ' - n u c l e o t i d a s e p r o d u c e s a d e n o s i n e f o r v a s o d i l a t i o n , a membrane bound enzyme woul d appear most s u i t a b l e f o r t h e e f f i c i e n t , d i s p o s a l o f t h i s f u n c t i o n . ' F o r m a t i o n o f a d e n o s i n e i n the v i c i n i t y o f th e c e l l membrane would p r o v i d e f o r t h e s h o r t e s t d i s t a n c e o f d i f f u s i o n t o t h e e f f e c t i v e s i t e ; i t would a l s o r e d u c e t o a minimum t h e p o s s i b i l i t y o f d e g r a d a t i o n by a d e n o s i n e deaminase o f t h e c y t o p l a s m (Baer e t a l . , 1966). T h e r e f o r e , s t u d i e s o f 5 ' - n u c l e o t i d a s e , w i t h r e f e r e n c e t o t h e a d e n o s i n e h y p o t h e s i s , s h o u l d i n v o l v e t h e membrane bound enzyme i n p r e f e r e n c e t o t h e s o l u b l e enzyme. Thus, a s t u d y o f membrane bound, c a r d i a c 5 ' - n u c l e o t i d a s e may be r e l e v a n t t o f u r t h e r i n g o ur knowledge of c o r o n a r y p e r f u s i o n . An u n d e r -s t a n d i n g o f t h e mechanisms w h i c h ^ e f f e c t r e g u l a t i o n o f c o r o n a r y b l o o d f l o w i s p e r t i n e n t t o b o t h t h e d e s i g n and p r e s c r i p t i o n o f p h a r m a c o l o g i c a l a g e n t s used i n t r e a t i n g d i s e a s e s o f c o r o n a r y o r i g i n . T h i s t h e s i s d e s c r i b e s a t t e m p t s t o c o n t r i b u t e t o such a p o o l o f knowledge by e x a m i n a t i o n o f : t h e l e v e l s o f 5 ' - n u c l e o t i d a s e i n h e a r t s o f s e v e r a l s p e c i e s , t h e l o c a l i z a t i o n o f 5 ' - n u c l e o t i d a s e i n c a r d i a c t i s s u e and t h e p r o p e r t i e s o f membrane bound 5 ' - n u c l e o t i d a s e i s o l a t e d f r o m h e a r t . 17. METHODS AND MATERIALS A. General 1. Phosphate Assay Determinations of inorganic phosphate were made by a modification of the Fiske and SubbaRow (1925) method as described below. Ac i d -molybdate reagent contained 2.5% (NH^^MoO^ i n 5N H^SO^. Fresh reducing s o l u t i o n was made every 4 or 5 days. It was prepared by d i s s o l v i n g 0.25 g of powder containing 7.70% l-amino-2-naphthol-4-sulfonic a c i d , 46.15% Na2S0^ and 46.15% NaHSO^ i n 10 ml water. An aliquot to be assayed was mixed with 50 u l acid-molybdate, 20 u l reducing s o l u t i o n and water i n a f i n a l volume of 1.0 ml. Aft e r incubation at room temperature for 10 minutes the absorbance was measured at 720 nm i n a Beckman DU spectro-photometer. The l i g h t path was 1.0 cm. 2. Protein Determination Protein concentrations of extracts were estimated by the biuret method except i n the case of the p a r t i a l l y p u r i f i e d 5'-nucleotidase, i n which the o p t i c a l assay was used (Laynej 1957). Ammonium ions were removed from protein preparations by d i a l y s i s against d i s t i l l e d water when required. B. Survey of Cardiac Enzymes which U t i l i z e 5'-AMP and Adenosine For a l l experiments described i n t h i s section, enzymes were prepared and assayed on the same day; preparation of enzymes was ca r r i e d out at 0-4°. In a l l cases, hearts were removed from animals immediately a f t e r s a c r i f i c e , washed to remove blood and used d i r e c t l y or stored at -80° for enzyme preparation at a l a t e r date. 1. 5'-Nucleotidase Fresh or frozen hearts were trimmed of n o n - v e n t r i c u l a r t i s s u e , minced and homogenized w i t h a Potter-Elvehjem homogenizer, i n 10 volumes of 50 mM 2-amino-2-methyl-l, 3-propanediol b u f f e r , pH 9.0 c o n t a i n i n g 2.8 mM MgC^ and 0.15 M KCl . The homogenate was s t r a i n e d through cheese c l o t h to remove connective t i s s u e . In order to remove endo-genous orthophosphate, one volume of saturated (NH^^SO^ s o l u t i o n was added sl o w l y to the- homogenate, w i t h constant s t i r r i n g . The mixture was c e n t r i f u g e d at 30,000 x g f o r 10 minutes, the supernatant discarded and the w a l l s of the c e n t r i f u g e tube c a r e f u l l y r i n s e d w i t h the above b u f f e r . The p e l l e t was dispersed i n b u f f e r , the pH was adjusted to 9.0 and the volume was adjusted to that of the o r i g i n a l homogenate. For the assay:, 20 y l of 0.10 M 5'-AMP was added to 230 y l of.the t i s s u e suspension, and t h i s mixture was incubated f o r 10 minutes at 37° i n a water bath w i t h mechanical shaking. C o n t r o l s were c a r r i e d out i n which 5'-AMP was replaced w i t h 3'-AMP, 3 -glycerophosphate or water. A l l assays were performed i n d u p l i c a t e . The r e a c t i o n was stopped by a d d i t i o n of 1.0 ml of' i c e - c o l d 3% 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 to remove denatured p r o t e i n , a l i q u o t s ' o f the supernatant f l u i d were assayed f o r i n o r g a n i c phosphate. A c t i v i t y i s expressed as ymoles per minute per mg p r o t e i n or per gram of t i s s u e wet weight. 2. Adenylate Deaminase Fresh or fr o z e n hearts were trimmed of. n o n - v e n t r i c u l a r t i s s u e , minced and homogenized, w i t h a Potter-Elvehjem homogenizer i n 10 volumes of 0.10 M potassium phosphate b u f f e r pH 7.5, c o n t a i n i n g 0.15 M KCl . The 19. homogenate was c e n t r i f u g e d a t 30,000 x g f o r 20 m i n u t e s and t h e c l e a r s u p e r n a t a n t was k e p t f o r a s s a y . The i n c u b a t i o n m i x t u r e c o n t a i n e d 20 p i o f 0.10 M 5'-AMP, an a p p r o p r i a t e amount o f enzyme s o l u t i o n and hom o g e n i z i n g b u f f e r i n a f i n a l volume o f 250 y l . The r e a c t i o n p r o c e e d e d a t 37° f o r 10 m i n u t e s and was s t o p p e d by t h e a d d i t i o n o f 250 y l o f 10% p e r c h l o r i c a c i d . A c o n t r o l tube was p r e p a r e d i n an i d e n t i c a l manner, e x c e p t t h a t t h e enzyme was added a f t e r p e r c h l o r i c a c i d . F o l l o w i n g r e m o v a l o f 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 , 20 VI a l i q u o t s o f each s u p e r n a t a n t were d i l u t e d w i t h b u f f e r t o a f i n a l volume of 1.0 m l . The ab s o r b a n c e a t 265 nm was measured i n a Unicam SP 825 s p e c t r o p h o t o m e t e r . The d e c r e a s e i n ab s o r b a n c e o f t h e e x p e r i m e n t a l t u b e s a t t h i s wave l e n g t h compared w i t h the c o n t r o l was used t o c a l c u l a t e t h e amount o f s u b s t r a t e deaminated t o IMP. The v a l i d i t y o f t h e a s s a y r e s u l t s f r o m t h e f a c t t h a t t h e m o l e c u l a r e x t i n c t i o n c o e f f i c i e n t of IMP i s 40% o f t h a t o f 5'-AMP under t h e s e c o n d i t i o n s (Ae 265 nm 3 8.1 x 10 ). The p o s s i b i l i t y o f an a r t i f a c t u a l d e c r e a s e i n a b s o r b a n c e due t o d e p h o s p h o r y l a t i o n o f 5'-AMP by a phosphate f o l l o w e d by d e a m i n a t i o n of a d e n o s i n e was e x c l u d e d , because no ph o s p h a t a s e c o u l d be d e t e c t e d . C h r o m a t o g r a p h i c a n a l y s i s .(paper; i s o p r o p a n o l : NH^OH: w a t e r , 7:1:2) d i s c l o s e d t h a t o n l y 5'-AMP and i t s d e a m i n a t i o n p r o d u c t , IMP, were p r e s e n t i n t h e r e a c t i o n m i x t u r e a t t h e end of i n c u b a t i o n . 3. A d e n y l a t e K i n a s e E x t r a c t s o f v e n t r i c l e were p r e p a r e d as f o r a d e n y l a t e deaminase d e t e r -m i n a t i o n s , e x c e p t t h a t t h e hom o g e n i z i n g b u f f e r was 0.1 M T r i s - H C l , pH 7.0, c o n t a i n i n g 10 mM M g C l 2 and 0.10 M KC1. The 30,000 x g s u p e r -n a t a n t was a s s a y e d u s i n g ADP as s u b s t r a t e and 5'-AMP formed was deaminated by c o u p l i n g w i t h an e x c e s s o f p u r i f i e d a d e n y l i c deaminase. 20. The reactions are: Adenylate Kinase 2 ADP ^ * ATP + 5'-AMP Excess Adenylate Deaminase 5'-AMP » 5'-IMP The assay was performed i n spectrophotometric c e l l s ( l i g h t path = 0.5 cm) which contained 20 y l of 10 mM ADP, 20 y l of adenylic deaminase s o l u t i o n (75 yg pr o t e i n ) , an appropriate amount of 30,000 x g supernatant and homogenizing buffer i n a f i n a l volume of 1.5 ml. The adenylic deaminase added was s u f f i c i e n t to deaminate 1.5 ymoles of 5'-AMP per minute under these conditions. A blank was prepared i n the same manner as the experimental, except ADP was omitted. The reaction was started by addition of heart extract (diluted with buffer as required) and the rate of decrease i n absorbance at 265 nm was determined using a Unicam SP 825 recording spectrophotometer with a 1.0 A attenuator (to allow use of high substrate concentrations). ' Assays were conducted at 30°. A c t i v i t y i s expressed as i n i t i a l v e l o c i t y i n ymoles ADP u t i l i z e d per minute per mg protein or per gram of tiss u e wet weight. 4. Adenosine Deaminase Adenosine deaminase assays were c a r r i e d out on extracts prepared for adenylate kinase assay. Reaction mixtures, i n spectrophotometric c e l l s ( l i g h t path = 0.5 cm), contained 50 y l of 10 mM adenosine, 5 or 10 y l of heart extract, and 0.10 M c i t r a t e buffer pH 6.5, containing 0.10 M KCl i n a f i n a l volume of 1.5 ml. The blank contained a l l components of the assay except adenosine. The reaction rate was determined from the decrease i n absorbance at 265 nm as for adenylate 21. deaminase. Assays were conducted at 30 C. A c t i v i t y i s expressed as i n i t i a l v e l o c i t y i n umoles per minute per mg protein. C. Perfusion o f Rat, Rabbit and T u r t l e Hearts Hearts were removed as quickly as possible a f t e r s a c r i f i c e and placed i n perfusion f l u i d . A f t e r extraneous connective ti s s u e was trimmed away, hearts were cannulated and perfused by the Langendorf technique. -Rat and rabbit hearts were cannulated by the aorta and perfused with Krebs-Ringer bicarbonate s o l u t i o n at 37°. This s o l u t i o n contained 118.5 mM NaCl, 2.75 mM KC1, 2.54 mM C a C l 2 , 118 mM KH 2P0 4, 1.18 mM MgSO^, 24.9 mM NaHC03, 1.8 g/1 glucose and was aerated with 95% 0 2 -5% C0 2. " T u r t l e hearts were cannulated through the r i g h t aorta (Robb, 1965) and were perfused with Mine's s o l u t i o n (Meester, Hardman and Barboriak, 1965) at room temperature. Mine's s o l u t i o n contained 101 mM NaCl, 5.0 mM H 3B0 3, 3.9 mM N a ^ H . ^ , 2.0 mM C a C l 2 , 3.0 mM KC1, 1.0 g/1 glucose and was aerated with 100% 0 2. The perfusion pressure i n a l l experiments was 50 cm water. The apices of the hearts were connected to a force transducer (Grass FT 03) which was coupled to a s t r i p chart recorder (Grass 5D). The d i a s t o l i c tension was 1 g. Flow rates were determined by c o l l e c t i n g the perfusate f o r several 1 minute periods. Rates of coronary perfusion, i n the absence and presence of adenosine, were recorded only a f t e r an e q u i l i b r a t i o n period; hearts were considered to be e q u i l i b r a t e d when both flow rate and c o n t r a c t i l e force had reached a steady state. Control flow rates were recorded while hearts were being perfused with the solutions described above; experi-mental rates were recorded while hearts were being perfused with solutions containing the desired concentration of adenosine. Rates of coronary perfusion are expressed as percent of the co n t r o l . 22. H i s t o c h e m i s t r y The l o c a l i z a t i o n o f 5 ' - n u c l e o t i d a s e was d e t e r m i n e d by t h e method o f W a c h s t e i n and M e i s e l (1957) i n w h i c h l i b e r a t e d p h osphate i s t r a p p e d a t t h e enzyme s i t e by p r e c i p i t a t i o n as t h e l e a d s a l t . Lead d e p o s i t i o n i s s u b s e q u e n t l y made v i s i b l e by c o n v e r s i o n t o t h e s u l f i d e w h i c h a p p e a r s as d a r k brown o r b l a c k d e p o s i t s i n t h e t i s s u e s e c t i o n . F r e s h t i s s u e was mounted on c r y o s t a t microtome p l a n c h e t t e s w i t h c o m m e r c i a l mounting medium (Ames) and q u i c k f r o z e n . S e c t i o n s , l O y t h i c k , were c u t , mounted on c o v e r s l i p s and a l l o w e d t o a i r d r y . These p r e p a -r a t i o n s were i n c u b a t e d f o r 45^minutes a t 37° i n a medium c o n t a i n i n g 4 ml of 0.2 M T r i s b u f f e r a d j u s t e d t o pH 7.2 w i t h m a l e i c a c i d , 1 ml o f 0.10 Mg S 0 4 , 0.6 ml o f 2% l e a d n i t r a t e , 0.4 ml H 20 and 4 ml o f 2.5 mM 5'-AMP as t h e n e u t r a l i z e d p o t a s s i u m s a l t . C o n t r o l p r e p a r a t i o n s were t r e a t e d i n e x a c t l y t h e same manner, e x c e p t 5'-AMP was r e p l a c e d w i t h 3'-AMP (2.5 mM) o r g - g l y c e r o p h o s p h a t e (4 mM) o r w a t e r . The r e a c t i o n was s t o p p e d by removing t h e s e c t i o n s f r o m t h e i n c u b a t i o n medium and p l a c i n g them i n 10% f o r m b l s a l i n e ( 4% f o r m a l d e h y d e i n 0.9% s a l i n e ) . A f t e r t h o r o u g h w a s h i n g i n w a t e r , s e c t i o n s were p l a c e d i n d i l u t e (NH^^S f o r 1 m i n u t e . F o l l o w i n g t h o r o u g h w a s h i n g i n w a t e r , t h e c o v e r s l i p s w i t h s e c t i o n s were mounted on m i c r o s c o p e s l i d e s w i t h g l y c e r i n e j e l l y and s e a l e d w i t h n a i l l a c q u e r . A s s a y o f P a r t i a l l y P u r i f i e d 5 ' - N u c l e o t i d a s e I n o r d e r t o d e t e r m i n e t h e p r o p e r t i e s o f 5 ' - n u c l e o t i d a s e , i t s a c t i v i t y was measured under a b r o a d range o f c o n d i t i o n s w h i c h a r e d e s c r i b e d i n d e t a i l i n c o n j u n c t i o n w i t h r e s u l t s . To accommodate a l l t h e s e c o n d i t i o n s , two d i f f e r e n t a s s a y p r o c e d u r e s were n e c e s s a r y . ' A t 23. substrate (5'-AMP) concentrations of 0.16 mM or higher, the a c t i v i t y was determined by the estimation of l i b e r a t e d phosphate as described previously. In general, the assay mixtures contained buffer s o l u t i o n , 5'-nucleotidase, substrate and i n some cases stimulators and/or i n h i b i t o r s . After incubation with mechanical shaking at 37° for 10 minutes, the reactions were stopped by the addition of ic e - c o l d t r i c h l o r o a c e t i c a c i d which denatured the enzyme; The denatured p r o t e i n was removed by ce n t r i f u g a t i o n and an aliquo t of the supernatant was assayed for phosphate content. Enzyme a c t i v i t y i s expressed as ymoles of orthophosphate l i b e r a t e d per minute. This method was l i m i t e d by the s e n s i t i v i t y of the phosphate assay. Hence, at 5'-AMP concentrations l e s s than 0.16 mM, 5'-nucleotidase was assayed by a more.sensitive, d i r e c t o p t i c a l method. This method depends on the degradation of newly formed adenosine to inosine by excess adenosine deaminase and measure-ment of the r e s u l t i n g change i n absorbance at 265 nm.. Incubations were ca r r i e d out i n spectrophotometric c e l l s maintained at 37° by a thermo-s t a t i c a l l y c o n t r o l l e d water jacket. The assay mixtures contained buffer s o l u t i o n , 5'-nucleotidase, 5'-AMP, excess adenosine deaminase and i n some experiments, stimulators and/or i n h i b i t o r s . The reactions are: 5'-nucleotidase 5'-AMP •—} adenosine excess adenosine deaminase adenosine •—•• • -> inosine 3 At 265 nm, the molecular e x t i n c t i o n c o e f f i c i e n t of adenosine i s 8.1 x 10 / cm greater than that of inosine. Therefore, the reaction rate was deter-mined from the decrease i n absorbance at th i s wavelength which was recorded with a Unicam SP 825 spectrophotometer and recorder. This assay was 24. limited by the low signal to noise r a t i o at high substrate concentration. Materials Sources of materials and tissues are l i s t e d below: Calbiochem, Los Angeles: IMP, creatine phosphate, glucose-1-phosphate, fructose-l-phosphate, fructose-1, 6-diphosphate, UMP, sodium deoxycholate and adenosine deaminase (calf i n t e s t i n a l mucosa). Sigma Chemical Co., St. Louis: 5'-AMP, 3'-AMP, 3-glycerophosphate, ADP, ATP, glucose-6-phosphate, dUMP, dCMP, dGMP and adenosine. N u t r i t i o n a l Biochemical Co., Cleveland: ribose-5-phosphate, ribulose-5-phosphate, and galactose-6-phosphate. Schwarz Bipresearch Inc., Mt. Vernon: CMP. Ames Co., Elkhart: Tissue-tek mounting medium. University of B r i t i s h Columbia Animal Sit e : Swiss albino mice, Wistar ra t s , albino rabbits and mongrel dogs. College B i o l o g i c a l Supplies, Seattle: t u r t l e s . Pigeons were obtained l o c a l l y . Human pa p i l l a r y muscle was obtained as biopsy material though the courtesy of Dr. Peter A l l a n , Vancouver General Hospital. Substrates were obtained as sodium or potassium sal t s or were converted to the potassium s a l t prior to use. A l l other materials were reagent grade from various sources. 25. RESULTS Preliminary One method for determination of 5'-nucleotidase a c t i v i t y depends on the estimation of inorganic phosphate l i b e r a t e d from added nucleo-t i d e . F i g . 1 shows the standard curve for determination of ortho-phosphate by a modification of the Fiske and SubbaRow (1925) method. Absorbance at 720 nm i s a l i n e a r function of orthophosphate up to 0.30 umoles. This standard curve was repeated several times over a 2 % year period, and a l l results...were i d e n t i c a l . When the l i g h t path was 1.0 cm, an absorbance of 0.380 represented 0.1 umole of ortho-phosphate; t h i s value was used i n the c a l c u l a t i o n of rates. The v a l i d i t y of the 5'-nucleotidase assay depends on more than the r e p r o d u c i b i l i t y of inorganic phosphate determination. The quantity of orthophosphate l i b e r a t e d i n the enzymatic reaction must be propor-t i o n a l to time of incubation and enzyme content. This l i n e a r r e l a t i o n -ship i s depicted i n F i g . 2, i n which enzyme a c t i v i t y was determined according to conditions•described f o r the survey experiments (Methods, section B. 1). Similar r e s u l t s were obtained from analogous experiments with the p a r t i a l l y p u r i f i e d enzyme; i n t h i s case l i n e a r i t y was observed w e l l beyond the time and amount of protein used r e g u l a r l y i n assays. JUMOLES ORTHOPHOSPHATE Figure 1. Standard curve for assay of inorganic phosphate. Graph of absorbance at ,720 nm versus ymoles of orthophosphate Mg PROTEIN (A) 0.8 1.2 1.6 L U I— < o_ to o Q_ O IE I— C£ o cn L U O 3 6 8 MINUTES (O) F i g u r e 2.. L i n e a r i t y of 5'^nucleotidase assay w i t h time and p r o t e i n . The enzyme p r e p a r a t i o n used was crude homogenate of r a t v e n t r i c l e . Experiments to show l i n e a r i t y w i t h time (O) were conducted using 1.5 mg of p r o t e i n ; those to show l i n e a r i t y w i t h p r o t e i n (A) were conducted using 10 minute i n c u b a t i o n p e r i o d . 26. B. Cardiac Enzymes which U t i l i z e 5'-AMP and Adenosine 1. 5'-Nucleotidase On the basis of the adenosine hypothesis, the l e v e l s of cardiac 5'-nucleotidase might be expected to vary from species to species and might also be expected to be a function of the^physical a c t i v i t y of the species. If the adenosine hypothesis i s applicable to a wide range of animals, one might expect a c o r r e l a t i o n between the a b i l i t y of animals to impose large loads and oxygen demands on t h e i r hearts and the a b i l i t y of t h e i r hearts to produce adenosine. Hence, l e v e l s of 5'-nucleotidase i n hearts might be re l a t e d to the p o t e n t i a l necessity f o r , and magnitude of, coronary v a s o d i l a t i o n . The t u r t l e , f o r example, was anticipated to have low l e v e l s of the cardiac enzyme, since i t can function anaerobically for long periods. Turtles survive for up to 17 hours i n t o t a l anoxia (Belkin, 1963) and apparently do not place large loads oh t h e i r hearts. On the other hand, b i r d s and mammals were expected to have r e l a t i v e l y high l e v e l s of cardiac 5'-nucleotidase, which would allow rapid production of adenosine. These animals would then be equipped to accommodate high cardiac demands imposed by increased a c t i v i t y . The rat increases oxygen consumption 3.5-fold by running (Taylor, Schmidt-Nielsen and Raab, 1970), and s i m i l a r r e s u l t s were obtained from human subjects (Bevegard and Shepherd, 1967). Energy consumption of pigeons during f l i g h t i s 8.5 times the r e s t i n g values (LeFebvre, 1964). If energy u t i l i z a t i o n and oxygen d e l i v e r y are i n d i c a t i v e of the amount of blood pumped by the heart, both rat and pigeon should have a well developed mechanism for the formation of adenosine. In f a c t j the adenosine hypothesis might predict a higher l e v e l of cardiac 5'-nucleo-tidase i n pigeons than i n r a t s . 27. The l e v e l s o f 5 ' - n u c l e o t i d a s e found i n homogenates o f h e a r t s f r o m v a r i o u s s p e c i e s a r e shown i n F i g . 3. A s s a y s were c a r r i e d o ut a t pH 9.0 to i n a c t i v a t e a d e n y l a t e deaminase w h i c h c o u l d compete f o r t h e s u b s t r a t e , 5'^AMP (Le e , 1957). Comparison o f v a l u e s i s s i m i l a r w hether a c t i v i t y i s e x p r e s s e d as umoles p e r m i n u t e p e r mg p r o t e i n o r p e r gram wet w e i g h t o f t i s s u e . There were marked d i f f e r e n c e s i n enzyme a c t i v i t y i n h e a r t s o f t h e v a r i o u s s p e c i e s s t u d i e d ; r a t h e a r t s d e m o n s t r a t e d t h e h i g h e s t a c t i v i t y f o l l o w e d by dog, mouse and r a b b i t . The 5 ' - n u c l e o t i d a s e a c t i v i t y o f r a b b i t v e n t r i c l e homogenates was l e s s t h a n 10% of r a t v e n t r i c l e . Homogenates o f t u r t l e v e n t r i c l e c o n t a i n e d no d e t e c t a b l e 5 ' - n u c l e o t i d a s e a c t i v i t y under t h e s e c o n d i t i o n s . But i n t h i s t i s s u e , a n o n - s p e c i f i c p h o s p h a t a s e was f o u n d , t h e s p e c i f i c a c t i v i t y o f w h i c h was 0.0054 umoles p e r m i n u t e p e r mg p r o t e i n when e i t h e r 3'-AMP, 5'-AMP o r B - g l y c e r o p h o s p h a t e was used as s u b s t r a t e . Under t h e c o n d i t i o n s o f th e a s s a y , p i g e o n v e n t r i c l e homogenates c o n t a i n e d no d e t e c t a b l e 5 ' - n u c l e o t i d a s e o r n o n - s p e c i f i c p h o s p h a t a s e a c t i v i t y . 0.03 o O 0.02 -o 0.01 -< \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ s o o Q \ \ \ \ \ CO ZD o \ \ BIT \ CO < \ \ o U J O 2.0 CO CO H 1.0 g LJJ ' I o Figure 3. Survey of ventricular 5"-nucleotidase. Bars depict 5'-nucleotidase a c t i v i t y of crude homogenates of hearts of various species. Enzyme a c t i v i t y i s represented on the basis of protein (open bars) and tissue wet weight (hatched bars). Lines represent standard error of the mean. Values for mouse v e n t r i c l e were obtained from 12 pooled hearts. 2. A d e n y l a t e Deaminase and A d e n y l a t e K i n a s e The s u b s t r a t e , 5'-AMP, fro m w h i c h a d e n o s i n e i s formed by t h e a c t i o n o f 5 ' - n u c l e o t i d a s e , can a l s o be u t i l i z e d by o t h e r enzymes. A d e n y l a t e deaminase c a t a l y z e s t h e c o n v e r s i o n o f 5'-AMP t o IMP. A d e n y l -a t e k i n a s e c a t a l y z e s a r e v e r s i b l e r e a c t i o n w h i c h c o n v e r t s one m o l e c u l e of 5'-AMP and one m o l e c u l e o f ATP t o two m o l e c u l e s o f ADP. I t f o l l o w s t h e n t h a t t h e f a t e o f 5'-AMP i n c e l l s c o u l d be d e t e r m i n e d by t h e r e l a t i v e a c t i v i t i e s o f t h e s e two enzymes as w e l l as 5 ' - n u c l e o t i d a s e . F o r t h i s r e a s o n , b o t h a d e n y l a t e deaminase and a d e n y l a t e k i n a s e were e s t i m a t e d i n h e a r t s o f t h e above s p e c i e s . S i n c e b o t h t h e s e enzymes fr o m h e a r t a r e s o l u b l e ( L e e , 1957a; C o l o w i c k and K a l c k a r , 1 9 4 3 ) , a s s a y s were p e r f o r m e d u s i n g 30,000 x g s u p e r n a t a n t s . A d e n y l a t e deaminase l e v e l s o f v e n t r i c u l a r s u p e r n a t a n t s o f v a r i o u s s p e c i e s a r e shown i n F i g . 4. I n a l l c a s e s , e x c e p t dog h e a r t s , enzyme a c t i v i t y was d e t e c t e d . T u r t l e v e n t r i c l e c o n t a i n e d a t l e a s t f i v e t i m e s as much a d e n y l a t e deaminase as t h a t f r o m r a t , r a b b i t o r p i g e o n . A c t i v i t y o f t h i s enzyme i n t h i s t i s s u e was c o n s i d e r a b l y l e s s t h a n t h a t o f r a b b i t s k e l e t a l m u s c l e , w h i c h i s shown i n F i g . 4 f o r c o m p a r a t i v e p u r p o s e s . A d e n y l a t e k i n a s e a c t i v i t y i n v e n t r i c u l a r s u p e r n a t a n t s o f v a r i o u s s p e c i e s i s g i v e n i n F i g . 5. P i g e o n v e n t r i c l e p o s s e s s e d h i g h e s t a c t i v i t y f o l l o w e d by r a b b i t , d o g , r a t and t u r t l e . W h i l e t u r t l e v e n t r i c l e was most s i m i l a r t o s k e l e t a l m u s c l e w i t h r e s p e c t t o a d e n y l a t e deaminase a c t i v i t y ( F i g . 4 ) , i t was l e a s t s i m i l a r w i t h r e s p e c t t o a d e n y l a t e k i n a s e ( F i g . 5 ) . Figure 4. Survey of ventricular adenylate deaminase. Adenylate deaminase was estimated i n the 30,000 x g supernatant of heart homogenates. Enzyme a c t i v i t y i s represented on the basis of protein (open bars) and tissue wet weight (hatched bars). Figure 5. Survey of ventricular adenylate kinase. Adenylate kinase of the 30,000 x g supernatant of ventricular homogenates. Enzyme a c t i v i t y i s represented on the basis of protein (open bars) and tissue wet weight (hatched bars). Lines represent standard error of the mean. 29. 3. A d e n o s i n e Deaminase I f a d e n o s i n e m e d i a t e s c o r o n a r y a u t o r e g u l a t i o n , v a s o t o n e must be a f u n c t i o n o f e x t r a c e l l u l a r a d e n o s i n e c o n c e n t r a t i o n i n t h e h e a r t . T h i s c o n c e n t r a t i o n must be a f u n c t i o n o f a d e n o s i n e d e g r a d a t i o n by a d e n o s i n e deaminase as w e l l as f o r m a t i o n by 5 1 - n u c l e o t i d a s e . F o r example, t h e e x t r a c e l l u l a r a d e n o s i n e c o n c e n t r a t i o n o f h e a r t s c o u l d a t t a i n h i g h v a l u e s i n s p i t e of low r a t e s of f o r m a t i o n p r o v i d e d t h a t t h e r a t e of d e g r a d a t i o n was s u f f i c i e n t l y s l o w . Hence i t was d e s i r a b l e t o e s t i m a t e t h e c o n t e n t of a d e n o s i n e deaminase i n h e a r t s o f v a r i o u s s p e c i e s . The a d e n o s i n e deaminase a c t i v i t i e s d e t e c t e d i n r a t , r a b b i t , dog, p i g e o n and t u r t l e h e a r t s i s l i s t e d i n T a b l e 1. O n l y m i n o r d i f f e r e n c e s were found between s p e c i e s . The most n o t a b l e o b s e r v a t i o n was t h e d e t e c t i o n o f s i g n i f i c a n t a d e n o s i n e deaminase i n p i g e o n h e a r t s . I n p r e v i o u s e x p e r i -ments ( 5 ' - n u c l e o t i d a s e s u r v e y ) , p i g e o n h e a r t s appeared u n a b l e t o form a d e n o s i n e , i . e . t h e y l a c k e d 5 ' - n u c l e o t i d a s e . I t was s u r p r i s i n g t h e r e f o r e , t h a t t h e y s h o u l d have a w e l l d e v e l o p e d means t o degrade t h i s n u c l e o s i d e . 29(a) Adenosine Deaminase from Hearts of Various Species ymoles/min/mg Protein s.e.m. number 3 3 XIO XIO Rat 9.00 0.27 10 Rabbit 3.38 0.95 3 Dog 8.28 2 Pigeon 4.03 0.31 6 Turtle 2.92 0.41 4 TABLE I. Survey of ventricular adenosine deaminase. Adenosine deaminase of the 30,000 x g supernatants of homogenates of ventricles from various species i s represented on the basis of.protein. Assays were conducted by the direct o p t i c a l method. Standard error of the mean was calculated i f more than two hearts were sampled. 30. C. E f f e c t o f A d e n o s i n e on C o r o n a r y F l o w S i n c e homogenates o f p i g e o n and t u r t l e v e n t r i c l e c o n t a i n e d no d e t e c t a b l e 5 ' - n u c l e o t i d a s e , i t seemed p o s s i b l e t h a t t h e s e t i s s u e s were i n c a p a b l e o f a d e n o s i n e f o r m a t i o n . I t was o f i n t e r e s t t h e n t o d e t e r m i n e whether a d e n o s i n e c o u l d m e d i a t e c o r o n a r y d i l a t i o n i n t h e s e h e a r t s . Some p r e l i m i n a r y e x p e r i m e n t s were p e r f o r m e d t o examine t h e e f f e c t s o f a d e n o s i n e on c o r o n a r y f l o w i n i s o l a t e d p e r f u s e d h e a r t s o f t u r t l e , r a b b i t and r a t s . P i g e o n h e a r t s c o u l d n o t be s u c c e s s f u l l y p e r f u s e d because t h e y d i d n o t s u r v i v e t h e i n t e r v a l f r o m s a c r i f i c e t o c a n n u l a t i o n . The r e s u l t s o f e x p e r i m e n t s u s i n g L a n g e n d o r f p r e p a r a t i o n s o f r a t and t u r t l e h e a r t s a r e r e p r e s e n t e d i n F i g . 6. A d e n o s i n e i n d u c e d a l a r g e i n c r e a s e i n r a t c o r o n a r y f l o w and i n c r e a s e d r a b b i t c o r o n a r y f l o w 54% a t 3 yM. The e f f e c t o f a d e n o s i n e on t u r t l e c o r o n a r y p e r f u s i o n r a t e was n o t s t a t i s t i c a l l y s i g n i f i c a n t . < O < o o 40 RAT A ~ 30 -20 — 10 0 s y \ b V * • -10 \ \ i TURTLE i • 1 1 1 2 2 6 8 10 ADENOSINE (juM) Figure 6. E f f e c t of adenosine on coronary flow. Experiments were conducted using Langendorf preparations of rat (A) and t u r t l e (O) hearts. Changes i n perfusion rate are expressed as percent of control rate determined immediately befpre experimental. V e r t i c l e l i n e s depict standard error of the mean where 4 or more values were obtained. 31. H i s t o c h e m i c a l . L o c a l i z a t i o n o f C a r d i a c 5 ' - N u c l e o t i d a s e A c c o r d i n g t o t h e a d e n o s i n e h y p o t h e s i s o f c o r o n a r y a u t o r e g u l a t i o n , a d e n o s i n e i s formed i n c a r d i a c m u s c l e c e l l s and t h e n d i f f u s e s i n t o t h e i n t e r s t i t i a l f l u i d (Rubio and Be r n e , 1969). The n u c l e o s i d e would t h e n d i f f u s e t o c o r o n a r y r e s i s t a n c e v e s s e l s , s u c h as p r e c a p i l l a r y s p h i n c t e r s ( P r o v e n z a and S c h e r l i s , 1 9 5 9 ) , i n d u c e v a s o d i l a t i o n and t h u s i n c r e a s e oxygen d e l i v e r y t o t h e myocardium. I f t h i s i s s o , t h e e n z y m a t i c m a c h i n e r y f o r a d e n o s i n e p r o d u c t i o n s h o u l d be found i n h e a r t m u s c l e c e l l s . Most o f t h e c a r d i a c 5 ' - n u c l e o t i d a s e i s a s s o c i a t e c i w i t h p a r t i c u -l a t e f r a c t i o n s o f h e a r t homogenates (Baer e t a l . , 1 9 6 6 ) , hence i t w o u l d / be r e a s o n a b l e t o e x p e c t t h i s enzyme t o be l o c a t e d on o r i n membranes of h e a r t m u s c l e c e l l s . T h i s l o c a t i o n would be most s u i t e d f o r r a p i d e f f l u x o f a d e n o s i n e . F o r m a t i o n o f a d e n o s i n e a t s i t e s e l s e w h e r e i n t h e s e c e l l s would r e s u l t i n s l o w e r e f f l u x because o f g r e a t e r d i f f u s i o n d i s t a n c e s ; i n a d d i t i o n a d e n o s i n e m i g h t be degraded by a d e n o s i n e deaminase o f t h e c y t o -p l a s m (Baer e t a l . , 1 966). Some h i s t o c h e m i c a l e v i d e n c e o f i n o r g a n i c p hosphate l i b e r a t i o n f r o m 5'-AMP by c a r d i a c m u s c l e c e l l s have a l r e a d y been r e p o r t e d ( R o s t g a a r d and Behnke, 1959; Sommer and Spach, 1964). R p s t g a a r d and Behnke (1959) found t h i s a c t i v i t y i n i n t e r c e l l u l a r s p a c e s , T-system' and e n d o t h e l i a l c e l l s . These w o r k e r s c a r r i e d o u t 1 e x a m i n a t i o n s o f t i s s u e s e c t i o n s by e l e c t r o n m i c r o s c o p y and u s i n g p r e f i x e d r a t h e a r t s . , I n t h e p r e s e n t s t u d i e s , the; s i t e o f 5 ' - n u c l e o t i d a s e i n h e a r t s was d e t e r m i n e d by t h e method o f W a c h s t e i n and M e i s e l (1957) i n w h i c h u n f i x e d s e c t i o n s a r e employed. These u n f i x e d , f r o z e n s e c t i o n s were chosen because i t i s g e n e r a l l y a c c e p t e d t h a t such p r e p a r a t i o n s p r o v i d e t h e b e s t enzyme p r e s e r -v a t i o n and s h o u l d be most s u i t a b l e f o r such s t u d i e s ( P e a r s e , 1 960). I n t h i s method, t h e l o c a l e o f 5 ' - n u c l e o t i d a s e i s c h a r a c t e r i z e d by b l a c k o r d a r k brown d e p o s i t s o f l e a d s u l f i d e . 32. U s i n g t h i s method, s e c t i o n s o f a l l mammalian h e a r t s examined, e s p e c i a l l y t h o s e o f r a t , c o n t a i n e d c o n s i d e r a b l e l e a d d e p o s i t i o n a f t e r i n c u b a t i o n w i t h 5'-AMP as t h e s u b s t r a t e • ( F i g . 7, p l a t e s A, B, C, D, G, H, and J ) . That t h i s d e p o s i t i o n was due t o s p e c i f i c 5 ' - n u c l e o t i d a s e r a t h e r : t h a n n o n - s p e c i f i c p h o s p h a t a s e s i s a p p a r e n t upon c o m p a r i s o n o f s e c t i o n s i n c u b a t e d i n t h e p r e s e n c e o f 5'-AMP t o t h o s e i n c u b a t e d w i t h 3'-AMP o r 3- g l y c e r o p h o s p h a t e . F o r example, compare F i g . 7 p l a t e D, w h i c h r e p r e s e n t s a s e c t i o n o f human p a p i l l a r y m u s c l e a f t e r i n c u b a t i o n w i t h 5'-AMP, t o p l a t e E f o r w h i c h 3'-AMP was used . S i n c e t h e o n l y d i f f e r e n c e i n t h e s e e x p e r i m e n t s was t h e p o s i t i o n o f t h e phosphate m o i e t y o f a d e n y l a t e j t h e d a r k brown and b l a c k a r e a s o f p l a t e D must r e p r e s e n t t h e a c t i o n o f s p e c i f i c 5 ' - n u c l e o t i d a s e . S i m i l a r l y , t h e p r e s e n c e o f s p e c i f i c 5 ' - n u c l e o t i d a s e i s seen on c o m p a r i s o n o f p l a t e G, w h i c h r e p r e s e n t s a s e c t i o n o f g u i n e a p i g h e a r t i n c u b a t e d w i t h 5'-AMP, to p l a t e H, w h i c h i s r e p r e s e n t a t i v e o f s i m i l a r s e c t i o n s i n c u b a t e d w i t h 3 - g l y c e r o p h o s p h a t e , 3'-AMP o r no phosphate e s t e r . I n c o n t r a s t t o mammalian h e a r t s e c t i o n s , p i g e o n v e n t r i c l e s e c t i o n s c o n t a i n e d , no l e a d d e p o s i t i o n w h a t s o e v e r a f t e r t r e a t m e n t by t h e method o f W a c h s t e i n and M e i s e l ( 1 9 5 7 ) . There was a co m p l e t e absence o f l e a d d e p o s i t i o n when e i t h e r 5'-AMP, 3'-AMP, 3-g l y c e r o p h o s p h a t e o r no phosphate e s t e r was p r e s e n t i n t h e i n c u b a t i o n medium ( p l a t e K ) . T u r t l e v e n t r i c l e i n t h e h i s t o c h e m i c a l t e s t showed f a i n t l e a d d e p o s i t i o n b u t t h i s o c c u r r e d whether 5'-AMP, o r 3'-AMP was t h e added phosphate e s t e r ( p l a t e L ) , i n d i c a t i n g t h e p r e s e n c e o f n o n - s p e c i f i c p h o s p h a t a s e as had been a s c e r t a i n e d f r o m d i r e c t .enzyme a s s a y ( F i g . 3 ) . I n f a c t , t h e i n t e n s i t y o f l e a d depo-s i t i o n i n v e n t r i c u l a r s e c t i o n s f r o m t h e v a r i o u s s p e c i e s appeared t o p a r a l l e l t h e 5 ' - n u c l e o t i d a s e a c t i v i t y d e t e r m i n e d by t e s t tube a s s a y ( F i g . 3 ) . The p a t t e r n o f l e a d d e p o s i t i o n i n mammalian h e a r t s e c t i o n s r e v e a l e d t h a t 5'-33. n u c l e o t i d a s e was d i s c r e t e l y l o c a l i z e d i n s p e c i f i c r e g i o n s t h r o u g h o u t t h e e n t i r e v e n t r i c l e . The u n i f o r m i t y o f t h i s d i s t r i b u t i o n i s i l l u s -t r a t e d i n F i g . 7, p l a t e A w h i c h i s a low m a g n i f i c a t i o n p h o t o m i c r o g r a p h o f r a t v e n t r i c l e s e c t i o n i n c u b a t e d w i t h 5'-AMP. I t i s c l e a r t h a t l e a d was d e p o s i t e d t h r o u g h o u t t h e s e c t i o n and t h a t t h i s d e p o s i t i o n was r e s t r i c t e d t o d i s t i n c t a r e a s . Upon e x a m i n a t i o n a t g r e a t e r m a g n i f i c a t i o n , no e v i d e n c e o f a c t i v i t y c o u l d be o b s e r v e d i n c a r d i a c m u s c l e c e l l s o r i n t h e plasma membrane o f c a r d i a c m u s c l e c e l l s ( p l a t e B ) . V i r t u a l l y a l l 5 ' - n u c l e o t i d a s e a c t i v i t y o f r a t h e a r t s e c t i o n s was found i n s t r u c t u r e s w h i c h s u r r o u n d m u s c l e c e l l s ; s u c h s t r u c t u r e s appeared t o be i d e n t i c a l w i t h e n d o t h e l i a l c e l l s . E v i d e n c e t h a t t h e r e g i o n s o f l e a d d e p o s i t i o n i n h e a r t s e c t i o n s were i n d e e d o c c u p i e d by e n d o t h e l i a l c e l l s was p r o v i d e d f r o m a d j a c e n t s e c t i o n s s t a i n e d f o r n u c l e i w i t h h e m a t o x y l i n and c o u n t e r s t a i n e d w i t h e o s i n ( p l a t e F ) . A r e a s h a v i n g 5 ' - n u c l e o t i d a s e were i d e n t i c a l t o n u c l e i c o n t a i n i n g r e g i o n s o f r o u t i n e l y s t a i n e d s e c t i o n s , w h i c h r e p r e s e n t e d e n d o t h e l i a l c e l l s o f t h e c o r o n a r y m i c r o c i r c u l a t i o n . Thus i t appears t h a t , by t h i s method, most o f t h e c a r d i a c 5 ' - n u c l e o t i d a s e was l o c a l i z e d i n e n d o t h e l i a l c e l l s o f c a p i l l a r i e s . T h i s d i s t r i b u t i o n was o b s e r v e d i n dog, human, g u i n e a p i g and mouse v e n t r i c l e s e c t i o n s w h i c h a r e r e p r e s e n t e d i n p l a t e s C, D, G and I , r e s p e c t i v e l y . I n each c a s e , h e a r t m u s c l e c e l l s a r e c l e a r l y v i s i b l e , b u t no d e p o s i t s o f l e a d c a n be d e t e c t e d w i t h i n them; l e a d d e p o s i t s were found i n d i s c r e t e a r e a s between m u s c l e c e l l s . I n c o n t r a s t , l e a d d e p o s i t i o n i n ' t u r t l e v e n t r i c l e , a l t h o u g h f a i n t , was n o t c o n f i n e d t o e n d o t h e l i a l c e l l s b u t appeared t o be g e n e r a l l y d i s t r i b u t e d i n a l l c e l l s . The p a t t e r n o f l e a d d e p o s i t i o n d e m o n s t r a t e d i n t h e s e p h o t o g r a p h s was h i g h l y r e p r o d u c i b l e ; t h r e e t o s i x h e a r t s were sampled i n d u p l i c a t e f r o m each s p e c i e s a l w a y s w i t h i d e n t i c a l r e s u l t s . . 34. A l t h o u g h no 5 ' - n u c l e o t i d a s e c o u l d be d e t e c t e d w i t h i n m u s c l e c e l l s by t h e h i s t o c h e m i c a l method u s e d , o t h e r l a b o r a t o r i e s have r e p o r t e d t h a t t h e enzyme was d e t e c t a b l e t h e r e . As p r e v i o u s l y m e n t i o n e d , R o s t g a a r d and Behnke ( 1 9 5 9 ) , who used enzyme h i s t o c h e m i s t r y and e l e c t r o n m i c r o s c o p y , o b s e r v e d a c t i v i t y i n t h e T-system o f r a t h e a r t . By s i m i l a r t e c h n i q u e s , Sommer and Spach (1964) d e m o n s t r a t e d t h e p r e s e n c e o f a p h o s p h a t a s e a t the t r i a d s and i n t e r c a l l a t e d d i s c s i n s e c t i o n s of p r e f i x e d dog h e a r t s . T h i s i n f o r m a t i o n may n o t be as p e r t i n e n t t o t h e a d e n o s i n e h y p o t h e s i s as t h a t o f R o s t g a a r d and Behnke (1959) because a n o n - s p e c i f i c phospha-t a s e was i n v o l v e d ; a c t i v i t y was d e m o n s t r a t e d i n t h e p r e s e n c e o f ATP, ADP, IDP, AMP, t h i a m i n e p y r o p h o s p h a t e and ^ - g l y c e r o p h o s p h a t e w h i c h i s i n d i c a t i v e o f a n o n - s p e c i f i c p h o s p h a t a s e . Hardpnk (1968) r e p o r t e d s m a l l amounts of 5 ' - n u c l e o t i d a s e i n m u s c l e c e l l s o f r a t h e a r t s e c t i o n s ; t h e W a c h s t e i n and M e i s e l (1957) t e c h n i q u e was used. Whether a c t i v i t y was extended t h r o u g h o u t t h e m u s c l e c e l l s o r l o c a l i z e d n e a r t h e s u r f a c e was n o t c l e a r . I t i s p o s s i b l e t h a t t h i s o b s e r v a t i o n of 5 ' - n u c l e o t i d a s e i n m u s c l e c e l l s was an a r t i f a c t o f t h e T r i s - H C l b u f f e r used by Hardonk (1968). When T r i s - H C l and T r i s - m a l e a t e b u f f e r s were compared i n t h e p r e s e n t s t u d y , t h e l a t t e r was f a r s u p e r i o r . T r i s - H C l was i n f e r i o r b ecause t h e r e was random a g g r e g a t i o n o f l e a d on t h e s u r f a c e s o f t i s s u e s e c t i o n s when i t was used. T h i s s u p e r f i c i a l l e a d p r e c i p i t a t i o n , i n t h e p r e s e n c e o f T r i s - H C l , made i t i m p o s s i b l e t o r e l i a b l y i d e n t i f y t h e s i t e o f 5 ' - n u c l e o t i d a s e . S e c t i o n s f r o m g u i n e a p i g v e n t r i c l e and human p a p i l l a r y m u s c l e c o n t a i n e d i n t e n s e l e a d d e p o s i t s w i t h i n t h e w a l l s o f l a r g e r b l o o d v e s s e l s , i n a d d i t i o n t o t h o s e d e p o s i t s i n e n d o t h e l i a l c e l l s . F i g . 7, p l a t e J i s a low m a g n i f i c a t i o n p h o t o m i c r o g r a p h o f a s e c t i o n from g u i n e a p i g h e a r t showing s u b s t a n t i a l 5 ' - n u c l e o t i d a s e a c t i v i t y i n the c e n t r e of t h e f i e l d . 35. Examination of such vessels at higher magnification revealed that lead deposition was largely confined to the intima and media; the adventia had only s l i g h t a c t i v i t y . F i g u r e 7. H i s t o c h e m i c a l l o c a l i z a t i s e c t i o n s . The enzyme i s l o c a t e d i n l e a d d e p o s i t i o n . P l a t e B. Rat v e n t r i c l e ; s u b s t r a t e 5'-AMP. M a g n i f i c a t i o n 900X, showing l o c a l i z a t i o n o f 5 ' - n u c l e o t i d a s e i n c a p i l l a r y e n d o t h e l i u m . n o f 5 1 - n u c l e o t i d a s e i n v e n t r i c l e t h e d a r k brown o r b l a c k a r e a s o f P l a t e A. Rat v e n t r i c l e ; s u b s t r a t e 5'-AMP M a g n i f i c a t i o n 90X, showing g e n e r a l i z e d h i g h a c t i v i t y between m u s c l e c e l l s . P l a t e C. Dog v e n t r i c l e ; s u b s t r a t e 5'-AMP M a g n i f i c a t i o n 900X, showing r e a c t i o n p r o d u c t l i m i t e d t o c a p i l l a r y e n d o t h e l i u m between m u s c l e c e l l s . P l a t e E. Human p a p i l l a r y muscle; c o n t r o l using 3'-AMP. M a g n i f i c a t i o n 360X. I d e n t i c a l r e s u l t s were obtained w i t h 3-glycerophosphate or water. P l a t e D. Human p a p i l l a r y muscle; substrate 5'-AMP. L o n g i t u d i n a l l y sectioned e n d o t h e l i a l c e l l s e x h i b i t high 5'-nucleotidase a c t i v i t y . M a g n i f i c a t i o n 900X. P l a t e F. Human p a p i l l a r y muscle; hema-t o x y l i n and eo s i n . M a g n i f i c a t i o n 360X. Note the v a s c u l a r -i z a t i o n adjacent to myocardial c e l l s i n v o l v i n g s t r u c t u r e s which c o n t a i n c l e a r l y v i s i b l e n u c l e i , p r o v i d i n g evidence that the s t r u c t u r e s c o n t a i n i n g lead deposits are e n d o t h e l i a l c e l l s . Plate H. Guinea pig v e n t r i c l e ; control. Water replaced 5'-AMP i n the incubation medium. Analogous resu l t s were obtained with human, r a t , dog and mouse v e n t r i c l e sections when water, g-glycero-phosphate or 3'-AMP replaced 5'-AMP i n the incubation medium. Magnification 360X. Plate G. Guinea pig v e n t r i c l e ; substrate 5'-AMP. Magnification 900X. Plate I. Mouse v e n t r i c l e ; substrate 5'-AMP. 5'-Nucleotidase a c t i v i t y l o c a l i z e d i n areas between myocardial c e l l s . Magni-f i c a t i o n 360X. Plate J . Guinea pig v e n t r i c l e ; substrate 5'-AMP. Magnification 90X, showing 5'-nucleo-tidase located i n large blood vessels i n addition to c a p i l l a r i e s . Plate K. Pigeon v e n t r i c l e ; substrate 5'-AMP. Magnification 360X. Note absence of lead deposition; results correlate with lack of 5'-nucleotidase measured by biochemical assay. Plate L. Turtle v e n t r i c l e ; substrate 5'-AMP. Magnification 90X. Diffuse lead deposition obtained with 3'-AMP as well as 5'-AMP indicating non-specific phosphatase a c t i v i t y . 36. P a r t i a l P u r i f i c a t i o n o f 5 1 - N u c l e o t i d a s e C o n s i d e r a b l e i n t e r e s t i n t h e p r o p e r t i e s o f c a r d i a c 5 ' - n u c l e o t i d a s e has a r i s e n r e c e n t l y because o f t h e p o s s i b l e i n v o l v e m e n t o f a d e n o s i n e i n r e g u l a t i o n o f c o r o n a r y b l o o d f l o w . A s t u d y o f 5 ' - n u c l e o t i d a s e from c a r d i a c t i s s u e was u n d e r t a k e n i n t h i s l a b o r a t o r y i n an a t t e m p t t o l e a r n more about t h e f o r m a t i o n o f a d e n o s i n e i n h e a r t . W h i l e t h i s work was i n p r o g r e s s , p a p e r s by Edwards and M a g u i r e (1970) and S u l l i v a n and A l p e r s (1971) were p u b l i s h e d i n w h i c h s e v e r a l c h a r a c t e r i s t i c s o f c a r d i a c 5 ' - n u c l e o t i d a s e were d e s c r i b e d . 5 ' - N u c l e o t i d a s e has been r e p o r t e d i n t h e s o l u b l e and membrane-c o n t a i n i n g , p a r t i c u l a t e f r a c t i o n s o f r a t h e a r t homogenates (Baer e t a l . , 1966). I n o r d e r t h a t t h e s e s t u d i e s m i g h t be r e l e v a n t t o t h e p o s s i b l e r o l e o f a d e n o s i n e i n c o r o n a r y a u t o r e g u l a t i o n , e x a m i n a t i o n o f t h e membrane bound 5 ' - n u c l e o t i d a s e was deemed more p e r t i n e n t t h a n e x a m i n a t i o n o f t h e s o l u b l e enzyme. Rat h e a r t s were chosen as t h e s o u r c e o f enzyme, because th e y c o n t a i n e d t h e h i g h e s t l e v e l o f 5 ' - n u c l e o t i d a s e ( F i g . 3) and t h e m a j o r i t y (83%) o f enzyme f r o m t h e s e h e a r t s was membrane bound (Baer eit a l . , 1,966) . Some p r o p e r t i e s o f t h e enzyme f r o m t h i s s o u r c e have a l r e a d y been e l u c i d a t e d (Baer e t a l . , 1 966). B e f o r e an e x t e n s i v e s t u d y o f 5'-n u c l e o t i d a s e c o u l d be c a r r i e d o u t , i t s p u r i f i c a t i o n was n e c e s s a r y . I n p a r t i c u l a r , i t was e s s e n t i a l t o remove a l l enzymes w h i c h c o u l d i n t e r f e r e w i t h t h e b a s i c a s s a y s f o r 5 ' - n u c l e o t i d a s e and t h e subsequent m o d i f i c a t i o n s o f t h e s e a s s a y s . I n a t y p i c a l p r e p a r a t i o n , 10-15 f r o z e n , washed r a t h e a r t s were trimmed o f n o n - v e n t r i c u l a r t i s s u e and minced. Mi n c e d v e n t r i c l e s were homogenized i n 10 volumes of a c e t o n e a t -15°. The homogenate was c e n t r i f u g e d a t 30,000 x g f o r 10 m i n u t e s a t -15°; t h e s u p e r n a t a n t 37. acetone was discarded. The p e l l e t was extracted twice with the same volume of acetone. A f t e r the t h i r d extraction, the p e l l e t was dried under vacuum, provided by water aspirator., f o r 1 hour, broken up into a powder and dri e d under o i l pump vacuum for 3 hours. This powder was stored at -80° u n t i l used. Procedures f o r extraction of 5'-nucleotidase from the acetone powder were c a r r i e d out at 4°. Acetone powder (1.5 g) was mixed with 10 ml of 50 mM Tris-HCl b u f f e r , pH 7.5 i n a c h i l l e d glass mortar u n t i l a smooth paste was obtained. The paste was transferred to a glass Potter-Elvehjem homogenizer, 40 ml of 50 mM Tris-HCl buffer pH 7.5 was added and the mixture homogenized thoroughly. The suspension was centrifuged at 30,000 x g for 10 minutes and the supernatant was discarded. The p e l l e t was extracted with a further 50 ml of 50 mM Tris-HCl buffer pH 7.5 and centrifuged at 30,000 x g for 10 minutes. The supernatant was discarded.- The p e l l e t was then extracted 9 times with 50 ml of 50 mM Tris-HCl buffer, pH 7.5, containing 2 M KBr. The c e n t r i f u g a t i o n was at 30,000. x g f o r 10 minutes and a l l supernatants were discarded. . The KBr remaining i n the p e l l e t was removed by extracting the p e l l e t twice with 50 ml of 50 mM Tris-HCl pH 7.5 as above. The p e l l e t was then homogenized i n 20 ml of-50 mM T r i s - H C l , pH 7.5 containing 1% Na deoxycholate. This was s t i r r e d f o r 3 hours and centrifuged at 30,000 x g for 10 minutes. The supernatant which contained the enzyme was saved. The p e l l e t was extracted with 3 ml of 50 mM Tris-HCl pH 7.5, centrifuged at 30,000 x g for 10 minutes and t h i s r e s u l t i n g p e l l e t was discarded. The supernatants were pooled for immediate use or could be stored at -20° without loss of a c t i v i t y . 38. The d e o x y c h o l a t e e x t r a c t was b r o u g h t t o 15% s a t u r a t i o n w i t h r e s p e c t t o (NH)^SO^ by a d d i t i o n o f a s a t u r a t e d s o l u t i o n . The m i x t u r e was c e n t r i f u g e d a t 30,000 x g f o r 10 m i n u t e s and t h e p r e c i p i t a t e was d i s c a r d e d . The r e s u l t i n g s u p e r n a t a n t was d i a l y z e d a g a i n s t 30 volumes o f 50 mM T r i s - H C l pH 7.2 f o r 3 h o u r s w i t h 2 changes o f d i a l y s i s b u f f e r . F o r e x p e r i m e n t s i n w h i c h i t was n e c e s s a r y t o remove e n d o g e n o u s . ; d i v a l e n t c a t i o n , t h e s u p e r n a t a n t was b r o u g h t t o 10 mM EDTA f o r 10 m i n u t e s p r i o r t o d i a l y s i s . T h i s p u r i f i c a t i o n scheme i s summarized i n F i g . 8 and t h e y i e l d s f r o m a t y p i c a l p r e p a r a t i o n a r e l i s t e d i n T a b l e 2. The enzyme was p u r i -f i e d 1 6 - f o l d f r o m t h e a c e t o n e powder w i t h a y i e l d o f 107%. T h i s h i g h y i e l d was p r o b a b l y due t o s o l u b i l i z a t i o n w i t h spdium d e o x y c h o l a t e , w h i c h may have enhanced the a v a i l a b i l i t y o f s u b s t r a t e t o t h e a c t i v e s i t e . The f i n a l enzyme p r e p a r a t i o n was o b t a i n e d i n s o l u b l e f o r m , w h i c h f a c i l i t a t e d a s s a y p r o c e d u r e s and p e r m i t t e d t h e use o f d i r e c t o p t i c a l a s s a y s . T h i s f i n a l p r e p a r a t i o n was used f o r enzyme c h a r a c t e r i z a t i o n s t u d i e s . I t was s u f f i c i e n t l y f r e e o f ATPase, n o n - s p e c i f i c p h o s p h a t a s e , a d e n y l a t e deaminase, a d e n y l a t e k i n a s e and a d e n o s i n e deaminase t h a t i n t e r f e r e n c e w i t h 5 ' - n u c l e o -t i d a s e a s s a y s were o b v i a t e d . F u r t h e r e f f o r t s t o p u r i f y t h e enzyme, i . e . t o o b t a i n a h i g h e r a c t i -v i t y p e r mg o f p r o t e i n ( s p e c i f i c a c t i v i t y ) , were u n s u c c e s s f u l . Dowex 1 and v a r i o u s forms o f c e l l u l o s e (DEAE-, CM-, phospho-, PAB-, ECTE0LA-) and Sephadex (DEAE-, QAE-) were used i n a t t e m p t s t o p u r i f y 5 ' - n u c l e o -t i d a s e on t h e b a s i s o f m o l e c u l a r c h a r g e . Sepharose 6-B and u l t r a -f i l t r a t i o n were used i n a t t e m p t s t o p u r i f y t h e enzyme on t h e b a s i s o f m o l e c u l a r s i z e . O ther t e c h n i q u e s t r i e d were e t h a n o l and i s o e l e c t r i c p r e c i p i t a t i o n . A l l t h e s e methods were abandoned because t h e i n c r e a s e o f s p e c i f i c a c t i v i t y was n o t s i g n i f i c a n t o r t h e r e was e x c e s s i v e l o s s o f a c t i v i t y . 38(a) S p e c i f i c A c t i v i t y ymole/min/mg P r o t e i n T o t a l P r o t e i n mg T o t a l A c t i v i t y ymole/min 1. A c e t o n e Powder 0.0093 1,190 11.1 2. Sodium D e o x y c h o l a t e E x t r a c t 0.085 200 17.1 15% Ammonium S u l f a t e S u p e r n a t a n t 0.149 79.8 11.9 T a b l e 2. Summary o f P u r i f i c a t i o n . Y i e l d and i n c r e a s e o f s p e c i f i c a c t i v i t y o b t a i n e d d u r i n g a t y p i c a l p r e p a r a t i o n o f 5 ' - n u c l e o t i d a s e s t a r t i n g f r o m t h e a c e t o n e powder. The a s s a y s were c o n d u c t e d by d e t e r m i n a t i o n o f o r t h o p h o s p h a t e l i b e r a t e d . The i n c u b a t i o n m i x t u r e s c o n t a i n e d enzyme, 5 mM 5'-AMP, 16 mM MgCl2 and 50 mM T r i s -H C l b u f f e r , pH 7.2 i n a f i n a l volume of 1.0 m l . F u r t h e r p u r i f i c a t i o n by c h r o m a t o g r a p h i c methods was u n s u c c e s s f u l . Figure 8. Summary of 5'-nucleotidase p u r i f i c a t i o n Ventricles a. Acetone Extraction Powder b. Aqueous Extraction P e l l e t c. 2 M KBr Extraction N1/ P e l l e t d. Sodium deoxycholate extraction Acetone Soluble material discarded -> Supernatant discarded Supernatant discarded •> P e l l e t discarded Supernatant e. 15% (NH 4) 2S0 4 -> P e l l e t discarded Supernatant, f i n a l enzyme preparation 39. P r o p e r t i e s of C a r d i a c 5 ' - N u c l e o t i d a s e I f a d e n o s i n e m e d i a t e s v a s o d i l a t i o n o f t h e c o r o n a r y v a s c u l a t u r e i n r e s p o n s e to h y p o x i a , i t s r a t e o f f o r m a t i o n must be enhanced under t h i s c o n d i t i o n . One m ight e x p e c t , t h e r e f o r e , t h a t t h e a c t i v i t y o f 5 ' - n u c l e o t i d a s e may be enhanced d u r i n g oxygen d e f i c i t . I t f o l l o w s t h a t 5 ' - n u c l e o t i d a s e m i g h t be s t i m u l a t e d by s u b s t a n c e s w h i c h a c c u -mmulate i n oxygen d e f i c i e n t c a r d i a c t i s s u e . C o n v e r s e l y , 5 ' - n u c l e o -t i d a s e might be i n h i b i t e d by s u b s t a n c e s w h i c h a r e p r e s e n t i n a d e q u a t e l y o x y g e n a t e d h e a r t s b u t a r e d e f i c i e n t i n h y p o x i c t i s s u e . For example, t h e c o n c e n t r a t i o n of ATP i s s u b s t a n t i a l l y g r e a t e r i n w e l l o x y g e n a t e d h e a r t s t h a n i n a n o x i c h e a r t s ( G e r l a c h e t a l . , 1963; W i l l i a m s o n , 1966). ATP i s a p o t e n t i n h i b i t o r o f 5 ' - n u c l e o t i d a s e (Baer e t a l . , 1 9 6 6 ) , t h e r e f o r e a r e d u c t i o n i n i t s c o n c e n t r a t i o n i n h y p o x i c h e a r t s may f a c i l i t a t e the f o r m a t i o n o f a d e n o s i n e . I t i s a l s o q u i t e p o s s i b l e t h a t 5 1 - n u c l e o t i d a s e c o u l d be r e g u l a t e d by o t h e r m e t a b o l i t e s whose l e v e l s change when t h e oxygen s u p p l y t o t h e h e a r t becomes i n a d e q u a t e . F o r example, c a r d i a c o r t h o p h o s p h a t e c o n t e n t i n c r e a s e s d u r i n g h y p o x i a ( I m a i , e t a l . , 1964; W i l l i a m s o n , 1 9 6 6 ) , and so s t i m u l a t i o n of t h e enzyme by p h o s p h a t e may be a p p r o p r i a t e f o r c t h e c o r r e c t i o n of t h i s c o n d i t i o n . O b v i o u s l y , a p r e c i s e u n d e r s t a n d i n g o f t h e p r o p e r t i e s o f 5 ' - n u c l e o t i d a s e and t h e ways i n w h i c h i t may be r e g u l a t e d c o u l d c o n t r i b u t e t o t h e e v a l u a t i o n of t h e r o l e o f a d e n o s i n e i n c o r o n a r y a u t o r e g u l a t i o n . The f o l l o w i n g e x p e r i m e n t s were d e s i g n e d t o i n v e s t i g a t e t h e s e f e a t u r e s w i t h p a r t i c u l a r emphasis on p o t e n t i a l p h y s i o l o g i c a l s i g n i f i c a n c e . 1. Dependence on pH 5 ' - N u c l e o t i d a s e dependence on pH was d e t e r m i n e d o v e r t h e range of pH 5 t o pH 10. A combined T r i s - m a l e a t e b u f f e r was used so t h a t t h e 40. v a r i o u s pH v a l u e s c o u l d be accommodated w i t h a minimum o f d i f f e r e n c e f r o m one a s s a y t o t h e n e x t . Hence t h e o n l y a l t e r a t i o n s were a d d i t i o n s o f HC1 o r KOH w h i c h were used t o a d j u s t a l l a d d i t i o n s t o t h e i n c u b a t i o n m i x t u r e t o t h e a p p r o p r i a t e pH v a l u e s . N e i t h e r K + i o n s n o r CI i o n s p e r s e . a p p e a r e d to have any e f f e c t on t h e a s s a y ; up t o 0.15 M KC1 had no e f f e c t on 5 ' - n u c l e o t i d a s e a c t i v i t y . The a c t i v i t y i s e x p r e s s e d as umoles per m i n u t e p e r mg p r o t e i n a f t e r s u b t r a c t i o n o f n o n - s p e c i f i c p h o s p h a t a s e c o n t r o l v a l u e s w h i c h were o b t a i n e d i n a manner i d e n t i c a l to t h e e x p e r i m e n t a l , e x c e p t t h a t B - g l y c e r o p h o s p h a t e (10 mM) r e p l a c e d 5'-AMP.. The pH p r o f i l e o f p a r t i a l l y p u r i f i e d r a t h e a r t 5 ' - n u c l e o t i d a s e i s shown i n F i g . 9. The enzyme was a c t i v e o v e r a b r o a d range of pH, p o s s e s s i n g 85% o r more o f maximal a c t i v i t y between pH 7 and pH 10. T h i s was s i m i l a r t o t h e r e s u l t s o f Edwards and M a g u i r e ( 1 9 7 0 ) , who f o u n d t h a t r a t h e a r t enzyme m a i n t a i n e d 50% o r more of maximal a c t i v i t y f r o m pH 6 t o 8.8. Baer e t a l . (1966) r e p o r t e d t h a t t h e enzyme p o s s e s s e d 50% o r more of maximum a c t i v i t y f r o m pH 6 t o 10.5. I n F i g . 9 t h e pH optimum i s shown t o be 8.5, w h i c h i s h i g h e r t h a n t h e v a l u e , 7.6, r e p o r t e d by Edwards and M a g u i r e (1970) and l o w e r t h a n t h a t o f 9.5 r e p o r t e d by Baer e t a l . (1966) The. pH. p r o f i l e o f r a t heart.; 5 ' - n u c l e o t i d a s e i s q u i t e d i f f e r e n t f r o m t h a t o f t h e enzyme i s o l a t e d f r o m s e v e r a l o t h e r s o u r c e s . F o r example, I t o h , M i t s u i and Tsushima (1967) f o u n d t h a t t h e enzyme f r o m c h i c k e n l i v e r was m a x i m a l l y a c t i v e a t pH 6.5 and p o s s e s s e d 50% o r more of maximal a c t i v i t y o v e r a n a r r o w range of o n l y 1.5 pH u n i t s . 5 ' - N u c l e o t i d a s e f r o m p i g i n t e s t i n a l smooth m u s c l e (Burger and L o w e n s t e i n , 1970) had two pH o p t i m a , one a t 5.5 and a n o t h e r a t 9.3. S i m i l a r l y t h e membrane bound 5 ' - n u c l e o -41. tidase from r a t l i v e r (Song and Bodansky, 1967) had pH optima at 7.5 and 9.3 The discrepancy between the data represented i n F i g . 9 and that of Edwards and Maguire (1970) and Baer et^ al_. (1966) may be due to differences i n i s o l a t i o n procedure or assay conditions. The assay I | mixtures of Edwards and Maguire (1970) contained no Mg , while the I j | | present assay employed 10 mM Mg and Baer et a l . (1966) used 8 mM Mg The high a c t i v i t y reported by Baer ejt al_. at pH values above 8.5 may have been contributed to by non-specific phosphatases. Examination of F i g . 9 reveals that the pH p r o f i l e would have been considerably d i f f e r e n t i f the rate of phosphate l i b e r a t i o n from g-glycerophosphate had not been subtracted from that from 5'-AMP. Although the pH optimum f o r 5'-nucleotidase was found to be 8.5, thi s pH was not used i n the remaining studies. A l l other experiments were conducted at pH 7.2 which decreased the p o s s i b i l i t y of error due to non-specific phosphatase a c t i v i t y (Fig. 9) and was considered to be more p h y s i o l o g i c a l . If the enzyme were to function i n t r a c e l l u l a r l y , pH 7.0 would have been a more appropriate choice (Woodbury, 1965). However, there i s some evidence that i t may function e x t r a c e l l u l a r l y . Baer and Drummond (1968) found that phosphate was removed from 5'-AMP a f t e r exposure of : only a few seconds to the vasculature of rat hearts perfused by the Langendorf Method.' When radioactive 5'-AMP was infused into the coronary c i r c u l a t i o n , most of the l a b e l was recovered i n the perfusate as adenosine or inosine, which are breakdown products of 5'-AMP. Since i t i s generally accepted that nucleoside phosphates do not r e a d i l y enter c e l l s , these workers suggested that the active s i t e of 5'-nucleotidase was accessible to e x t r a c e l l u l a r substrate. If t h i s enzyme functioned e x t r a c e l l u l a r l y , the Figure 9. A c t i v i t y of 5'-nucleotidase as-a function-of pH. Rate of phosphate liberated from 5'-AMP by sp e c i f i c 5'-nucleotidase (O) a n d from 3 _glycerophosphate by non-specific phosphatase (^) plotted against pH. Points represent values obtained i n duplicate. F i n a l concentrations i n the incubation were Tris-maleate, 40 mM; MgCl2, 10 mM; 5'-AMP, 10 mM; enzyme, 0.62 mg/ml i n a f i n a l volume of 0.25 ml. 42. pH of i t s natural milieu could be 7.4 (Spector, 1956). Since the exact disposition of 5'-nucleotidase was not known, the pH value chosen for further characterization studies was a compromise of 7.2. F. 2. Effects of Divalent Cations In determining the effects of divalent cations on 5'-nucleotidase, I | one source of a r t i f a c t could be endogenous metal ions such as Mg . In order to reduce this problem to a minimum, the enzyme was treated with 10 mM EDTA, a chelating agent which binds divalent cations. The EDTA-cation chelate and excess EDTA were removed from enzyme by d i a l y s i s . Enzyme a c t i v i t y was determined by estimation of phosphate liberated. A l l values of Fig. 10 are represented as the percent of maximal a c t i v i t y i n the presence of UgCl^ after subtraction of. control values obtained by substitution of 3-glycerophosphate for 5'-AMP i n the incubation mixture. Addition of divalent cation was not essential to the demonstration of 5'-nucleotidase a c t i v i t y ; t h i s enzyme from rat heart was active even after EDTA treatment to remove divalent metal ions (Fig. 10). This obser-vation was similar to those of Edwards and Maguire (1970) and Sullivan and Alpers (1971), who also found that t h i s enzyme was active i n the absence of added Mg . 5'-Nucleotidase from rat heart was similar to that from rat cerebellum (Bosmann and Pike, 1971) which was also active i n the absence of divalent metal ion. In contrast, the soluble 5'-nucleo-tidases of rat (Fritzson, 1969) and chicken l i v e r (Itoh, et a l . , 1967) I | had absolute requirements for Mg ; no 5'-nucleotidase a c t i v i t y could be observed unless Mg was added. Although the results of the present study and those of Edwards and Maguire (1970) and Sullivan and Alpers (1971) a l l showed that 5'-nucleotidase was active i n the absence of divalent 43. c a t i o n s , t h e r e were s u b s t a n t i a l d i f f e r e n c e s between r e s u l t s o b t a i n e d i n t h e p r e s e n c e o f such i o n s . I n t h i s s t u d y 16 mM M g C ^ was a b l e t o s t i m u l a t e t h e enzyme up to 5 - f o l d ( F i g . 10) w h i l e Edwards and M a g u i r e (1970) r e p o r t e d i n h i b i t i o n by b o t h Mg and Ca . S u l l i v a n and A l p e r s (1971) r e p o r t e d no s t i m u l a t i o n by d i v a l e n t c a t i o n but c l o s e r e x a m i n a t i o n o f t h e i r d a t a r a i s e s t h e p o s s i b i l i t y o f a n o t h e r i n t e r p r e t a t i o n . They I | j | | | | | r e p o r t e d t h a t a f t e r EDTA t r e a t m e n t , Ca , Mn , Co and Mg i n c r e a s e d t h e enzyme a c t i v i t y . The p r e p a r a t i o n may have c o n t a i n e d some d i v a l e n t c a t i o n w h i c h was removed by t h i s t r e a t m e n t ; hence a d d i t i o n o f d i v a l e n t c a t i o n c o u l d c a use enzyme s t i m u l a t i o n . I n b o t h t h e p r e s e n t s t u d y and t h a t o f S u l l i v a n and A l p e r s ( 1 9 7 1 ) , EDTA t r e a t m e n t d i d n o t e l i m i n a t e a l l a c t i v i t y even i f EDTA were p r e s e n t i n t h e a s s a y . I t would appear t h a t t h e enzyme does n o t have an a b s o l u t e r e q u i r e m e n t f o r d i v a l e n t c a t i o n o r t h a t i t b i n d s t h e s e i o n s more t i g h t l y t h a n does EDTA. The f i r s t a l t e r n a t i v e seems more l i k e l y because p r o l o n g e d e x p o s u r e t o EDTA (60 m i n u t e s ) d i d n o t cause r e d u c t i o n o f a c t i v i t y g r e a t e r t h a n 10 m i n u t e s e x p o s u r e . -H- -H- ++ The s t i m u l a t i o n o f 5 ' - n u c l e o t i d a s e by Mg , N i and Mn i s d e p i c t e d i n F i g . 10. Enzyme a c t i v i t y was maximal a t 16 mM i n t h e c a s e o f Mg w i t h no d e c l i n e i n a c t i v i t y a t 80 mM. T h i s i s d i f f e r e n t f r o m t h e s o l u b l e 5 ' - n u c l e o t i d a s e o f r a t l i v e r w h i c h was m a x i m a l l y a c t i v e a t j | 0.1 M Mg and d e c l i n e d t o l e s s t h a n h a l f maximal a t 0.5 M ( F r i t z s o n , I j | | 1969). The s t i m u l a t i o n o f r a t h e a r t 5 ' - n u c l e o t i d a s e by Mn and N i ( F i g . 10) was maximal a t much l o w e r c o n c e n t r a t i o n s , 2 mM and 1 mM r e s p e c t i v e l y . The K f o r Mg , o b t a i n e d f r o m a H o f s t e e p l o t o f t h e ct | [ d a t a r e p r e s e n t e d i n F i g . 10 . ( v e l o c i t y v e r s u s v e l o c i t y / M g c o n c e n t r a t i o n ) I | | [, was 1.9 mM. Mn was a more e f f e c t i v e s t i m u l a t o r o f t h e enzyme t h a n Mg ; 4 3 ( a ) M g C l 2 (mM) C a C l 2 (mM) A c t i v i t y (%) 16 - 100 ( c o n t r o l ) - 15 10 28 16 5 94 4 - 73 4 5- 70 TABLE 3. E f f e c t o f Ca on 5 ' - n u c l e o t i d a s e . E f f e c t s o f C a C l 2 on EDTA t r e a t e d 5 ' - n u c l e o t i d a s e w i t h and w i t h o u t s t i m u l a t i o n by MgCl2->• > I— o < X o 140 -120 •• 100 80 -60 r 40 20 CATION (mM) F i g u r e 10. E f f e c t o f Mg , Mn , and N i on 5 ' - n u c l e o t i d a s e . S t i m u l a t i o n o f EDTA p r e - t r e a t e d 5 ' - n u c l e o t i d a s e was e f f e c t e d by a d d i n g MgCl2 (O). MnCl2 (A), and N i C l 2 ( • ) . V a l u e s , o b t a i n e d i n d u p l i c a t e a r e r e p r e s e n t e d as t h e p e r c e n t o f maximum o b t a i n e d i n the p r e s e n c e o f MgC^. The f i n a l c o n c e n t r a t i o n s i n t h e r e a c t i o n m i x t u r e were T r i s - H C l , 50 mM, pH 7.2; 5'-AMP, 10 mM; enzyme 0.21 mg/ml and m e t a l c h l o r i d e i n 0.25 m l . 44. maximal a c t i v i t y i n t h e p r e s e n c e o f Mn was 154% of t h a t o b t a i n e d i n the p r e s e n c e o f Mg . I n t h e c a s e s o f b o t h Mn and N i , 5 ' - n u c l e o -t i d a s e a c t i v i t y r e a c h e d an optimum a t low c o n c e n t r a t i o n s and d e c r e a s e d I | a t h i g h e r c o n c e n t r a t i o n s . Ca (10 mM) had o n l y a s l i g h t s t i m u l a t o r y I | e f f e c t ( T a b l e 3 ) ; i n t h e p r e s e n c e o f Mg i t d i d n o t a l t e r a c t i v i t y a t a l l . These r e s u l t s a r e s t r i k i n g l y d i f f e r e n t from t h o s e of Edwards I | and M a g u i r e ( 1 9 7 0 ) ; t h e y found t h a t 10 mM Mg r e d u c e d a c t i v i t y t o I [ 30% o f t h a t i n t h e absence o f Mg . There does n o t seem t o be an.. ap p a r e n t r e a s o n f o r t h i s d i s c r e p a n c y . The d a t a r e p r e s e n t e d i n F i g . 10 a r e a l s o q u i t e d i f f e r e n t f r o m t h o s e o b t a i n e d f r o m s t u d i e s o f sheep I | b r a i n 5 ' - n u c l e o t i d a s e ( I p a t a , 1968). W h i l e N i enhanced r a t h e a r t 5 ' - n u c l e o t i d a s e a c t i v i t y by about 4 - f o l d , i t i n h i b i t e d t h e enzyme from sheep b r a i n by 58%. 3. S u b s t r a t e S p e c i f i c i t y S u b s t r a t e s p e c i f i c i t y s t u d i e s were p e r f o r m e d w i t h E D T A - t r e a t e d enzyme. A c t i v i t y was e s t i m a t e d by m e a s u r i n g o r t h o p h o s p h a t e l i b e r a t e d f r o m t h e v a r i o u s phosphate e s t e r s . The r a t e o f phosphate r e l e a s e from each e s t e r was d e t e r m i n e d i n t h e p r e s e n c e o f 16 mM MgC^; i n a d d i t i o n , r a t e s f o r most o f t h e s e e s t e r s were d e t e r m i n e d i n t h e absence o f MgC^. S i n c e s e v e r a l p h o s p h a t e e s t e r s t e s t e d were a c i d l a b i l e , c o n t r o l v a l u e s t o a c c o u n t f o r a c i d h y d r o l y s i s d u r i n g c o l o u r development were s u b t r a c t e d f r o m e x p e r i m e n t a l v a l u e s . A c t i v i t y i s r e p r e s e n t e d as p e r c e n t o f a c t i v i t y i n t h e p r e s e n c e o f 16 mM M g C ^ w i t h 5'-AMP as s u b s t r a t e . The enzyme p o s s e s s e d a b r o a d s u b s t r a t e s p e c i f i c i t y f o r n u c l e o s i d e 5'-monophosphates; t h i s i s t y p i c a l o f most 5 ' - n u c l e o t i d a s e s ( f o r r e v i e w , see Drummond and Yamamoto, 1971). P h o s p h a t e was l i b e r a t e d f r o m each o f a v a r i e t y o f n u c l e o s i d e 5'-monophosphates t e s t e d ( F i g . 1 1 ) . 5'-AMP was 120 >- 100 o < X < 80 60 £ 40 20 UMP 5 'AMP s Open Bars +Mg Hatched Bars: -Mg CMP IMP 1 dUMP dCMP dGMP 0 3'AMP El /3GP J Z O . Figure 11. Substrate S p e c i f i c i t y . Open bars represent orthophosphate liberated i n the presence of 16 mM MgCl2 and hatched bars i n the absence of MgCl2-The following were not substrates: p-nitrophenylphosphate, pyrophosphate, ribose-5-phosphate, glucose-6-phosphate, fructose-1, 6-diphosphate, fructose-l-phosphate, ribulose-5-phosphate and galactose-6-phosphate: The f i n a l concen-trations i n the incubation mixtures were Tris-HCl, 50 mM, pH 7.2;. MgCl2» 16 mM; enzyme, 0.49 or 0.77 mg/ml and test compound, 10 mM i n a f i n a l volume of 1.0 ml. 45. not the best substrate; UMP was hydrolyzed more r a p i d l y . The 2'-deoxy-ribonucleoside 5'-monophosphates were u t i l i z e d at about half the rate of the corresponding ribonucleoside 5'-monophosphates. Although a c t i v i t y was observed when a number of substrates containing d i f f e r e n t bases were tested, no enzyme a c t i v i t y was found i f the base was absent. For example the following sugar phosphates di d not act as substrates, ribose-5-phosphate, glucose-6-phosphate, fructose-l-phosphate, fructose-1, 6-diphosphate, ribulose-5-phosphate and galactose-6-phosphate. In addition t h i s enzyme did not release orthophosphate :from either p-nitrophenyl-phosphate or pyrophosphate. The broad substrate s p e c i f i c i t y does not I | appear to be due to the presence of two enzymes; one requiring Mg I | and one independent of Mg . The r e l a t i v e rates of hydrolysis of substrates I | were s i m i l a r i n both the presence and absence of Mg . Further evidence for the existence of a sing l e enzyme can be in f e r r e d from the fact that the pyrimidine nucleotide, UMP, competitively i n h i b i t e d the dephosphory-l a t i o n of 5'-AMP (this w i l l be described l a t e r ) . F. 4. E f f e c t of 5'-AMP Concentration The enzyme^preparation used was pretreated with the chelating agent EDTA, followed by d i a l y s i s to remove endogenous divalent c a t i o n . With such a preparation, the stimulatory e f f e c t of Mg on 5'-nucleotidase could be determined over the f u l l extent of substrate concentration. Two methods of assay were required because the broad range of 5'-AMP concen-t r a t i o n tested (0.0033 mM to 10 mM) could not be accommodated by either assay alone. For concentrations of 5'-AMP from 0.16 mM to 10 mM, 5'-nucleotidase a c t i v i t y was determined by estimation of phosphate l i b e r a t e d . The incubation mixture contained.Tris-HCl buffer, 50 mM, pH 7.2; MgCl„, 16 mM; 46. enzyme, 0.17 mg/ml and 5'-AMP i n a f i n a l volume o f 1.0 m l . A c t i v i t y i s r e p r e s e n t e d a s umoles p e r m i n u t e p e r mg p r o t e i n . I n t h e range o f 5'-AMP c o n c e n t r a t i o n f r o m 0.0033 mM t o 0.16 mM, t h e d i r e c t o p t i c a l a s s a y was use d . The r e a c t i o n m i x t u r e c o n t a i n e d T r i s - H C l , 50 mM, pH 7.2; M g C l 2 , 16 mM; enzyme 0.17 mg; e x c e s s a d e n o s i n e deaminase and 15 1-AMP i n a f i n a l volume o f 3.0 m l . A c o r r e s p o n d i n g s e r i e s w i t h o u t M g C l 2 was c a r r i e d o u t f o r t h e f u l l r a nge o f 5'-AMP c o n c e n t r a t i o n . L a c k o f n o n - s p e c i f i c phospha-t a s e was a s c e r t a i n e d by r e p l a c i n g 5'-AMP w i t h 3'-AMP.-The a c t i v i t y o f 5 ' - n u c l e o t i d a s e as a f u n c t i o n o f 5'-AMP c o n c e n t r a t i o n i s seen, i n F i g . 12. Rate v a l u e s o b t a i n e d by d i r e c t o p t i c a l a s s a y ( p a n e l A) a r e comparable t o t h o s e o b t a i n e d by t e s t tube a s s a y ( p a n e l B ) . 5'-Nucleo-t i d a s e d i s p l a y e d t y p i c a l M i c h a e l i s ^ - M e n t b n dependence on s u b s t r a t e c o n c e n -I | t r a t i o n i n t h e p r e s e n c e and absence o f Mg ov e r t h e range 0.0033 mM t o 0.16 mM 5'-AMP. I n t h i s r a n g e , t h e a c t i v i t y was s t i m u l a t e d l e s s t h a n 2 - f o l d by 16 mM M g C l 2 - The K m i n t h e absence o f M g C l 2 was 2.1 x 1 0 ~ 5 M —5 | | and i n t h e p r e s e n c e o f M g C l 2 was 2.3 x 10 M w h i c h i n d i c a t e d t h a t Mg s t i m u l a t e d t h e enzyme by i n c r e a s i n g maximum v e l o c i t y r a t h e r t h a n by ch a n g i n g a f f i n i t y f o r s u b s t r a t e ( F i g . 14 and'15). These K v a l u e s a r e comparable t o t h o s e o b t a i n e d by o t h e r w o r k e r s . Edwards and M a g u i r e (1970) and S u l l i v a n and A l p e r s (1971) r e p o r t e d v a l u e s o f 1.45 x 10 ~* M and 2.3 x 10 ^ M, r e s p e c t i v e l y when e x p e r i m e n t s were c o n d u c t e d i n t h e I | absence o f Mg 5 ' - N u c l e o t i d a s e a c t i v i t y d i d n o t i n c r e a s e a t c o n c e n t r a t i o n s o f I | 5'-AMP g r e a t e r t h a n 0.16 mM i f Mg was n o t p r e s e n t i n t h e i n c u b a t i o n m i x t u r e . I n t h e p r e s e n c e o f M g C l 2 enzyme a c t i v i t y was maximal a t 4 mM 5'-AMP. A t t h i s c o n c e n t r a t i o n t h e a c t i v i t y was 0.08 0.16 2 4 AMP (mM) 8 Figure 12. Effect of 5'-AMP. concentration. 5'-Nucleotidase a c t i v i t y was determined i n the" absence (A,V) and presence (P,0) of -MgCl.2 (16 mM) . Panel A represents enzyme a c t i v i t y at low concen-trations of 5'-AMP; assays were performed by the direct o p t i c a l method. Panel B shows a c t i v i t y at high 5'-AMP concentrations; a c t i v i t y was estimated by determination of phosphate l i b e r a t i o n . stimulated 4.4-fold by 16 mM MgC^, i n contrast to less than 2-fold at lower 5'-AMP concentrations. This high a c t i v i t y i n the presence of 16 mM MgCl 2 appeared to involve-a second estimated to be about -4 10 M. This second apparent K m may indicate more than one enzyme or i t may represent one enzyme with two a f f i n i t i e s for 5'-AMP. Similar observations of multiple a f f i n i t i e s have been made for nucleoside-3' , 5'-cyclic phosphodiesterase from bovine heart (Beavo, Hardman and Sutherland, 1970) and rat brain (Thompson and Appleman, 1971). Beavo et a l . (1970) suggested that this enzyme could have multiple binding s i t e s for substrate and that binding at one s i t e might affect binding at another. IR v i t r o Regulation of 5'-Nucleotidase As described previously, i t was expected that the a c t i v i t y of cardiac 5'-nucleotidase might be regulated by various metabolites and thus by the metabolic state of the heart. Baer et a l . (1966) have already shown that t h i s enzyme was markedly inhibited by ATP and suggested that the decrease i n ATP levels during hypoxia could result i n greater 5'-nucleotidase a c t i v i t y and increased adenosine formation. If metabolic state regulates this enzyme, a c t i v i t y could be a function of more.than cardiac ATP,.concentration alone. More appropriately i t could be a function of energy charge. Atkinson and Walton (1967) defined energy charge of the adenylate system as "half the average number of anhydride-bound phosphate groups per adenosine moiety". This may also be written (ATP + ^ADP) energy charge = : (ATP + ADP + AMP) 48. Recently Burger and Lowenstein (1970) showed that 5 1-nucleotidase of pig i n t e s t i n a l smooth muscle was not only i n h i b i t e d by ATP but was more powerfully i n h i b i t e d by ADP. On the basis of the above.information, i t seemed important to examine the e f f e c t s of these and other metabolites on rat heart 5'-nucleotidase. The p a r t i c u l a r substances studied were chosen because t h e i r concentrations i n cardiac tissue are subject to considerable change during hypoxia (Imai et a l . , 1964; Williamson, 1966). Thus the e f f e c t s of ATP, ADP, creatine phosphate and inorganic phosphate on the enzyme were examined under a v a r i e t y of conditions. I [ F. 5. a. E f f e c t s of ADP and ATP i n the Presence of Mg at High Concentrations of 5'-AMP. In the presence of 16 mM MgC^ and at concentrations of 5'-AMP 0.15 mM or greater, 5'-nucleotidase was i n h i b i t e d by both ADP and ATP (Fig. 13). I t i s u n l i k e l y that t h i s i n h i b i t i o n was due to chelation I [ of Mg because the concentration of MgCl 2 was 16 or 32 times greater than that of i n h i b i t o r . ADP was considerably more e f f e c t i v e as an i n h i b i t o r of 5'-nucleotidase. At 2 mM 5'-AMP, the i n h i b i t i o n by 0.5 mM ADP was 41% while i n h i b i t i o n by; 1.0 mM ATP was only 12%. No attempts were made to determine the type of i n h i b i t i o n or i n h i b i t o r y constants using data obtained at high 5'-AMP concentrations (0.16 mM or greater; F i g . 13). b. E f f e c t s of ADP, ATP and Creatine Phosphate i n the Absence [ | of Mg I | The i n h i b i t i o n of 5'-nucleotidase by ADP i n the absence of Mg i s shown i n F i g . 14, 16 and 18. Examination of the Hofstee (1952) plo t i n F i g . 14 reveals that the enzyme was i n h i b i t e d i n a mixed I 2 3 4 AMP (mM) F i g u r e 13. I n h i b i t i o n by ATP and ADP a t h i g h c o n c e n t r a t i o n s o f 5'-AMP. A s s a y s were p e r f o r m e d as f o r the e f f e c t o f 5'-AMP c o n c e n -t r a t i o n (range 0.16 mM t o 10 mM 5'-AMP), e x c e p t t h a t t h e enzyme was n o t t r e a t e d w i t h EDTA. The f i n a l i n c u b a t i o n m i x t u r e c o n t a i n e d T r i s - H C l , 50 mM, pH 7.2; MgCl2, 16 mM; enzyme, 0.019 mg; 5'-AMP "and ATP, 1.0 mM (A) o r ADP, .0.5 mM (Q). P o i n t s r e p r e s e n t v a l u e s o b t a i n e d i n d u p l i c a t e and a f t e r s u b t r a c t i o n o f c o n t r o l s t o a c c o u n t f o r p o s s i b l e h y d r o l y s i s o f i n h i b i t o r d u r i n g c o l o u r development. The i n h i b i t i o n of 5'--nucleotidase by ADP i s represented i n this Hofstee plot. Assays were conducted using an EDTA treated preparation i n the absence (O) and presence (Q) of ADP. The incubation mixture contained Tris-HCl, 50 mM, pH 7.2; ADP, 0.0033 mM when required; . enzyme, 0.179 mg, excess adenosine deaminase and 5'-AMP i n a f i n a l volume of 3.0 ml. Reaction rates were determined by the direct o p t i c a l assay. ( c o m p e t i t i v e - n o n - c o m p e t i t i v e ) manner. The maximum v e l o c i t y was : d e c r e a s e d by 25% w h i l e t h e a p p a r e n t was i n c r e a s e d f r o m 2.1 x 10 ^ M to 5.0 x 10 ^ M.. The e f f e c t o f i n c r e a s i n g c o n c e n t r a t i o n o f ADP on t h e e x t e n t o f i n h i b i t i o n i s shown i n F i g . 16. I f t h e s e d a t a a r e r e p l o t t e d by t h e method o f D i x o n ( 1 9 5 3 ) , two s t r a i g h t l i n e s o f d i f f e r e n t s l o p e a r e o b t a i n e d ; t h i s i s i n t e r p r e t e d t o mean t h a t ADP i n h i b i t e d by b i n d i n g a t two s i t e s . The o b s e r v a t i o n o f mixed i n h i b i t i o n i s s i m i l a r t o t h e mixed i n h i b i t i o n by n u c l e o s i d e t r i p h o s p h a t e s r e p o r t e d by Edwards and Ma g u i r e (1970) . These w o r k e r s found t h a t ATP, UTP, GTP and ITP a l l i n h i b i t e d by a l t e r a t i o n o f b o t h maximum v e l o c i t y and K . I n c o n t r a s t , J m S u l l i v a n and A l p e r s (1971) r e p o r t e d t h a t b o t h ADP and ATP i n h i b i t e d t h e enzyme f r o m t h e same s o u r c e , r a t h e a r t , i n o n l y a c o m p e t i t i v e manner. F i g . 18 shows t h a t t h i s enzyme was v i r t u a l l y c o m p l e t e l y i n h i b i t e d by 0.033 mM ATP o r 0.033 mM ADP when Mg i s n o t p r e s e n t i n t h e r e a c t i o n m i x t u r e . C r e a t i n e phosphate was found t o i n h i b i t 18% a t 0.067 mM and no t a t a l l a t 0.0067 mM when t h e c o n c e n t r a t i o n o f 5'-AMP was 0.0083 mM. The i n h i b i t i o n by d i - and t r i p h o s p h a t e e s t e r s o f n u c l e o s i d e s i s n o t l i m i t e d t o 5 ' - n u c l e o t i d a s e from c a r d i a c t i s s u e . F o r example, t h e enzyme fr o m sheep b r a i n was i n h i b i t e d by ATP, UTP and CTP i n a mixed c o m p e t i t i v e n o n - c o m p e t i t i v e way ( I p a t a , 1 9 6 8 ) . I n a d d i t i o n , 5 ' - n u c l e o t i d a s e from r a t c e r e b e l l u m was i n h i b i t e d by ATP, UTP, I T P , CTP, and GTP (Bosmann and P i k e , 1 9 7 1 ) . [ | c. E f f e c t s o f ADP, UMP and Or t h o p h o s p h a t e i n t h e P r e s e n c e o f Mg I | I n h i b i t i o n by ADP, UMP and o r t h o p h o s p h a t e i n t h e p r e s e n c e o f Mg was examined u s i n g enzyme w h i c h had n o t been t r e a t e d w i t h EDTA. The i n c u b a t i o n m i x t u r e c o n t a i n e d T r i s - H C l , 50 mM, pH 7.2; M g C ^ j 16 mM; enzyme, 0.185 mg; e x c e s s a d e n o s i n e deaminase; 5'-AMP and ADP, 0.033 mM; UMP, 50. 0.067 mM o r o r t h o p h o s p h a t e , AO mM i n a f i n a l volume of 3.0 m l . A c o n t r o l s e r i e s was a l s o c a r r i e d out i n t h e absence o f i n h i b i t o r s . R e a c t i o n r a t e s were d e t e r m i n e d by t h e d i r e c t o p t i c a l method. The i n h i b i t i o n o f 5 1 - n u c l e o t i d a s e by ADP i n t h e p r e s e n c e o f 16 mM M g C ^ i s shown i n F i g . 15 and 16; F i g . 16 d e m o n s t r a t e s t h a t i n h i b i t i o n i s a f u n c t i o n o f ADP c o n c e n t r a t i o n . That t h e i n h i b i t i o n by ADP was e f f e c t e d i n a n o n - c o m p e t i t i v e manner i s seen i n F i g . 15, i n w h i c h t h e d a t a have been p l o t t e d by t h e method o f H o f s t e e ( 1 9 5 2 ) . The maximum v e l o c i t y was d e c r e a s e d 45% by 0.033 mM ADP w h i l e t h e a f f i n i t y f o r 5'-AMP d i d n o t appear t o be a f f e c t e d . The K o f t h e u n i n h i b i t e d enzyme was 2;-16 x 10 ^ M w h i l e t h e a p p a r e n t i n t h e p r e s e n c e o f ADP was 2.58 x 1 0 ~ 5 M. The c a l c u l a t e d v a l u e o f K. f o r ADP was 4 x 1 0 ~ 5 M. These I r e s u l t s a r e q u i t e d i f f e r e n t f r o m t h o s e o f S u l l i v a n and A l p e r s ( 1 9 7 1 ) . These w o r k e r s found no i n h i b i t i o n by ADP i n t h e p r e s e n c e o f 8 mM MgC^, however o n l y 0.0017 mM ADP was used compared t o 0.033 mM used to o b t a i n t h e d a t a o f F i g . 15.. When t h e y used t w i c e as much ADP (0.0034 mM), I | s l i g h t i n h i b i t i o n was o b s e r v e d i n t h e p r e s e n c e o f Mg ATP i n t h e p r e s e n c e o f 16 mM M g C l 2 d i d n o t i n h i b i t t o t h e same e x t e n t as ADP. When t h e c o n c e n t r a t i o n o f 5'-AMP was 0.033 mM, ATP (0.033 mM) o n l y i n h i b i t e d 11% w h i l e ADP (0.033. mM) i n h i b i t e d 35% ( F i g . 1 8 ) . These d a t a a r e n o t c o n s i s t e n t w i t h t h e f i n d i n g s o f S u l l i v a n and A l p e r s who r e p o r t e d no i n h i b i t i o n o f 5 ' - n u c l e o t i d a s e when the" c o n c e n t r a t i o n of ATP was 0.02 mM and t h a t o f MgSO^ was 10 mM. 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 . UMP i n h i b i t e d t h e enzyme i n a c o m p e t i t i v e manner ( F i g . 1 7 ) . The a p p a r e n t K was 5.2 x 10 ^ M i n t h e p r e s e n c e o f UMP compared t o t h e m c o n t r o l v a l u e o f 2.4 x 10 ^ M; maximum v e l o c i t y was unchanged. These i i I I : I i _L I 2 3 4 5 6 7 SPECIFIC ACTIVITY (S) mM Figure 15. Inh i b i t i o n by ADP i n the presence of MgC^. This Hofstee plot shows the i n h i b i t i o n of 5'-nucleotidase by ADP. Assays for the uninhibited (O) and ADP inhibited (Q) reactions were conducted i n the presence of 16 mM MgCl 2 > by the direct o p t i c a l method. A I L I I I I I 0.01 0.02 0.03 0.04 0.05 0.06 ADP (mM) Figure 16. Effect of ADP concentration on i n h i b i t i o n . I n h i b i t i o n by ADP was ascertained i n the absence (A) and presence (O) °f 16 mM MgCl2- Values are depicted as percent of uninhibited control. 5'-AMP concentration was always 0.033 mM. by the direct o p t i c a l method. 51. r e s u l t s should not be unexpected, since UMP was also found to be a substrate (Fig. 11). The observation of competitive i n h i b i t i o n i n d i -cates that one enzyme was responsible f o r hydrolysis of 5'-AMP and UMP. If there had been two enzymes, one s p e c i f i c f o r purine nucleotides and another s p e c i f i c f o r pyrimidine nucleotides, i t i s u n l i k e l y that competi-t i v e i n h i b i t i o n would have been observed. Orthophosphate, unlike UMP, i n h i b i t e d 5'-nucleotidase i n a non-competitive rather than competitive manner (Fig.- 17). The K m determined i n the presence of 40 mM ortho-phosphate was 2.3 x 10 ^  M which was unchanged from 2.4 x 10 ^ M i n i t s absence. The value of K. was calculated to be 73 mM. The e f f e c t s of I orthophosphate on 5'-nucleotidase was quite d i f f e r e n t from i t s e f f e c t on enzyme from sheep brain. Ipata (1968) found that the ATP-induced i n h i b i t i o n of sheep brain 5'-nucleotidase was reversed by the addit i o n of orthophosphate. d. E f f e c t of Mg"1"1" on I n h i b i t i o n by ADP and ATP. Both ADP and ATP v i r t u a l l y eliminated a l l 5'-nucleotidase a c t i v i t y at a concentration of 0.033 mM i n the absence of MgC^. Addition of MgCl2 to 16 mM restored the a c t i v i t y of the ADP i n h i b i t e d enzyme to 63% of uninhibited control and restored the a c t i v i t y of the ATP i n h i b i t e d enzyme to 89% of control (Fig. 18). In comparison, S u l l i v a n and Alpers (1971) reported that the addit i o n of MgSO^ completely reversed the i n h i b i t i o n due to ATP. Examination of F i g . 18 reveals that the a c t i v i t y of the ADP i n h i b i t e d enzyme was maximally enhanced by 4 mM MgCl^; 11 mM MgCl 2 was required f o r maximum stimulation of the ATP i n h i b i t e d enzyme. The K f o r stimulation of ADP and ATP i n h i b i t e d enzyme was about -3 -3 1 x 10 M MgCl 9 compared to 1.9 x 10 M for the uninhibited enzyme. 100 80 P 60 o < o 40 on 20 ATP (0.033 mM) ADP (0.033 mM) o — o o 12 14 MgCI 2 (mM) 16 Figure 18. E f f e c t of MgCl2 concentration on i n h i b i t i o n by ADP and ATP. A c t i v i t y of ADP (O) and ATP (•) i n h i b i t e d 5'-nucleotidase as a function of MgCl2 concentration i s represented as percent of uninhibited control i n the presence of 16 mM MgCl2« Assays were performed by the d i r e c t o p t i c a l method and enzyme which had been treated with EDTA was used. The f i n a l incubation mixture contained Tris-HCl,. 50 mM, pH 7.2; 5'-AMP, 0.033 mM; enzyme, 0.21 mg; excess adenosine deaminase; ADP or ATP, 0.033 mM and MgCl2 i n a f i n a l volume of 3.0 ml. DISCUSSION The o b s e r v a t i o n s made i n t h e c o u r s e o f t h i s s t u d y p r o v i d e some measure of s u p p o r t f o r t h e r o l e o f a d e n o s i n e i n t h e m e d i a t i o n o f c o r o n a r y a u t o r e g u l a t i o n . The r e s u l t s o f c e r t a i n e x p e r i m e n t s , however, w a r r a n t some s k e p t i c i s m o f t h i s p o s s i b i l i t y . D a t a a c c u m u l a t e d f r o m the s u r v e y o f v e n t r i c u l a r enzymes i n v o l v i n g 5'-AMP and fr o m c a r d i a c p e r f u s i o n e x p e r i m e n t s s u g g e s t t h a t a d e n o s i n e may be i n v o l v e d i n a u t o - . r e g u l a t i o n o f c o r o n a r y b l o o d f l o w o f mammals b u t t h a t i t m ight n o t o p e r a t e i n t h i s c a p a c i t y i n o t h e r a n i m a l s . The o b s e r v a t i o n t h a t most c a r d i a c 5 ' - n u c l e o t i d a s e was p r e s e n t i n e n d o t h e l i a l c e l l s o f t h e c o r o n a r y c i r c u l a t i o n and n o t i n h e a r t muscle, c e l l s may s u g g e s t a s l i g h t m o d i f i -c a t i o n o f t h e model o f Rubio and Berne (1969) . A d e n o s i n e formed d u r i n g h y p o x i a may n o t be d e r i v e d f r o m m u s c l e c e l l s b u t may come fr o m e n d o t h e l i a l c e l l s . C e r t a i n p r o p e r t i e s o f r a t v e n t r i c l e 5 ' - n u c l e o t i d a s e , I | s u c h as i n h i b i t i o n by ATP and s t i m u l a t i o n by Mg , i n d i c a t e t h a t t h e p r o d u c t i o n o f a d e n o s i n e by t h i s enzyme may be enhanced d u r i n g h y p o x i a . C e r t a i n o t h e r p r o p e r t i e s , however, i n d i c a t e j u s t t h e o p p o s i t e . The r e s u l t s o f a s u r v e y o f 5 ' - n u c l e o t i d a s e l e v e l s o f h e a r t s o f v a r i o u s s p e c i e s g e n e r a l l y s u p p o r t s t h e a d e n o s i n e h y p o t h e s i s . The amounts of 5 ' - n u c l e o t i d a s e found i n mammalian and t u r t l e h e a r t s a r e i n harmony w i t h t h o s e e x p e c t e d on t h e b a s i s o f t h i s h y p o t h e s i s . H e a r t s o f mammals were p r e d i c t e d t o p o s s e s s a w e l l d e v e l o p e d means f o r t h e p r o d u c t i o n o f a d e n o s i n e and t h u s s u b s t a n t i a l l e v e l s o f 5 ' - n u c l e o t i d a s e . T h i s p r e d i c t i o n was made because mammals a r e c a p a b l e o f q u i c k l y p l a c i n g l a r g e l o a d s on t h e i r h e a r t s . F o r example, when r a t s and dogs were f o r c e d t o r u n , oxygen c o n s u m p t i o n i n c r e a s e d 3 . 5 - f o l d and 2 . 5 - f o l d , r e s p e c t i v e l y ( T a y l o r e t a l . , 1 9 7 0 ) ; t h e i n c r e a s e was s i m i l a r i n humans 53. (Rushmer et^ a l _ . , 1963). T h i s would r e s u l t i n i n c r e a s e d h e a r t work and c o n s e q u e n t l y c o r o n a r y v a s o d i l a t i o n i n o r d e r t o f u l f i l l a g r e a t e r oxygen demand (Gregg, 1 9 6 3 ) . I f a d e n o s i n e were r e s p o n s i b l e f o r t h i s v a s o -d i l a t i o n , s u b s t a n t i a l amounts of 5 ' - n u c l e o t i d a s e would be n e c e s s a r y f o r i t s , p r o d u c t i o n . That c o n s i d e r a b l e 5 ' - n u c l e o t i d a s e was found i n mam-m a l i a n h e a r t s i s a p p a r e n t from F i g . 3. T u r t l e v e n t r i c l e s were e x p e c t e d to have a p o o r l y d e v e l o p e d means f o r t h e p r o d u c t i o n of a d e n o s i n e because v a s o d i l a t i o n i n t h e s e h e a r t s i s p r o b a b l y u n n e c e s s a r y . C a r d i a c energy d e f i c i e n c i e s due t o a n o x i a appear t o be f u l f i l l e d q u i t e a d e q u a t e l y by a n a e r o b i c means. F o r example, t u r t l e s can s u r v i v e 17 h o u r s of t o t a l a n o x i a ( B e l k i n , 1963) and i s o l a t e d t u r t l e h e a r t s were found t o f u n c t i o n f o r s e v e r a l h ours i n unoxygenated Mines s o l u t i o n ( p e r s o n a l o b s e r v a t i o n ) . The need f o r oxygen and thus a d e n o s i n e and v a s o d i l a t i o n may n o t be as c r i t i c a l i n t u r t l e h e a r t s as i n mammalian h e a r t s . The l a c k o f d e t e c t a b l e 5 ' - n u c l e o t i d a s e i n t u r t l e v e n t r i c l e s would seem to s u p p o r t t h e a d e n o s i n e h y p o t h e s i s . To t h i s p o i n t , i t has been assumed t h a t t h e v a s o d i l a t o r y e f f e c t s o f a d e n o s i n e a r e e q u a l i n t u r t l e and mammalian h e a r t s . The p r e c e d i n g c o n c l u s i o n c o u l d be r e p u d i a t e d i f t h e c o r o n a r y c i r c u l a t i o n o f t u r t l e were much more s e n s i t i v e t o a d e n o s i n e t h a n .that of mammals. I f t h i s were t h e c a s e ^ u n d e t e c t a b l e amounts o f 5 ' - n u c l e o t i d a s e m ight be a b l e t o produce s u f f i c i e n t a d e n o s i n e f o r v a s o d i l a t i o n . T h i s p o s s i b i l i t y c a n be e l i m i n a t e d because a d e n o s i n e d i d n o t i n c r e a s e f l o w t h r o u g h p e r -f u s e d t u r t l e h e a r t s , i n amounts s u f f i c i e n t t o i n d u c e s u b s t a n t i a l v a s o -d i l a t i o n i n h e a r t s o f r a b b i t and r a t s ( F i g . 6 ) . These p e r f u s i o n s t u d i e s , a l t h o u g h n o t e x t e n s i v e , s u g g e s t t h a t w h i l e a d e n o s i n e may m e d i a t e a u t o -r e g u l a t i o n o f t h e mammalian c o r o n a r y c i r c u l a t i o n , i t does n o t do so i n t u r t l e h e a r t s . I f a d e n o s i n e were t o m e d i a t e c o r o n a r y a u t o r e g u l a t i o n i n 54. b i r d s , the mechanism f o r i t s formation might be expected to be well developed. For example, pigeons use 8.5 times as much oxygen during f l i g h t as at res t (LeFebvre, 1964). The rate of energy output of g u l l s and budgerigars while f l y i n g may be as high as 20 times the res t i n g values (Tucker, 1969). It seems reasonable to expect that hearts of these birds would require s i m i l a r increases i n oxygen supply. If 5'-nucleotidase produced adenosine to induce v a s o d i l a t i o n and thus f a c i l i t a t e oxygen d e l i v e r y to these hearts, substantial l e v e l s of t h i s enzyme might be expected. This was not the case; i n f a c t , no 5'-nucleotidase was detected i n pigeon hearts under the conditions of the d i r e c t assay or by histochemical examination. This observation may indica t e that pigeons use some means, other than adenosine, to regulate coronary flow.• Since the l e v e l s of cardiac 5'-nucleotidase exhibited large v a r i a t i o n s amongst species, i t seemed that the o v e r a l l metabolism of 5'-AMP by hearts of these species probably d i f f e r e d s i g n i f i c a n t l y . This was supported by r e s u l t s of a survey of adenylate kinase and adenylate deaminase. The catabolism of 5'-AMP i n hearts which are poorly equipped for adenosine production could be channeled through IMP by adenylate deaminase. Conversely, anabolic processes could be involved and 5'-AMP could be converted to ADP by the action of adenylate kinase. The difference i n the cardiac l e v e l s of these enzymes may r e f l e c t c e r t a i n s p e c i f i c functions of nucleosides and nucleotides and the manner i n which these hearts respond to p a r t i c u l a r demands. For example, t u r t l e v e n t r i c l e i s well equipped for. deamination of 5'-AMP. Hence, i t may metabolize 5'-AMP l a r g e l y by conversion to IMP and produce only small amounts of adenosine, a s i t u a t i o n s i m i l a r to that i n s k e l e t a l muscle 55. ( I m a i e t a l _ . , 1964). A d e n o s i n e m i g h t n o t f u n c t i o n i n a r e g u l a t o r y c a p a c i t y i n t u r t l e h e a r t s . I n c o n t r a s t , t h e r e l a t i v e l y l o w e r l e v e l s o f a d e n y l a t e deaminase i n mammalian h e a r t s would tend t o a l l o w a s i g n i -f i c a n t c a t a b o l i s m of a d e n y l a t e t h r o u g h a d e n o s i n e r a t h e r t h a n IMP. T h i s would be p a r t i c u l a r l y a p p r o p r i a t e i f t h e s e a n i m a l s use a d e n o s i n e t o m e d i a t e r e g u l a t i o n of c o r o n a r y b l o o d f l o w because c o m p e t i t i o n f o r t h e s o u r c e o f a d e n o s i n e would tend t o be r e s t r i c t e d . The h i g h l e v e l s o f . a d e n y l a t e k i n a s e i n p i g e o n v e n t r i c l e p r o v i d e s a w e l l d e v e l o p e d mechanism whereby ATP c a n be r e s y n t h e s i z e d f r o m ADP. Thus, t h i s t i s s u e seems w e l l e q u i p p e d t o use b o t h h i g h energy bonds o f ATP by i t s a b i l i t y t o m a i n t a i n a d e n i n e n u c l e o t i d e s i n a f o r m s u i t a b l e f o r energy r e l e a s e . I n t h i s way, t h e p i g e o n would be w e l l s u i t e d t o cope w i t h r a p i d i n c r e a s e s i n c a r d i a c work. A t t h e o p p o s i t e end o f t h e s p e c t r u m , t u r t l e v e n t r i c l e may need o n l y s m a l l amounts o f a d e n y l a t e k i n a s e , s i n c e t h e i r l o w e r energy demands ( S p e c t o r , 1956) might be s a t i s f i e d by a n a e r o b i c m e t a b o l i s m . T h i s i s s u p p o r t e d by t h e o b s e r v a t i o n t h a t t u r t l e h e a r t s f u n c t i o n f o r s e v e r a l h o u r s i n unoxygenated Mine's s o l u t i o n and t h a t - t u r t l e s s u r v i v e p r o l o n g e d p e r i o d s of a n o x i a ( B e l k i n , 1 9 6 3 ) . Mammals w i t h t h e i r i n t e r m e d i a t e l e v e l s o f a d e n y l a t e k i n a s e appear l e s s w e l l e q u i p p e d t h a n p i g e o n s t o cope w i t h a r a p i d o n s e t of h e a r t work. A c o m p a r i s o n of t h e l e v e l s o f a d e n o s i n e deaminase i n t h e h e a r t s t e s t e d i s n o t p a r t i c u l a r l y r e v e a l i n g , but t h e p r e s e n c e o f t h i s enzyme i n p i g e o n v e n t r i c l e i s i m p o r t a n t . A mechanism f o r t h e c a t a b o l i s m of a d e n o s i n e i m p l i e s a means f o r t h e f o r m a t i o n o f a d e n o s i n e . T h i s r a i s e d t h e p o s s i b i l i t y t h a t p i g e o n v e n t r i c l e d i d p o s s e s s 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 was n o t d e t e c t a b l e under t h e c o n d i t i o n s d e s c r i b e d h e r e i n . T h i s was t h e main s t i m u l u s f o r the subsequent s u c c e s s f u l s e a r c h f o r 56. 5 ' - n u c l e o t i d a s e i n p i g e o n h e a r t s ( G i b s o n and Drummond, i n p r e p a r a t i o n ) . T h i s enzyme was found t o be q u i t e d i f f e r e n t f r o m t h e membrane bound 5 ' - n u c l e o t i d a s e o f r a t h e a r t . P i g e o n v e n t r i c l e 5 ' - n u c l e o t i d a s e was s o l u b l e r a t h e r t h a n p a r t i c u l a t e ; v i r t u a l l y a l l t h e enzyme was found i n t h e 100,000 x g s u p e r n a t a n t . I t showed a h i g h s p e c i f i c i t y f o r 5'-AMP and had an a b s o l u t e r e q u i r e m e n t f o r Mg . The K was h i g h , 3 about 10 t i m e s g r e a t e r t h a n t h e K m o f r a t h e a r t enzyme. The d i s c o v e r y o f 5 ' - n u c l e o t i d a s e i n p i g e o n h e a r t c a n be i n t e r p r e t e d as s u p p o r t f o r t h e a d e n o s i n e h y p o t h e s i s a l t h o u g h i t s p r e s e n c e as a s o l u b l e enzyme i s n o t e n t i r e l y c o n s i s t e n t . The g r e a t e s t p r o p o r t i o n o f c a r d i a c 5 ' - n u c l e o t i d a s e has been found a t s i t e s o t h e r t h a n t h e s u r f a c e membrane o f m u s c l e c e l l s . I n h e a r t t i s s u e , 5 ' - n u c l e o t i d a s e has been r e p o r t e d i n i n t e r c e l l u l a r s p a c e s , T-s ystem, e n d o t h e l i a l c e l l s ( R o s t g a a r d and Behnke, 1 9 5 9 ) , and m u s c l e c e i l s (Hardonk; 1968). The predominance of a c t i v i t y i n e n d o t h e l i a l c e l l s and t h e a p p a r e n t absence of a c t i v i t y i n m u s c l e c e l l s ( F i g . 7) p r o v i d e s an i n d i c a t i o n of t h e p r o p o r t i o n o f enzyme and t h e p o t e n t i a l f o r a d e n o s i n e f o r m a t i o n a t each l o c a t i o n . On t h e b a s i s o f t h e s e f i n d i n g s i t i s s u g g e s t e d t h a t a m i n o r m o d i f i c a t i o n of t h e a d e n o s i n e h y p o t h e s i s m i g h t be a p p r o p r i a t e . S p e c i f i c a l l y , t h e s o u r c e o f a d e n o s i n e f o r t h e r e g u l a t i o n o f c o r o n a r y f l o w may be e n d o t h e l i a l c e l l s o f c a p i l l a r i e s and n o t m u s c l e c e l l s . Hence, o f h e a r t muscle c e l l s may p l a y an i n d i r e c t r a t h e r t h a n a d i r e c t r o l e as s t i m u l u s f o r a d e n o s i n e r e l e a s e . M u s c l e c e l l . h y p o x i a c o u l d r e s u l t i n a s t e e p e r oxygen g r a d i e n t f r o m c a p i l l a r i e s t o m u s c l e and cause a r e d u c t i o n i n p 0 2 o f t h e c a p i l l a r y e n d o t h e l i u m . " H y p o x i c " e n d o t h e l i a l c e l l s may r e l e a s e a d e n o s i n e t o t h e i n t e r s t i t i a l f l u i d . T h i s n u c l e o s i d e c o u l d t h e n d i f f u s e i n t o t h e r e g i o n of p r e c a p i l l a r y 57. resistance vessels and induce vasodilation. This would increase the blood and oxygen supply to the myocardium and correct the hypoxia as previously described (Berne, 1963). In addition to i t s potential significance with respect to the role of adenosine i n coronary auto-regulation, this d i s t r i b u t i o n of 5'-nucleotidase carries implications pertinent to other areas of research such as membrane biochemistry and physiology. 5'-Nucleotidase has often been used as a membrane marker but i t s value as such should be questioned for two reasons. F i r s t l y , the presence of 5'-nucleotidase i n a given sample does not assure the presence of mem-brane. For example, i f a f r a c t i o n of rat l i v e r homogenate were found to contain this enzyme, i t might be concluded that membranes were also present. Since rat l i v e r contains a soluble 5'-nucleotidase (Fritzson, 1969) contamination by this soluble enzyme could give false positive r e s u l t s . Secondly, the absence of '5'-nucleotidase does not necessarily indicate the absence of membranes. An i l l u s t r a t i o n of this point may be seen i n Fig. 7, plate B which shows cardiac muscle c e l l membranes without 5'-nucleotidase a c t i v i t y . I t i s possible then that heart muscle c e l l membranes could be studied by the examination of a preparation i n which no 5'-nucleotidase could be detected. While demonstration of the presence or absence of membranes i s not the theme of this thesis, an . examination of the properties of membrane 5'-nucleotidase i s relevant. Analysis of,certain properties of rat heart 5'-nucleotidase i n d i -cates that enzyme a c t i v i t y and adenosine production may be enhanced during cardiac hypoxia and diminished during adequate oxygenation; such regulation by the metabolic state of the heart supports the adenosine hypothesis. On the basis of these properties, 5'-nucleotidase appears 58. w e l l s u i t e d f o r , t h e a p p r o p r i a t e f o r m a t i o n o f a d e n o s i n e f o r a u t o r e g u l a t i o n of c o r o n a r y b l o o d f l o w . One c h a r a c t e r i s t i c w h i c h may be i n t e r p r e t e d as s u p p o r t f o r t h e a d e n o s i n e h y p o t h e s i s i s t h e p o s i t i v e c o r r e l a t i o n between Mg c o n c e n t r a t i o n and enzyme a c t i v i t y . S i n c e ATP i s known t o be an I | . . . e x c e l l e n t c h e l a t o r o f Mg (Walaas, 1 9 5 8 ) , * t h e c e l l u l a r c o n c e n t r a t i o n ++ o f ATP c o u l d r e g u l a t e t h e amount o f Mg a v a i l a b l e f o r s t i m u l a t i o n o f t h e enzyme. Thus, a t h i g h energy c h a r g e ( A t k i n s o n , 1 9 6 4 ) , a h i g h l e v e l I | o f c e l l u l a r ATP would l i m i t t h e amount of uncomplexed Mg and r e s u l t I j i n a l o w enzyme a c t i v i t y . . A t l o w energy c h a r g e , l i t t l e Mg would be n u c l e o t i d e bound and a g r e a t e r q u a n t i t y would be a v a i l a b l e t o enhance 5 ' - n u c l e o t i d a s e a c t i v i t y . Thus, s t i m u l a t i o n o f 5 ' - n u c l e o t i d a s e by Mg c o u l d f a c i l i t a t e p r o d u c t i o n o f a d e n o s i n e d u r i n g p e r i o d s o f h y p o x i a when energy c h a r g e i s r e d u c e d . A l t h o u g h Mg i s an e x c e l l e n t s t i m u l a t o r o f 5 ' - n u c l e o t i d a s e , i t I | may n o t n e c e s s a r i l y be t h e c a t i o n i n v o l v e d i n v i v o . S i n c e Mn i s more p o t e n t and more e f f e c t i v e as a s t i m u l a t o r o f t h i s enzyme, and i s a l s o p r e s e n t i n t h e h e a r t ( S p e c t o r , 1 9 5 6 ) , i t s h o u l d a l s o be c o n s i d e r e d as a c a n d i d a t e f o r the i n v i v o c o f a c t o r . I n c o m p a r i s o n , Ca has l i t t l e e f f e c t on t h e enzyme and p r o b a b l y i s n o t i n v o l v e d i n i t s a c t i o n . The f i n d i n g t h a t N i s t i m u l a t e s t h e enzyme has some- m e t h o d o l o g i c a l s i g n i -f i c a n c e , S p e c i f i c 5 ' - n u c l e o t i d a s e a c t i v i t y has been d e t e r m i n e d u s i n g I | N i as a s p e c i f i c i n h i b i t o r ; t h e v a l u e f o r 5 ' - n u c l e o t i d a s e c a t a l y z e d p h o s p h a t e r e l e a s e was c a l c u l a t e d by s u b t r a c t i o n o f p h o s p h a t a s e a c t i v i t y I | i n the. p r e s e n c e o f N i from t h a t . i n i t s absence ( C a m p b e l l , 1 9 6 2 ) . The n i c k e l s t i m u l a t i o n o f t h i s enzyme and t h e n i c k e l i n d u c e d i n c r e a s e o f l e a d d e p o s i t i o n i n t i s s u e s e c t i o n s i n d i c a t e t h e da n g e r s i n h e r e n t i n t h e I | above method. I n d e e d , i n c a s e s i n w h i c h N i i s w i t h o u t i n h i b i t o r y 59. e f f e c t o r i s s t i m u l a t o r y , f a l s e n e g a t i v e r e s u l t s would be o b t a i n e d i f t h i s method were used t o e s t i m a t e 5 ' - n u c l e o t i d a s e . The r e g u l a t i o n o f 5 ' - n u c l e o t i d a s e by energy c h a r g e i s p r o b a b l y p e r f o r m e d more e f f e c t i v e l y by a d i r e c t a c t i o n t h a n by c h e l a t i o n o f d i v a l e n t c a t i o n . ATP i n h i b i t s t h e enzyme; c r e a t i n e p hosphate a l s o i n h i b i t s b u t n o t as w e l l as ATP. When t h e energy c h a r g e o f c a r d i a c t i s s u e i s h i g h , t h e 5'-AMP c o n t e n t i s d e c r e a s e d ( W i l l i a m s o n , 1 9 6 6 ) ; under i n v i t r o s i m u l a t i o n of t h e s e c o n d i t i o n s , 5 ' - n u c l e o t i d a s e a c t i v i t y was d i m i n i s h e d . D u r i n g a n o x i a when energy c h a r g e i s l o w , i t i s e x p e c t e d t h a t enzyme a c t i v i t y would be enhanced by r e m o v a l o f ATP and c r e a t i n e p h osphate i n h i b i t i o n and by i n c r e a s e d s u b s t r a t e c o n c e n t r a t i o n . Hence, a d e n o s i n e f o r m a t i o n c o u l d be enhanced d u r i n g h y p o x i a . The r e g u l a t i o n of 5 ' - n u c l e o t i d a s e by ATP, c r e a t i n e p h o s p h a t e , d i v a l e n t c a t i o n and 5'-AMP s u p p o r t t he a d e n o s i n e h y p o t h e s i s because t h i s r e g u l a t i o n a l l o w s p r o d u c t i o n o f a d e n o s i n e d u r i n g h y p o x i a when v a s o d i l a t i o n i s r e q u i r e d and v i c e - v e r s a . T h i s i s i n agreement w i t h Baer e t a l . ' (1966) who s p e c u l a t e d t h a t , i n w e l l o x y genated t i s s u e , ATP c o u l d m a i n t a i n 5'-n u c l e o t i d a s e i n an i n h i b i t e d s t a t e and t h a t t h i s i n h i b i t i o n was removed'-!! d u r i n g h y p o x i a . Some p r o p e r t i e s o f 5 ' - n u c l e o t i d a s e a r e n o t c o n s i s t e n t w i t h i t s s t i m u l a t i o n a t low energy c h a r g e . Such c h a r a c t e r i s t i c s i n d i c a t e t h a t t h i s enzyme may n o t be s u i t a b l e f o r a d e n o s i n e p r o d u c t i o n d u r i n g h y p o x i a when energy c h a r g e i s r e d u c e d . I n d e e d , t h e s e p r o p e r t i e s may even p r o v i d e a b a s i s f o r some s k e p t i c i s m o f t h e a d e n o s i n e h y p o t h e s i s . F o r example, b o t h ADP and o r t h o p h o s p h a t e a r e e f f e c t i v e i n h i b i t o r s o f r a t h e a r t 5'-n u c l e o t i d a s e and t h e c o n c e n t r a t i o n s o f b o t h i n c r e a s e d u r i n g h y p o x i a ( W i l l i a m s o n , 1966). Thus i n c r e a s e d ADP and o r t h o p h o s p h a t e would tend 60. to reduce adenosine production i n hypoxic cardiac ti s s u e i n which energy charge i s decreased. I t must be emphasized that because ADP has been found to be a more potent i n h i b i t o r of 5'-nucleotidase than ATP, the proposed regulatory function of the l a t t e r nucleotide (Baer et a l . , 1966) must be considered doubtful. Two other c h a r a c t e r i s t i c s of 5'-nucleotidase which f o s t e r questioning of i t s proposed r o l e i n the formation of adenosine for coronary autpregulation are i t s pH dependence and i t s broad substrate s p e c i f i c i t y . At pH values l e s s than optimum (8.5), 5'-nucleotidase a c t i v i t y decreased with increasing a c i d i t y . This implies that enzyme a c t i v i t y could a c t u a l l y decrease during the acidosis of anoxia. If the function of cardiac 5'-nucleotidase i s to produce adenosine for v a s o d i l a t i o n when there i s an oxygen d e f i c i t , an a c i d i c pH optimum would appear more advantageous because i t would favour increased ade- • nosine formation during hypoxia. This i n t e r p r e t a t i o n should be tempered with the observation that the enzyme was active over a broad pH range; thus pH control may not be an important regulatory mechanism. The substrate s p e c i f i c i t y of ra t heart 5'-nucleotidase was such that a l l nucleoside 5'-monophosphates tested were hydrolyzed; some were better substrates than 5'-AMP. If t h i s enzyme i s to catalyze the con-version of 5 '-AMP to adenosine during hypoxia, a greater s p e c i f i c i t y f o r '5'-AMP might have been expected. .Such a broad substrate s p e c i f i c i t y would be a hindrance to adenosine formation during periods of low energy charge i f substrates other than 5'-AMP were being hydrolyzed during t h i s time. Under these conditions adenylate dephosphorylation could be competi-t i v e l y i n h i b i t e d (Fig. 16) and adenosine production consequently diminished. To r e c a p i t u l a t e , t h e c h a r a c t e r i s t i c s o f 5 ' - n u c l e o t i d a s e w h i c h seem i n c o n s i s t e n t w i t h a r o l e f o r a d e n o s i n e i n c o r o n a r y a u t o r e g u l a t i o n a r e i t s i n h i b i t i o n by ADP and o r t h o p h o s p h a t e , pH p r o f i l e and b r o a d s u b s t r a t e s p e c i f i c i t y . Those c h a r a c t e r i s t i c s w h i c h s u p p o r t t h e ade-n o s i n e h y p o t h e s i s a r e t h e i n h i b i t i o n by ATP and c r e a t i n e phosphate and t h e p o s i t i v e r e l a t i o n s h i p between r e a c t i o n r a t e and c o n c e n t r a t i o n I | o f b o t h s u b s t r a t e and Mg The f o r e g o i n g d i s c u s s i o n of t h e p r o p e r t i e s o f c a r d i a c 5'-nucleo-t i d a s e and i t s p o s s i b l e r o l e i n t h e r e g u l a t i o n c o r o n a r y b l o o d f l o w was based on two a s s u m p t i o n s . S p e c i f i c a l l y , i „ i : t was assumed t h a t t h e 5'-n u c l e o t i d a s e w h i c h may f o r m a d e n o s i n e f o r c o r o n a r y v a s o d i l a t i o n was i d e n t i c a l w i t h . t h e enzyme s t u d i e d h e r e i n . I f t h i s p r o v e s t o be i n c o r r e c t , i t would be i n a p p r o p r i a t e t o d i s c u s s t h e enzyme i n terms o f t h e a d e n o s i n e h y p o t h e s i s and i t s h o u l d be c o n s i d e r e d i n a d i f f e r e n t framework. I t was a l s o assumed t h a t 5 ' - n u c l e o t i d a s e , i n v i v o , i s a c c e s s i b l e to t h e v a r i o u s m e t a b o l i t e s s t u d i e d . A p o s s i b i l i t y s t i l l t o - b e c o n s i d e r e d i s t h a t a d e n o s i n e f o r m a t i o n f o r v a s c u l a r r e g u l a t i o n c o u l d o c c u r a t a d i s c r e t e s i t e i n t h e myocardium and m i ght o n l y be r e g u l a t e d by s p e c i f i c m e t a b o l i t e s . T h i s seems p a r t i c u l a r l y p e r t i n e n t w i t h r e s p e c t t o ATP and ADP i n h i b i t i o n . - To f u r t h e r i n v e s t i g a t e t h e f i r s t a s s u m p t i o n , i t may be p r o f i t a b l e t o c h a r a c t e r i z e t h e enzyme f r o m a n o t h e r s o u r c e . I t would be d e s i r a b l e t o examine c a r d i a c 5 ' - n u c l e o t i d a s e from a s p e c i e s o t h e r t h a n r a t because c o n s i d e r a t i o n o f t h e r a t h e a r t enzyme may have been i n a p p r o p r i a t e f o r t h e s t u d y of- c o r o n a r y a u t o r e g u l a t i o n . There may be two 5 ' - n u c l e o t i d a s e s i n r a t v e n t r i c l e ; one i n abundant s u p p l y w h i c h does n o t p l a y a r o l e i n c o r o n a r y a u t o r e g u l a t i o n and a n o t h e r i n l i m i t e d s u p p l y w h i c h p r o d u c e s a d e n o s i n e a t a s i t e a p p r o p r i a t e f o r 62. v a s o d i l a t i o n . The i s o l a t i o n p r o c e d u r e s used t o d a t e may have r e s u l t e d i n l o s s o r d e s t r u c t i o n of t h e l a t t e r o r i t may have been masked by a g r e a t e r a c t i v i t y o f t h e f o r m e r . The l a r g e v a r i a t i o n of 5 ' - n u c l e o t i d a s e l e v e l s amongst s p e c i e s may r e f l e c t l e v e l s o f enzyme n o t i n v o l v e d i n v a s o d i l a t i o n . T h e r e f o r e , h e a r t s of a s p e c i e s such as r a b b i t , w h i c h has l i t t l e t o t a l 5 ' - n u c l e o t i d a s e , may be an e x c e l l e n t s o u r c e because t h i s enzyme might be i n v o l v e d p r i m a r i l y i n c o r o n a r y a u t o r e g u l a t i o n . I t i s p o s s i b l e t h a t t h e c h a r a c t e r i s t i c s o f 5 ' - n u c l e o t i d a s e from t h i s s o u r c e c o u l d be q u i t e d i f f e r e n t f r o m t h e r a t h e a r t enzyme.• I t s p r o p e r t i e s m i ght be more a p p r o p r i a t e f o r a d e n o s i n e f o r m a t i o n i n h y p o x i c t i s s u e o r i n c e l l s w i t h d i m i n i s h e d energy c h a r g e . A n o t h e r a r e a f o r f u t u r e endeavour s h o u l d be t h e s t u d y o f s p e c i f i c t y p e s of. h e a r t c e l l s t o f u r t h e r d e t e r m i n e t h e i r p o t e n t i a l f o r a d e n o s i n e p r o d u c t i o n . A l t h o u g h 5 ' - n u c l e o t i d a s e c a t a l y z e s t h e c o n v e r s i o n of 5'-AMP to a d e n o s i n e , t h i s does n o t g u a r a n t e e t h a t a d e n o s i n e i s formed wherever t h i s enzyme i s f o u n d . I t can a l s o c o n v e r t IMP t o i n o s i n e . : Of p r i m e i m p o r t a n c e i n t h e d e t e r m i n a t i o n of p r o d u c t formed by 5 ' - n u c l e o t i d a s e i s the a c t i v i t y o f a d e n y l a t e deaminase because t h i s enzyme can d i c t a t e t h e n a t u r e of s u b s t r a t e p r e s e n t e d t o 5 ' - n u c l e o t i d a s e . F o r i n s t a n c e , i f v e n t r i c u l a r e n d o t h e l i a l c e l l s a r e r i c h i n a d e n y l a t e deaminase t h e y may p r o d u c e l a r g e amounts o f i n o s i n e b u t o n l y s m a l l amounts o f a d e n o s i n e . C o n v e r s e l y , e n d o t h e l i a l c e l l s may o n l y have a low l e v e l o f a d e n y l a t e deaminase and a d e n o s i n e may be t h e p r i m a r y p r o d u c t o f 5 ' - n u c l e o t i d a s e . I f the l a t t e r i s o b s e r v e d , e n d o t h e l i a l c e l l s must be g i v e n s e r i o u s c o n s i d e r a t i o n as t h e s o u r c e of a d e n o s i n e i n h e a r t . S i m i l a r l y , a d e n y l a t e deaminase c o u l d d e t e r m i n e whether a d e n o s i n e i s formed i n s i g n i f i c a n t q u a n t i t i e s by c a r d i a c m u s c l e c e l l s . T h e r e f o r e , t h e c o n t e n t o f a d e n y l a t e 63. deaminase of h e a r t m u s c l e c e l l s and e n d o t h e l i a l c e l l s s h o u l d be d e t e r -mined. A p r e r e q u i s i t e t o t h e s e s t u d i e s i s an adequate s u p p l y o f a homogeneous p o p u l a t i o n of c e l l s . Thus t i s s u e c u l t u r e m i ght be employed t o o b t a i n t h e r e q u i r e d q u a n t i t i e s of p u r e h e a r t m u s c l e c e l l s o r p u r e e n d o t h e l i a l c e l l s . A n o t h e r t e c h n i q u e w h i c h has p o t e n t i a l f o r i s o l a t i o n o f p a r t i c u l a r cell'.otypes i n v o l v e s t i s s u e d i s i n t e g r a t i o n w i t h p r o t e o l y t i c enzymes as d e s c r i b e d by B e r r y , F r i e n d and Scheuer ;.(1970). W i t h t h e use o f t h e s e and s t a n d a r d b i o c h e m i c a l methods, one might be a b l e t o e s t a b l i s h t h e n a t u r e and s i t e o f a d e n o s i n e f o r m a t i o n i n c a r d i a c t i s s u e w i t h more c e r t a i n t y than!has been p o s s i b l e . C h a r a c t e r i z a t i o n o f b o t h c r u d e and 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 of 5 ' - n u c l e o t i d a s e o b t a i n e d f r o m t h e above s o u r c e s may be p e r t i n e n t . A n a l y s i s o f t h e c r u d e p r e p a r a t i o n may r e v e a l r e g u l a t o r y p r o p e r t i e s d i f f e r e n t f r o m t h o s e o f a p r e p a r a t i o n w h i c h has been exposed to a c e t o n e and sodium d e o x y c h o l a t e . I n t h e s e experiments:,analogues of b o t h ADP and ATP c o u l d be e x p l o i t e d . I n o r d e r t o use v e r y c r u d e p r e p a r a t i o n s , a n a l o g u e s r e s i s t a n t t o h y d r o l y s i s by p h o s p h a t a s e s would be r e q u i r e d . I n a d d i t i o n , a n a l o g u e s i n c a p a b l e of c a t i o n c h e l a t i o n would.be advantageous. I f t h e g e n e r a l c h a r a c t e r i s t i c s o f c r u d e and h i g h l y p u r i f i e d 5 ' - n u c l e o -t i d a s e p r e p a r a t i o n s were s i m i l a r , an e x t e n s i v e e x a m i n a t i o n of t h e p u r i -f i e d enzyme woul d be w a r r a n t e d ; H o p e f u l l y , an a n a l y s i s such as t h i s m i g ht c o n t r i b u t e t o a b e t t e r u n d e r s t a n d i n g of t h e i n v i v o p r o p e r t i e s and p h y s i o l o g i c a l f u n c t i o n o f 5 ' - n u c l e o t i d a s e i n h e a r t . N a t u r e o f ADP I n h i b i t i o n These s t u d i e s and t h o s e of S u l l i v a n and A l p e r s (1971) make i t c l e a r t h a t c a r d i a c 5 ' - n u c l e o t i d a s e i s i n h i b i t e d by ATP b u t i s i n h i b i t e d more p o w e r f u l l y by ADP. Thus t h e r o l e of ATP as a r e g u l a t o r of a d e n o s i n e 64. f o r m a t i o n d u r i n g h y p o x i a must be r e c o n s i d e r e d . I n a d d i t i o n some c o n f u s i o n e x i s t s r e g a r d i n g t h e k i n e t i c s of ADP and ATP i n h i b i t i o n . The a b i l i t y o f I | ADP t o i n h i b i t t h i s enzyme was r e d u c e d by t h e p r e s e n c e o f Mg ; t h e r e a s o n f o r t h i s i s n o t c l e a r a l t h o u g h t h e r e a r e a t l e a s t two p o s s i b i l i t i e s . S u l l i v a n and A l p e r s (1971) have s u g g e s t e d t h a t t h e f r e e forms o f ADP and ATP a r e s t r o n g l y i n h i b i t o r y w h i l e t h e i r complexes w i t h Mg a r e e i t h e r much l e s s p o t e n t o r i m p o t e n t . The a b i l i t y o f M g C ^ t o r e v e r s e ADP and ATP i n d u c e d i n h i b i t i o n o f r a t h e a r t 5 ' - n u c l e o t i d a s e was a s c r i b e d t o I | . . . complex f o r m a t i o n . S p e c i f i c a l l y , Mg was thought t o b i n d t h e s e n u c l e o -t i d e s so t h a t t h e y were e f f e c t i v e l y removed. However, a n o t h e r i n t e r -I | p r e t a t i o n i s p o s s i b l e . Mg - a n t a g o n i s m o f ADP and ATP i n h i b i t i o n may I | have i n v o l v e d t h e enzyme r a t h e r t h a n t h e n u c l e o t i d e s . S i n c e Mg s t i m u l a t e d 5 ' - n u c l e o t i d a s e ( F i g . 9 ) , t h e i n c r e a s e i n a c t i v i t y due t o I | a d d i t i o n o f MgCl^ ( F i g . 18) m i g h t s i m p l y be caused by Mg s t i m u l a t i o n o f t h e i n h i b i t e d enzyme. T h i s a l t e r n a t i v e e x p l a n a t i o n i s s u p p o r t e d by ADP a n a l y s i s o f t h e d a t a o f F i g . 18. The K f o r M g C l 0 s t i m u l a t i o n o f o r ATP i n h i b i t e d 5 ' - n u c l e o t i d a s e was about,10 M, v e r y s i m i l a r t o t h a t _3 f o r t h e u n i n h i b i t e d enzyme (1.9 x 10 M). The f o r m a t i o n c o n s t a n t f o r I | t h e Mg-ATP complex, i . e . (Mg-ATP)/(Mg ) ( A T P ) , i s about 8 t i m e s t h a t f o r t h e Mg-ADP complex (Walaas, 1 9 5 8 ) . I f t h e MgCl^ e f f e c t were s i m p l y due t o complex f o r m a t i o n , t h e K f o r s t i m u l a t i o n o f t h e ATP i n h i b i t e d r a p r e p a r a t i o n s h o u l d have been s u b s t a n t i a l l y l o w e r t h a n t h e f o r t h e . I | ADP i n h i b i t e d p r e p a r a t i o n . C o n t r o l o f 5 ' - n u c l e o t i d a s e by Mg r e g u l a t i o n o f a d e n i n e n u c l e o t i d e i n h i b i t i o n as d e s c r i b e d by S u l l i v a n and A l p e r s (1971) -H-seems cumbersome because a mechanism f o r t h e b i n d i n g and r e l e a s e o f Mg must be h y p o t h e s i z e d . But i f t h e enzyme were r e g u l a t e d by a d e n i n e n i i c l e o -I | t i d e m o d u l a t i o n o f Mg s t i m u l a t i o n , no such mechanism need be h y p o t h e s i z e d . 65. A means f o r t h e s y n t h e s i s and d e g r a d a t i o n o f t h e s e Mg - c o m p l e x i n g a g e n t s i s w e l l known. I t i s q u i t e p o s s i b l e t h a t 5 ' - n u c l e o t i d a s e i n a c e l l r i c h i n ADP and ATP c o u l d be i n h i b i t e d d i r e c t l y by t h e s e s u b s t a n c e s and I j i n d i r e c t l y by c h e l a t i o n of Mg ; t h e r e v e r s e m ight be e x p e c t e d i n a c e l l poor i n t h e s e s u b s t a n c e s . S i n c e t h e r e i s t h e p o s s i b i l i t y t h a t two s e p a r a t e , s y n e r g i s t i c I | a c t i o n s o f Mg work to i n c r e a s e t h e a c t i v i t y o f ADP o r ATP i n h i b i t e d enzyme, t h e e f f e c t of each s e p a r a t e a c t i o n would be d i f f i c u l t t o a s s e s s . I n o r d e r t o r e s o l v e t h i s p r o b l e m i t m i g h t be p r o f i t a b l e t o employ ana-l o g u e s o f t h e s e i n h i b i t o r s . Those a n a l o g u e s w h i c h would n o t complex I | Mg b u t i n h i b i t t h e enzyme would a l l o w d e t e r m i n a t i o n of t h e s t i m u l a t o r y I | e f f e c t of Mg on i n h i b i t e d 5 ' - n u c l e o t i d a s e w i t h o u t i n t e r f e r e n c e by c h e l a t i o n . There appears to be c o n s i d e r a b l e d i s a g r e e m e n t c o n c e r n i n g t h e manner i n w h i c h r a t h e a r t 5 ' - n u c l e o t i d a s e . i s i n h i b i t e d by ATP and ADP. I n h i b i t i o n by t h e s e s u b s t a n c e s has been v a r i o u s l y r e p o r t e d as c o m p e t i t i v e (Baer e t a l . ; 1966; S u l l i v a n and A l p e r s , 1971) :and mixed c o m p e t i t i v e -n o n - c o m p e t i t i v e (Edwards and M a g u i r e , 1 9 7 0 ) . I n a d d i t i o n t h e r e s u l t s f r o m t h e s e l a b o r a t o r i e s a r e c o m p l i c a t e d by t h e p r e s e n c e o r absence o f Mg i n t h e r e a c t i o n m i x t u r e s . I n t h e p r e s e n t s t u d y ADP i n d u c e d i n h i -b i t i o n was s u b j e c t e d t o f u r t h e r a n a l y s i s i n an a t t e m p t t o e x p l a i n t h e s e d i s c r e p a n c i e s . S u l l i v a n and A l p e r s (1971) r e p o r t e d t h a t 0.0017 mM ADP i n h i b i t e d 5 ' - n u c l e o t i d a s e i n a s t r i c t l y c o m p e t i t i v e manner i n t h e l [ absence o f Mg . I n c o m p a r i s o n , F i g . 14 shows t h a t , i n t h e absence o f I | Mg , 0.0033 mM ADP i n h i b i t e d i n a mixed c o m p e t i t i v e - n o n - c o m p e t i t i v e .manner. I f t h e d a t a of F i g . 1 6.are p l o t t e d by t h e method of D i x o n ( 1 9 5 3 ) , two b i n d i n g s i t e s f o r ADP a r e i n d i c a t e d . I f one s i t e o f b i n d i n g were t h e 66. a c t i v e s i t e o f t h e enzyme t h i s would a c c o u n t f o r t h e c o m p e t i t i v e component o f i n h i b i t i o n . B i n d i n g a t a n o t h e r c o u l d a c c o u n t f o r t h e n o n - c o m p e t i t i v e component. When 16 mM M g C ^ was p r e s e n t i n t h e r e a c t i o n m i x t u r e s t h e r e s u l t s were q u i t e d i f f e r e n t ; o n l y n o n - c o m p e t i t i v e i n h i b i t i o n was o b s e r v e d and o n l y one ADP b i n d i n g s i t e was i n d i c a t e d . On t h e b a s i s o f t h e s e o b s e r v a t i o n s , t h e f o l l o w i n g model i s p r o p o s e d . The enzyme e x i s t s i n two f o r m s ; one i n w h i c h ADP i n h i b i t s c o m p e t i t i v e l y and has a low f o r ADP and a n o t h e r i n w h i c h ADP i n h i b i t s n o n - c o m p e t i t i v e l y and has I | a h i g h e r f o r ADP. Mg s t a b i l i z e s t h e enzyme i n the l a t t e r f o r m so t h a t o n l y n o n - c o m p e t i t i v e i n h i b i t i o n i s o b s e r v a b l e when t h e enzyme i s j | s a t u r a t e d w i t h Mg . 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