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A study of the adenyl cyclase activity in testis of maturing chinook salmon (Oncorhynchus tshawytscha) Bendix, Marie Elaine 1974

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A STUDY OF THE ADENYL CYCLASE ACTIVITY IN TESTIS OF MATURING CHINOOK SALMON (Oncorhynchus tshawytscha) fey MARIE ELAINE BENDIX B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of , Biochem i s t r y 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 J u l y 1974 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia Vancouver 8 , Canada i . ABSTRACT Some p r o p e r t i e s of the adenyl c y c l a s e a c t i v i t y i n the maturing t e s t i s of Oncorhynchus tshawytscha (chinook salmon) were c h a r a c t e r i z e d . The enzymic r e a c t i o n was l i n e a r a t 30° and at 15° f o r at l e a s t 60 min. The d i v a -l e n t c a t i o n requirement of the salmon t e s t i s enzyme was reexamined ( 3 3 ) . The optimal c o n c e n t r a t i o n of Mg was about 10 mM and of Mn was 5 mM; Mn^ c o n c e n t r a t i o n s above 15 mM caused a marked decrease i n enzyme a c t i v i t y . A h igher maximal a c t i v i t y was achieved i n the presence of Mn^ than i n the presence of Mg . S t i m u l a t i o n of the enzyme w i t h the optimal c o n c e n t r a t i o n of P~, 12 mM, r e s u l t e d i n a 7 - f o l d i n c r e a s e i n the r e a c t i o n r a t e over the b a s a l a c t i v i t y . I n e f f o r t s to s o l u b i l i z e the enzyme, i t was found t h a t L u b r o l PX and T r i t o n X-100 destroyed enzymic a c t i v i -t y but Nonidet P4-0 and Tween 80 d i d not. The adenyl c y c l a s e a c t i v i t y i n salmon t e s t i s homog-enates was s t a b l e f o r at l e a s t 6 hours at 0° to 4° but was very unstable a t 24°; storage of the homogenate f o r 24 hours at e i t h e r 0° to 4° or 24° r e s u l t e d i n a t o t a l l o s s of a c t i v i t y . D i f f e r e n t i a l c e n t r i f u g a t i o n of salmon t e s t i s homog-enates which were prepared i n i s o t o n i c medium re v e a l e d t n a t a l l s u b c e l l u l a r f r a c t i o n s contained some adenyl c y c l a s e a c t i v i t y . About 55$ of the a c t i v i t y sedimented at 600g whi l e only 10% of the a c t i v i t y was recovered i n the 105,000g supernatant. The 6300g sediment had a very h i g h s p e c i f i c a c t i v i t y compared w i t h the s p e c i f i c a c t i v -i t y of the other f r a c t i o n s . The ATP analogue, a d e n y l y l imidodipho3phate (AMP-PNP), t r i t i u m l a b e l e d i n adenosine, was sy n t h e s i z e d from t r i -butylammonium imidodiphosphate and adenosine-5* phosphor-i m i d a z o l a t e . Salmon t e s t i s adenyl c y c l a s e c a t a l y z e d the conversion of AMP-PNP to c y c l i c AMP. i i i TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i i i LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS v i i i ABBREVIATIONS ix INTRODUCTION V I. Testicular Maturation in Oncorhynchus Tshawytscha . . . . . . . 5 A Spermatogenesis . 5 B Supporting Cells of the Salmonid Testis 11 C Hormones and Testicular Development in 0. Tshawytscha . . . . . 13 II. Adenyl Cyclase 21 MATERIALS AND METHODS 32 I. Materials 32 II. Methods 3 4 A Synthesis of Tritium Labeled Adenylyl Imidodiphosphate 34 B Adenyl Cyclase Assay 36 RESULTS 40 I. Synthesis of Tritium Labeled Adenylyl Imidodiphosphate 40 i v Page A M o l e c u l a r Weight and P u r i t y of Sodium Imidodiphosphate 40 B P u r i f i c a t i o n of T r i t i u m Labeled A d e n y l y l Imidodiphosphate 40 I I . Adenyl Cyclase A c t i v i t y i n 0. Tshawytscha T e s t i s 45 A The ATP-Regenerating System 45 B E f f e c t of Time, Temperature, and Substrate 45 C E f f e c t of D i v a l e n t Cations 47 D E f f e c t of F l u o r i d e I o n 49 E E f f e c t of Detergents and Some Other Reagents 49 F S t a b i l i t y 52 G S u b c e l l u l a r D i s t r i b u t i o n 54 DISCUSSION 57 I . The Adenyl Cyclase Assay 57 A ATP-Regeneration System 57 B E f f e c t of Time and Temperature . . . . 58 C E f f e c t of Mg 2 + and Mn 2 + 59 D E f f e c t of F" 61 I I . S o l u b i l i z a t i o n of Salmon T e s t i s Adenyl Cyclase 63 A S u b c e l l u l a r D i s t r i b u t i o n 63 B E f f e c t of Detergents 64 Page I I I . T r i t i u m Labeled A d e n y l y l Imidodiphosphate 68 A Synthesis 68 B AMP-PNP as a Substrate f o r Salmon T e s t i s Adenyl Cyclase 69 CONCLUSIONS 71 BIBLIOGRAPHY 75 v i LIST OF TABLES Page Table 1. C h a r a c t e r i z a t i o n of A d e n y l y l Imidodiphosphate by Paper Chromatog-raphy 41 Table 2. C h a r a c t e r i z a t i o n of A d e n y l y l Imidodiphosphate by Paper Chromatog-raphy (taken from Yount et a l (46)) . . 42 Table 3. E f f e c t of a V a r i e t y of Reagents on the Adenyl Cyclase A c t i v i t y of Salmon T e s t i s Homogenates 51 Table 4. S u b c e l l u l a r D i s t r i b u t i o n of Adenyl Cyclase i n Salmon T e s t i s Homogenates . . 5 5 v i i LIST OF FIGURES Page F i g u r e 1 . Chromatographic P u r i f i c a t i o n of 5H-AMP-PNP on DEAE C e l l u l o s e 43 Fi g u r e 2. E f f e c t of Time, Temperature, and Substrate on the Adenyl Cyclase A c t i v i t y of Salmon T e s t i s Homogenates 46 Fi g u r e 3. The E f f e c t of Changes i n Magnesium and Manganese Ion Concentration on the A c t i v i t y of Adenyl Cyclase i n Salmon T e s t i s Homogenates . . 48 Fi g u r e 4. The E f f e c t of Changes i n F l u o r i d e Ion Conc e n t r a t i o n on the A c t i v i t y of Adenyl Cyclase i n Salmon T e s t i s Homogenates . 50 F i g u r e 5. S t a b i l i t y of Adenyl Cyclase i n a Salmon T e s t i s Homogenate 53 v i i i ACKNOWLEDGEMENTS I wish to thank my re s e a r c h s u p r e v i s o r Dr. Michael Smith f o r h i s h e l p f u l advice and encouragement which I r e c e i v e d throughout the course of my l a b o r a t o r y work and the w r i t i n g of t h i s T h e s i s . I a l s o wish to thank Drs. S h i r l e y G i l l a m , Ian G i l l a m and Michael Doel and A l b e r t Fung, Graham H i l l , P a t r i c i a Jahanke, and V i v i a n Wylie f o r o f t e n p r o v i d i n g a s s i s t a n c e and , encouragement. The chemical sample r e c e i v e d from Dr. Ralph Yount was ap p r e c i a t e d . The author was the r e c i p i e n t of a Medical Research C o u n c i l of Canada Studentship ( 1 9 7 1 - 1 9 7 3 ) . i x ABBREVIATIONS ADP ADP-NH2 5 *-AMP AMP-POP AMP-PNP ATP 0 c y l c i c AMP c y c l i c G-MP c y c l i c IMP DEAE-cellulose DMF DNA dpm EDTA g O.D. adenosine 5'-diphosphate a d e n y l y l phosphoramidate adenosine 5'-monophosphate a d e n y l y l methylenediphosphate a d e n y l y l imidodiphosphate adenosine 5'-triphosphate C u r i e ; one Curie = t h a t q u a n t i t y of amr r a d i o a c t i v e isotope undergoing 3.7 10 d i s i n t e g r a t i o n s per second. adenosine 3 ' , 5 ' - c y c l i c phosphate guanosine 3 ' , 5 ' - c y c l i c phosphate i n o s i n e 3 ' , 5 , - c y c l i c phosphate O-( d i e t h y l a m i n o e t h y l ) c e l l u l o s e d i m e t h y l f o rmami d e d e o x y r i b o n u c l e i c a c i d d i s i n t e g r a t i o n s per minute e t h y l e n e d i a m i n e t e t r a a c e t a t e g r a v i t a t i o n a l constant (980 cm/sec ) o p t i c a l d e n s i t y or absorbance. One o p t i c a l d e n s i t y u n i t of a substance i s the amount of i t which when d i s s o l v e d i n 1 ml of a s o l v e n t has an absorbance of 1.0 when measured i n a c e l l w i t h a l i g h t p a t h - l e n g t h of 1.0 cm. i n o r g a n i c orthophosphate methylenediphosphonate imidodiphosphate inorganic pyrophosphate mobility r e l a t i v e to the solvent r i b o n u c l e i c acid tris(hydroxymethyl) aminomethane 1. INTRODUCTION This Thesis d e s c r i b e s some p r o p e r t i e s of the adenyl cyc l a s e a c t i v i t y present i n the maturing t e s t i s of the P a c i f i c chinook salmon, Oncorhynchus tshawytscha. This l a b o r a t o r y has been i n v e s t i g a t i n g v a r i o u s aspects of n u c l e i c a c i d metabolism w i t h i n the t e s t i s of s e x u a l l y maturing salmonids (7,33,45), and a study on the adenyl c y c l a s e of salmon t e s t i s was i n keeping w i t h t h i s l i n e of r e s e a r c h . The t e s t i s i s a p a r t i c u l a r l y i n t e r e s t i n g organ be-cause there are many d e t a i l s of spermatogenesis which are s t i l l u n resolved. The mechanism of DNA r e p l i c a t i o n dur-i n g m i t o s i s and meiosis ( 7 ) , the mechanism of c e l l u l a r d i f f e r e n t i a t i o n ( 1 2 ) , and the exact nature of the c o n t r o l exerted by the gonadotropins and the t e s t i c u l a r androgens on the c e l l s of the t e s t i s (20) are unknown. Because spermatogenesis i s under the d i r e c t hormonal c o n t r o l of the p i t u i t a r y gland (20,29) the p o s s i b l e involvement of the 'second messenger', c y c l i c AMP, i n t h i s process can-not be ignored. There are a l r e a d y a number of r e p o r t s of hormonal s t i m u l a t i o n of adenyl c y c l a s e w i t h i n mamma-l i a n t e s t e s (25,34) and r e p o r t s of c y c l i c AMP s t i m u l a t e d p r o t e i n k i n a s e s i n t r o u t t e s t i s (22) and i n mammalian 2. t e s t e s (28,38). I n view of the f i n d i n g t h a t a salmon p i t u i t a r y e x t r a c t w i t h gonadotropic a c t i v i t y s t i m u l a t e d the adenyl c y c l a s e of salmon t e s t i s s l i c e s ( 3 3 ) , t h i s enzyme i s thought to be of some importance i n mediating or r e g u l a t i n g spermatogenesis i n salmon t e s t i s . The enzyme adenyl c y c l a s e , although e x t e n s i v e l y s t u d i e d i n many organisms and t i s s u e s , i n a l l but a few cases (37) has eluded p u r i f i c a t i o n . Adenyl c y c l a s e i s u s u a l l y membrane bound (37) and t h e r e f o r e , i s d i f f i c u l t to s o l u b i l i z e . Because a s i g n i f i c a n t p o r t i o n of the aden-y l c y c l a s e i n crude homogenates of 0. tshawytscha t e s t i s appeared to be s o l u b l e ( 3 3 ) , i t seemed p o s s i b l e t h a t a l l of t h i s enzyme a c t i v i t y might be s o l u b i l i z e d . S o l u b i l i z a -t i o n - would be a f i r s t step t o p u r i f i c a t i o n and the sub-sequent d e t a i l e d c h a r a c t e r i z a t i o n of the enzyme. In the experiments r e l a t e d w i t h i n t h i s T h e s i s , the adenyl c y c l a s e a c t i v i t y of crude homogenates of salmon t e s t i s was monitored by r e c o r d i n g the p r o d u c t i o n of ^ H-c y c l i c AMP from ^H-ATP. The presence of ATPases w i t h i n t h i s t i s s u e , as i n many other t i s s u e s from other organ-isms, i n t e r f e r e s w i t h t h i s assay. The ATP r e g e n e r a t i n g system of phosphoenolpyruvate and pyruvate kinase was r o u t i n e l y i n c l u d e d i n the assay to m a i n t a i n the i n i t i a l ^H-ATP c o n c e n t r a t i o n throughout the i n c u b a t i o n p e r i o d . 3 . This system i s , however, cumbersome and i n c r e a s e s the com-p l e x i t y of the adenyl c y c l a s e assay; an a l t e r n a t i v e method of assay was t h e r e f o r e , i n v e s t i g a t e d . I t has been repor-ted t h a t the ATP analogue, a d e n y l y l imidodiphosphate (AMP-PNP) behaves i d e n t i c a l l y to ATP as a s u b s t r a t e f o r the adenyl c y c l a s e of r a t l i v e r plasma membranes ( 4 0 ) . I n a d d i t i o n , the analogue has the advantage of being a poor s u b s t r a t e f o r the ATPases i n t h i s t i s s u e ( 4 0 ) , and thus, no system f o r r e g e n e r a t i o n of the s u b s t r a t e i s r e q u i r e d when adenyl c y c l a s e a c t i v i t y i s measured by the p r o d u c t i o n of c y c l i c AMP from AMP-PNP. In t h i s Thesis a new method f o r the s y n t h e s i s of AMP-PNP from tributylammonium imidodiphosphate and adenosine-5' phosphorimidazolate was devised; t h i s method was based on the general procedure of Hoard and Ott (21) f o r the s y n t h e s i s of d e o x y o l i g o n u c l e o t i d e t r i p h o s p h a t e s . AMP-PNP, t r i t i u m l a b e l e d i n adenosine was s y n t h e s i z e d and was then t e s t e d as a s u b s t r a t e f o r the salmon t e s t i s adenyl c y c l a s e a c t i v i t y . I n summary, the o b j e c t i v e s of t h i s Thesis were to i n v e s t i g a t e the optimum c o n d i t i o n s f o r the assay of 0. tshawytscha t e s t i s adenyl c y c l a s e , to determine whether t h i s enzyme might be r e a d i l y s o l u b i l i z e d , to s y n t h e s i z e ^H-AIvtP-PNP by a new method and then to use t h i s n u c l e o t i d e 4. i n developing a method of assaying salmon t e s t i s adenyl c y c l a s e which would e l i m i n a t e the n e c e s s i t y of i n c l u d i n g an ATP r e g e n e r a t i n g system i n the assay medium. 5 . I . T e s t i c u l a r M a t u r a t i o n i n Oncorhynchus Tshawytscha A Spermatogenesis M i g r a t o r y f i s h of the genus Oncorhynchus e v e n t u a l l y r e t u r n to t h e i r f r e s h water place of "birth to spawn and then d i e . The age of the spawning Chinook salmon aver-ages 4 years but v a r i e s from 3 to 7 years ( 1 4 ) . The Chi-nook or k i n g salmon which migrate up the F r a s e r R i v e r un-dergo t e s t i c u l a r development d u r i n g the months of A p r i l to October. I n A p r i l the t e s t e s are immature and represent 0.2 t o 0.5$> of the body weight ( 4 5 ) . The mature t e s t i s i s f i l l e d w i t h sperm or spermatozoa which have been pro-duced by a process c a l l e d spermatogenesis. The c y t o l o g i -c a l changes i n v o l v e d i n spermatogenesis are somewhat sim-i l a r f o r a l l v e r t e b r a t e s ( 2 0 ) . The p r i m o r d i a l germ c e l l s which are the i n i t i a l pre-c u r s o r s of the sperm d i f f e r e n t i a t e through an ordered se-quence o f c e l l types to become sperm c e l l s . The d i f f e r -ent c e l l types i n v o l v e d i n t h i s pathway have been charac-t e r i z e d by t h e i r s i z e , t h e i r time of appearance d u r i n g t e s t i s development, the appearance of t h e i r chromatin, nucleus and cytoplasmic components ( 5 ) , t h e i r DNA and RNA co n t e n t s , t h e i r complement of n u c l e a r p r o t e i n s , (30,45), and t h e i r r e l a t i v e r a t e s of DNA, h i s t o n e and protamine 6. s y n t h e s i s ( 3 0 ) . The sequence of spermatogenic events i s as f o l l o w s . The i n f a n t i l e t e s t i s c o n t a i n s gonocytes which have a r i s e n from p r i m o r d i a l germ c e l l s ( 5 ) . At some p o i n t i n the development o f the animal the gonocytes mul-t i p l y and then are transformed i n t o primary spermatogonia, or A-type s p e r m a t o g o n i a 2 as they are c a l l e d i n mammals. The primary spermatogonia m u l t i p l y "by a s e r i e s o f m i t o t i c d i v i s i o n s and then d i f f e r e n t i a t e i n t o secondary or B-type spermatogonia. The number o f spe r m a t o g o n i a l d i v i s i o n s v a r i e s from s p e c i e s to s p e c i e s ( 5 ) . Up to 1 2 spermatogo-n i a l d i v i s i o n s are counted i n the guppy (5) and 1 0 or 1 1 are e s t i m a t e d i n the rainbow t r o u t (30); whereas there are only 4 to 6 d i v i s i o n s i n mammals depending on the s p e c i e s , and t h e r e are even fewer d i v i s i o n s i n b i r d s . The secondary spermatogonia are the d i r e c t p r e c u r s o r s o f the primary spermatocytes which are d e s t i n e d to d i v i d e by m e i o s i s . The primary spermatocytes double t h e i r n u c l e a r DNA content to a 4n amount, have a l o n g prophase d u r i n g which g e n e t i c r e c o m b i n a t i o n p r o b a b l y o c c u r s and then undergo the f i r s t 1 . That the gonocytes are the p r e c u r s o r s o f the sperma-t o g o n i a i s the p o p u l a r b e l i e f , but some i n v e s t i g a t o r s suggest t h a t i t i s the s u p p o r t i n g c e l l s which d i f f e r e n t i -ate i n t o the a d u l t germ c e l l s ( 5,43). 2 . The presence of an a d d i t i o n a l type o f sp e r m a t o g o n i a l c e l l , the prespermatogonium, which e x i s t s as a t r a n s i t i o n between the development o f the gonocyte i n t o an a d u l t A-type spermatogonium has been noted i n some mammals (5,43). 7. r e d u c t i o n d i v i s i o n to give two secondary spermatocytes, each c o n t a i n i n g a 2n amount of DNA. The secondary sperma-to c y t e s are short l i v e d and each soon d i v i d e s by the sec-ond r e d u c t i o n d i v i s i o n to give two e a r l y spermatids. C y t o l o g i c a l changes o c c u r r i n g d u r i n g meiosis are w e l l docu-mented ( 4 ) . E a r l y spermatids which c o n t a i n a h a p l o i d amount of n u c l e a r DNA do not d i v i d e , but d i f f e r e n t i a t e i n -to spermatozoa by a lengthy process c a l l e d spermiogenesis. There are a number of changes which occur i n the sperma-t i d s d u r i n g spermiogenesis. The volume of the spermatid i s g r e a t l y reduced; the morphology of the nucleus changes; the n u c l e a r DNA becomes i n c r e a s i n g l y dense; the s y n t h e s i s of RNA g r a d u a l l y d e c l i n e s to zero; and there i s a change i n the a c i d s o l u b l e p r o t e i n s attached to the DNA ( 3 0 ) . I n salmonids the removal of h i s t o n e s from DNA and t h e i r r e -placement by protamines i s a w e l l documented event ( 3 0 ) . I n mammals the stages of spermiogenesis are w e l l d e f i n e d a c c o r d i n g to changes i n the appearance of the acrosome ( 5 ) , but i n salmonids no acrosome i s formed ( 3 0 ) . There are two broad c a t e g o r i e s i n t o which the method of sperm pro d u c t i o n can be d i v i d e d , these are c y s t i c and n o n - c y s t i c . Most anamniotic s p e c i e s , i n c l u d i n g 0. tshawy-t s c h a , produce sperm c y s t i c a l l y . The t e s t e s of salmonids are s t r u c t u r e d by connective t i s s u e i n t o l o b u l e s each of 8. which contains 2 to 6 or more membrane w a l l e d c y s t s ( 2 0 ) . When the t e s t e s are immature the c y s t s are e s s e n t i a l l y empty and as t e s t i c u l a r maturation begins the gonocytes d i f f e r e n t i a t e i n t o primary spermatogonia. By a s e r i e s of m i t o t i c d i v i s i o n s the c y s t becomes populated w i t h a l a r g e number of primary spermatogonia which a l l s i m u l t a -neously d i f f e r e n t i a t e i n t o secondary spermatogonia. W i t h i n each c y s t a l l c e l l s are at the same stage of spermato-genic development and are maturing a t the same r a t e but w i t h i n a t e s t i s the v a r i o u s c y s t s may be at d i f f e r e n t stages of development ( 2 9 ) . Testes f o r the present study were obtained from Chi-nook salmon which were captured from the F r a s e r R i v e r d u r i n g the month of June. The t e s t e s from these f i s h were undergoing maturation and have been defined as stage 2 t e s t e s . The stage 2 t e s t i s contains mainly c y s t s of p r i -mary and secondary spermatocytes and some c y s t s of sperma-t i d s ( 4 5). A l l the c y s t s w i t h i n the mature t e s t i s are f i l l e d w i t h spermatozoa which are r e l e a s e d s i m u l t a n e o u s l y at the time of spawning. I n most f i s h the events of t e s -t i c u l a r m aturation and spermatozoan p r o d u c t i o n are c y c l i c because these processes are repeated each year ( 2 9 ) . P a c i f i c salmon of the genus Oncorhynchus t however l i v e through only one c y c l e of t e s t i c u l a r maturation (23). 9. The t e s t e s of amniotic species are not s t r u c t u r e d i n t o c y s t s , but i n t o t u b u l e s . Each spermatogonial c e l l develops i n d i v i d u a l l y and i s never surrounded by a c y s t w a l l . The e n t i r e l e n g t h of the seminiferous tubule i s c o n t i n u a l l y populated w i t h germ c e l l s at v a r i o u s stages of maturation. The c e l l u l a r a s s o c i a t i o n s between adjacent " germ c e l l s are f i x e d as a r e s u l t of r e g u l a r and c o o r d i -nated development of these c e l l s ( 5 ) . Once sperm produc-t i o n i s begun i t i s c a r r i e d out c o n t i n u o u s l y d u r i n g the l i f e of the animal. As mentioned e a r l i e r , w i t h i n the developing sperma-t i d s h i s t o n e s are r e p l a c e d by a group of a r g i n i n e - r i c h , sperm s p e c i f i c p r o t e i n s , the protamines. L i k e the h i s t o n e s , the protamines become i n t i m a t e l y bound to the DNA by i o n i c and p o s s i b l y hydrophobic i n t e r a c t i o n s . During the r e p l a c e -ment of the h i s t o n e s by protamines the template a c t i v i t y of DNA decreases d r a s t i c a l l y and the chromatin condenses. The r o l e s which have been proposed f o r the protamines are summarized by Louie (30): the protamines "(a) cause the condensation of chromatin, shrinkage of the n u c l e u s , and consequent s t r e a m l i n i n g of the c e l l , (b) i n h i b i t gene t r a n s c r i p t i o n , (c) erase the developmental h i s t o r y of the c e l l thus p r o v i d i n g a t o t i p o t e n t n u c l e u s , and (d) p r o t e c t the chromosome from adverse e f f e c t s of the environment." 10. The protamines are sy n t h e s i z e d w i t h i n the cytoplasm of middle stage spermatids. Between the time of t h e i r syn-t h e s i s and t h e i r secure b i n d i n g to the DNA the protamines are e x t e n s i v e l y phosphorylated and dephosphorylated. These m o d i f i c a t i o n s are thought to be of great importance to the c o r r e c t and f i n a l b i n d i n g of the protamines to the DNA ( 3 0 ) . The d i s c o v e r y of a c y c l i c AMP s t i m u l a t e d p r o t -amine s p e c i f i c p r o t e i n kinase w i t h i n the t e s t i s of r a i n -bow t r o u t (22) makes the b i o l o g y of the h i s t o n e s and p r o t -amines d u r i n g spermatogenesis a t o p i c of much i n t e r e s t i n r e l a t i o n to the adenyl c y c l a s e of salmonid t e s t e s . M o d i f i c a t i o n s of h i s t o n e s are a l s o of i n t e r e s t i n t h i s r e s p e c t . The h i s t o n e s are attached to the DNA of most somatic c e l l s and of a l l of the spermatogonial c e l l s except l a t e spermatids and spermatozoa ( 3 0 ) . E x t e n s i v e a c e t y l a t i o n , d e a c e t y l a t i o n , p h o s p h o r y l a t i o n and dephos-p h o r y l a t i o n of these p r o t e i n s are observed and i t i s pos-t u l a t e d that these m o d i f i c a t i o n s are i n v o l v e d i n the regu-l a t i o n of RNA polymerase and DNA polymerase r e a c t i o n s ( 3 8 ) . C y c l i c AMP s t i m u l a t e d p r o t e i n k i n a s e s which c a t a -tones have been observed i n t r o u t t e s t i s ( 2 2 ) , r a t t e s t i s (28,38), c a l f l i v e r and other c e l l types ( 2 2 ) . For f u r -t h e r i n f o r m a t i o n on the h i s t o n e s and protamines of s a l -l y z e the t r a n s f e r of the 1 1 . monid t e s t e s r e f e r to Louie ( 3 0 ) , Dixon ( 6 ) , and Urquhart (4 5 ) . B Supporting C e l l s of the Salmonid T e s t i s In a d d i t i o n to the t e s t i c u l a r germ c e l l s there are two other types of t e s t i s s p e c i f i c c e l l s - L e y d i g c e l l s and S e r t o l i c e l l s . 'i'he ^Leydig c e l l s are i n t e r s t i t i a l c e l l s which are g e n e r a l l y considered to be the major source of the t e s -t i c u l a r androgens ( 2 0 ) . T y p i c a l l y these c e l l s are seen as w e l l - v a s c u l a r i z e d aggregations of modified connective t i s s u e which l i e between the seminiferous c y s t s or tubules (29 ) . L e y d i g c e l l s have been i d e n t i f i e d i n 0. k e t a (chum salmon) (20) and are therefore probably present i n t e s t e s of 0. tshawytscha as w e l l . I n a d d i t i o n , a homologue of the L e y d i g c e l l has been i d e n t i f i e d w i t h i n the t e s t i s of s e x u a l l y mature A t l a n t i c salmon (Salmo s a l a r ) ( 4 7 ) . The types of androgens found w i t h i n the blood and t e s t i s of salmonids are reviewed by Hoar ( 2 0 ) . The de-velopment of secondary sex c h a r a c t e r i s t i c s p r i o r to breed-i n g ; i s dependent on the p r o d u c t i o n of t e s t i c u l a r andro-gens ( 2 0 ) . Experimentation w i t h hypophysectomized t e l e -osts showed t h a t a d m i n i s t r a t i o n of androgens s t i m u l a t e d 12. spermatogenesis or an in c r e a s e i n gonad s i z e , and i t seems p o s s i b l e t h a t these, e f f e c t s were the r e s u l t of an a c c e l e r a t i o n of post-spermatogonial development ( 2 9 ) . T e s t i c u l a r androgens may have an a d d i t i o n a l f u n c t i o n i n Oncorhynchus of modulating, to some e x t e n t , the p h y s i o l -ogy and behavior of spawning m i g r a t i o n (20). The process of s t e r o i d o g e n e s i s w i t h i n the t e s t e s of a l l v e r t e b r a t e s i s s u b j e c t to seasonal v a r i a t i o n which seems to be under the c o n t r o l of the gonadotropic hormone(s). I n the con-t e x t of t h i s Thesis i t i s of i n t e r e s t to note t h a t i n mammalian t e s t e s there i s evidence to support the hypoth-e s i s t h a t gonadotropic s t i m u l a t i o n of s t e r o i d p r o d u c t i o n i s mediated by an in c r e a s e i n c y c l i c AMP (17,34). Although the e x i s t e n c e o f S e r t o l i ; c e l l s or nurse c e l l s has been reporte d i n r e p r e s e n t a t i v e s of each v e r t e -brate c l a s s and i n a l l groups of f i s h e s (20,29) i n many f i s h there i s no evidence of these c e l l s ( 3 0). There has been no refe r e n c e to the presence of S e r t o l i c e l l s i n On-corhynchus , but they have been reporte d i n some t e l e o s t s (29). When observed i n anamniotes, S e r t o l i c e l l s are found as s t r u c t u r a l elements of the c y s t w a l l s . T h e i r or-i g i n i s b e l i e v e d to be the connective t i s s u e surrounding the primary germ c e l l s . S e r t o l i c e l l s e x i s t i n c l o s e as-13. s o c i a t i o n w i t h the germ c e l l s and m u l t i p l y as the germ c e l l s mature. Spermatids are p a r t i a l l y implanted w i t h i n the S e r t o l i c e l l s and are b e l i e v e d to d e r i v e n u t r i e n t s from them ( 5 ) . Two a d d i t i o n a l f u n c t i o n s of S e r t o l i c e l l s t h a t are proposed are the phagocytosis of unused sperm (20) and the p r o d u c t i o n of s t e r o i d hormones ( 2 9 ) . The l a t t e r f u n c t i o n may be under the c o n t r o l of a gonadotro-p i c hormone ( 2 9 ) . I n a d d i t i o n to germ c e l l s , L e y d i g c e l l s and S e r t o l i c e l l s , homogenates of whole t e s t i s c o n t a i n elements of v a s c u l a r , nervous and connective t i s s u e ( 3 4 ) . C Hormones and T e s t i c u l a r Development i n 0 . Tshawytscha I t has been s a i d t h a t "the most i n t e r e s t i n g proper-t y of adenyl c y c l a s e i s i t s responsiveness to hormones." (37) . I t i s w i t h t h i s statement i n mind th a t the hormonal c o n t r o l s of t e s t i c u l a r development i n 0 . tshawytscha are d i s c u s s e d . The p i t u i t a r y gland of a l l v e r t e b r a t e s i s of key importance to both aspects of gonadal development, gameto-genesis and s t e r o i d o g e n e s i s . Hypophysectomies of a range of v e r t e b r a t e species i n d i c a t e t h a t the p i t u i t a r y has a d i f f e r e n t degree of c o n t r o l over t e s t i c u l a r development H . w i t h i n d i f f e r e n t s p e c i e s . I n a p r i m i t i v e species of f i s h , Lampetra f l u v i a t i l i s , hypophysectomy r e t a r d s , hut does not a b o l i s h spermatogenesis and s p e r m i a t i o n ( 1 8 ) . I n mammalia t h i s o p e r a t i o n leads to degeneration of the sem-i n i f e r o u s e p i t h e l i u m and spermatogenic a r r e s t at the p r i -mary spermatocyte stage (43). I n t e l e o s t s which have been s u c c e s s f u l l y hypophysec-tomized the t r a n s f o r m a t i o n of secondary spermatogonia i n -to primary spermatocytes i s b l o c k e d , but i f primary sper-matocytes of a c e r t a i n age (3) are present i n the t e s t i s at the time of hypophysectomy they can develop i n t o sper-matids. There i s a l s o evidence t h a t m i t o s i s of spermato-gonia i s p a r t i a l l y i n h i b i t e d by hypophysectomy ( 3 ) . Sper-m i a t i o n u s u a l l y does not occur i n hypophysectomized t e l e -o s t s . Gases where sp e r m i a t i o n i s observed may r e f l e c t an advanced s t a t e of t e s t i c u l a r development at the time of hypophysectomy ( 2 0 ) . Removal of the p i t u i t a r y leads a l s o "to atrophy of the L e y d i g c e l l s and a l o s s of secon-dary sex s t r u c t u r e s and c h a r a c t e r i s t i c s ( 2 0). I n r e l a t i o n to p i t u i t a r y c o n t r o l of the spermatogo-n i a - spermatocyte t r a n s f o r m a t i o n , Louie (30) suggests t h a t as gonadotropin output by the p i t u i t a r y i n c r e a s e s the number of spermatogonia! c e l l s which are committed to d i f -1 5 . f e r e n t i a t e i n t o primary spermatocytes i n c r e a s e s , thereby s t i m u l a t i n g the spermatogenic process. The main e x t e r n a l f a c t o r which t r i g g e r s s e x u a l matu-r a t i o n of 0. tshawytscha i s photoperiod ( 7 ) . There i s evidence t h a t i n lower v e r t e b r a t e s , as i n mammalia, t h i s s t i m u l u s reaches the c e l l s of the p i t u i t a r y v i a the hypo-thalamus ( 2 9 ) . By h i s t o l o g i c a l examination of the adeno-hypophyseal c e l l s of 0. tshawytscha, 0. nerka and 0. k e t a d u r i n g the p e r i o d of gonadal maturation and spawning i t was concluded t h a t the gonadotropes are the b a s o p h i l s w i t h -i n the meso-adenohypophysis (35,36). Thus, i t i s pro-posed t h a t only a s i n g l e type of hypophyseal c e l l elabo-r a t e s g o n a d o t r o p i n ( s ) . However, there i s some s p e c u l a t i o n t h a t another type of adenohypophyseal c e l l producesigona^ d o t r o p i n ( s ) , but Overbeeke and McBride (36) do not agree. In a l l tetrapods i t appears that there are two d i s -t i n c t gonadotropic hormones, l u t e i n i z i n g hormone (LH) or i n t e r s t i t i a l c e l l s t i m u l a t i n g hormone (ICSH) and f o l l i c l e -s t i m u l a t i n g hormone (FSH) ( 2 0 ) . The nature of the gona-d o t r o p i c hormone(s) produced w i t h i n the p i t u i t a r y of 0. tshawytscha has been i n v e s t i g a t e d by Donaldson and co-work-ers, ( 8 ) . They have prepared, from the p i t u i t a r y of ma-t u r e chinook slamon, a p a r t i a l l y pure proteinaceous sam-ple capable of i n d u c i n g (a) precocious maturation of the 16. t e s t e s of immature rainbow t r o u t , (b) s p e r m i a t i o n i n g o l d f i s h , (c) gametogenesis i n 0. tshawytscha, (d) i n c o r -32 p o r a t i o n of P i n t o day old chick t e s t e s , and (e) other p o s i t i v e gametogenic responses. The extent of response v a r i e s from species to species and i s g r e a t e s t i n f i s h , l e s s i n R e p t i l i a and even l e s s i n Aves. Measurements of some p h y s i c a l p r o p e r t i e s of the p a r t i a l l y p u r i f i e d p i t u -i t a r y e x t r a c t i n d i c a t e t h a t 0. tshawytscha p i t u i t a r i e s produce a s i n g l e gonadotropic hormone ( 8 ) . Other s t u d i e s a l s o support t h i s h y p o t h e s i s . The r e -gressed t e s t e s of a number of species of hypophysectomized f i s h could be r e s t o r e d by a d m i n i s t r a t i o n of mammalian LH but mammalian FSH, alone or i n c o n j u n c t i o n w i t h LH, had no e f f e c t ( 2 4 ) . The mammalian LH was capable of e l i c i -t i n g both gametogenic and s t e r o i d o g e n i c a c t i o n s ( 2 0 ). There i s some evidence which i n d i c a t e s t h a t f i s h p i t u -i t a r y e x t r a c t s can e l i c i t both FSH-like and L H - l i k e r e -sponses ( 4 1 ) . A salmon p i t u i t a r y e x t r a c t gave a p o s i t i v e response to the r a t v a g i n a l c o r n i f i c a t i o n t e s t f o r FSH and a l s o to the weaver-finch f e a t h e r r e a c t i o n f o r LH, but the FSH content was very low (2 0 ) . U s i n g the immature female mouse as an assay animal, FSH-like and L H - l i k e components were i d e n t i f i e d i n p i t u i t a r y e x t r a c t s of mature, chum salmon (41). An obvious c a u t i o n r e l a t i n g to species 17. v a r i a b i l i t y must be e x e r c i s e d when a n a l y z i n g the e f f e c t s of p i t u i t a r y e x t r a c t s from one species on the development of the gonads of another s p e c i e s . Summing a l l evidence, Hoar (20) suggests t h a t t e l e -ost p i t u i t a r i e s produce a s i n g l e proteinaceous gonadotro-p i c f a c t o r which i s d i s t i n c t from the FSH or LH produced by tetrapods but more c l o s e l y resembles LH i n i t s a c t i o n . The primary t a r g e t c e l l s on which mammalian hormones exe r t t h e i r gametokinetic e f f e c t i s not c l e a r but i t ap-pears t h a t LH a c t s w i t h i n the t e s t i s by s t i m u l a t i n g the L e y d i g or i n t e r s t i t i a l c e l l s to s y n t h e s i z e androgens, and FSH s t i m u l a t e s spermatogenesis w i t h i n the seminiferous tubules ( 4 3 ) . There i s , however, much u n c e r t a i n t y about which t e s t i c u l a r c e l l s are the t a r g e t of FSH. This hor-mone may act d i r e c t l y on the c e l l s of the seminiferous tubules or act i n d i r e c t l y v i a the S e r t o l i c e l l s ( 1 2 ) . There i s as yet no w i d e l y h e l d hypothesis about which c e l l ( s ) w i t h i n f i s h t e s t i s i s the t a r g e t of f i s h gonadotropic a c t i o n . C y c l i c AMP, the second messenger, i s being i m p l i -cated i n the mechanism by which gonadotropins s t i m u l a t e t e s t i c u l a r development. Because almost a l l of the exper-im e n t a l data r e l a t i n g to t h i s i d e a i s being obtained from work on mammalian systems, r e s u l t s from mammalian 1 8 . s t u d i e s w i l l be d i s c u s s e d i n s p i t e of the p h y l o g e n e t i c remoteness of f i s h and mammals. Con s i d e r i n g the u b i q u i -tous occurrence of c y c l i c AMP i n the r o l e of a second mes-senger, the occurrence of f u n c t i o n a l l y s i m i l a r gonadotro-pic s) i n a l l v e r t e b r a t e s , the con s i d e r a b l e age of the t e s t i s ( 2 9 ) , and the s i m i l a r f u n c t i o n of a l l v e r t e b r a t e t e s t e s - the p r o d u c t i o n of gametes and androgens - some aspects of t e s t i c u l a r adenyl c y c l a s e a c t i v i t y may be com-mon to a l l v e r t e b r a t e s . The adenyl c y c l a s e a c t i v i t y i n homogenates and washed p a r t i c l e , p r e p a r a t i o n s of dog t e s t i s was s t i m u l a t e d by both ovine PSH and ovine LH (34). These r e s u l t s and the knowledge t h a t t e s t i s homogenates c o n t a i n a mixture of c e l l types l e d Murad et a l (34) to speculate t h a t there may be more than one type of adenyl c y c l a s e a c t i v i t y i n the t e s t i s homogenates. S i m i l a r l y , i n c r e a s e d l e v e l s of c y c l i c AMP were r e -corded i n whole r a t t e s t i s p r e p a r a t i o n s which had been exposed to ovine LH, but i n c r e a s e s i n c y c l i c AMP r e s u l t i n g from i n c u b a t i o n w i t h ovine FSH were observed only i f the r a t had been p r e v i o u s l y hypophysectomized ( 9 ) . I s o l a t e d seminiferous tubules from hypophysectomized r a t s contained e l e v a t e d l e v e l s of c y c l i c AMP a f t e r being incubated w i t h FSH but not w i t h LH. F r i t z suggests t h a t the i n c r e a s e d 19 . l e v e l s of c y c l i c AMP r e s u l t i n g from FSH treatment are con-t a i n e d w i t h i n the S e r t o l i c e l l s ( 1 2 ) . Another s i g n i f i c a n t o b s e r v a t i o n i s t h a t c y c l i c AMP can mimic the a b i l i t y of LH to s t i m u l a t e the i n c o r p o r a t i o n of acetate and c h o l e s t e r o l i n t o t e s t o s t e r o n e by t e s t i s ( 3 4 ) . H a l l (17) has proposed a pathway by which c y c l i c AMP might s t i m u l a t e t e s t i c u l a r s t e r o i d o g e n e s i s . The e x i s t e n c e of c y c l i c AMP s t i m u l a t e d p r o t e i n kinases w i t h i n . t e s t i s p r e p a r a t i o n s h.ave been b r i e f l y d i s c u s s e d . A c h a i n of events l i n k i n g hormonal s t i m u l a t i o n of the t e s -t i s and c y c l i c AMP a c t i v a t i o n of these kin a s e s to phosphoryl-"ate , h i s t o n e s and protamines has not been found but the p o s s i b i l i t y of such a pathway has been suggested ( 2 8 ) . An a n a l y s i s of the i n c r e a s e i n t e s t i c u l a r l e v e l s of adenyl c y c l a s e d u r i n g the maturation of r a t s leads to the assumption t h a t t h i s enzyme i s present i n mature sperma-to c y t e s and spermatids ( 1 2 ) . I n summary, there i s suggestive evidence f o r the presence of adenyl c y c l a s e a c t i v i t y i n L e y d i g c e l l s , Ser-t o l i c e l l s , spermatocytes and spermatids of mammals. Fur-thermore i t ' i s p o s s i b l e t h a t LH s t i m u l a t e s adenyl c y c l a s e i n L e y d i g c e l l s and FSH s t i m u l a t e s t h i s enzyme i n S e r t o l i c e l l s . There i s as yet no hypothesis about what hormone(s), i f any, might s t i m u l a t e an adenyl cyclase a c t i v i t y i n 20. spermatocytes and spermatids ( 1 2 ) . As p r e v i o u s l y s t a t e d , experiments r e l a t i n g to adenyl c y c l a s e a c i v i t y i n f i s h t e s t i s are s c a r c e . Two r e p o r t s a l r e a d y mentioned are the s t i m u l a t i o n of adenyl c y c l a s e a c t i v i t y i n t e s t i s s l i c e s of 0 . tshawytscha by 0 . tshawy-t s c h a p i t u i t a r y e x t r a c t s ( 3 3 ) , and the presence of a c y c l i c AMP s t i m u l a t e d protamine and h i s t o n e kinase a c t i v i t y i n Salmo g a i r d n e r i i (rainbow t r o u t ) t e s t i s ( 2 2 ) . 21 . I I . Adenyl Cyclase Since the d i s c o v e r y i n 1956 of the heat s t a b l e f a c t o r , c y c l i c AMP, t h i s n u c l e o t i d e has been detected i n b a c t e r i a , yeasts and i n almost every metazoan t i s s u e examined ( 3 7 ) . Adenyl c y c l a s e , the enzyme which c a t a l y z e d the s y n t h e s i s of c y c l i c AMP, has not been assayed as e x t e n s i v e l y as c y c l i c AMP, but t h i s enzyme has been detected i n b a c t e r i a and i n every metazoan s p e c i e s examined ( 3 7 ) . Despite the r e l a t i v e -l y s h o r t time span of c y c l i c n u c l e o t i d e r e s e a r c h , there i s a v a s t amount of l i t e r a t u r e on the su b j e c t i n c l u d i n g many ex t e n s i v e reviews (1,2,16,26,37,40). The f o l l o w i n g d i s c u s -s i o n of adenyl c y c l a s e i s t h e r e f o r e b r i e f and o r i e n t e d to. the problems of t h i s T h e s i s . The r e a c t i o n which adenyl c y c l a s e c a t a l y z e s i s : ATP ^ v c y c l i c AMP + P P i The enzyme r e q u i r e s a d i v a l e n t c a t i o n , u s u a l l y Mg or Mn , f o r i t s a c t i v i t y and works best i n the pH range of 7.0 to 8.0 ( 3 3 ) . The true s u b s t r a t e f o r the enzyme i s probably 2+ the ATP-Mg complex (10,37). Under standard r e a c t i o n con-d i t i o n s ( r e a c t a n t s and products at 1 M) the r e a c t i o n i s r e v e r s i b l e and f a v o r s ATP for m a t i o n , but at p h y s i o l o g i c a l c o n c e n t r a t i o n s of ATP, c y c l i c AMP^ and PPj^ ATP f o r m a t i o n i s not l i k e l y ( 3 7 ) . 22. C y c l i c - 3 ' , 5 ' - A M P has an enthalpy of h y d r o l y s i s of -H.1 k c a l per mole and Greengard s t a t e d i n 1971 t h a t t h i s was "the h i g h e s t enthalpy of h y d r o l y s i s of any of the c y c l i c n u c l e o t i d e s i n v e s t i g a t e d to date."(15) No 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 has been attached to the h i g h energy of h y d r o l -y s i s of the 3'-ester bond of c y c l i c A M P . There are many methods f o r a s s a y i n g adenyl c y c l a s e a c t i v i t y and t h i s s u b j e c t has been d e a l t w i t h i n the 1972 p u b l i c a t i o n "New Assay Methods f o r C y c l i c N u c l e o t i d e s . " ( 1 6 ) I n the experiments d e s c r i b e d i n t h i s Thesis the a c t i v i t y i n salmon t e s t i s homogenates was determined by measuring the pr o d u c t i o n of ^ H - c y c l i c A M P from ^ H - A T P . The c y c l i c A M P was separated from other r a d i o a c t i v e l y l a b e l e d n u c l e o t i d e s i n the assay mixture by paper chromatography i n the et h a n o l ; 1 M ammonium acetate (pH 7.5) (7:3,by volume) s o l v e n t sys-tem (10,33). This method of i s o l a t i n g r a d i o a c t i v e l y l a b e l e d c y c l i c A M P from the adenyl c y c l a s e assay mixture has been compared w i t h the s e p a r a t i o n achieved by chromatog-raphy on Dowex 50-H +, f o l l o w e d by treatment of the e l u a t e w i t h ZnSO^j. and Ba(0H) 2 ( 1 0 ) . The r e s u l t s were e s s e n t i a l l y i d e n t i c a l even f o r s m a l l amounts of r a d i o a c t i v i t y ( l e s s ..' than 5000 dpm of 1 ^ C - c y c l i c A M P ) . Drummond and Duncan ( 1 0 ) , however, p r e f e r e d the paper chromatography method because A T P degradation products can be monitored and because a 23. l a r g e r p o r t i o n of the e n t i r e y i e l d of r a d i o a c t i v e l y l a -beled c y c l i c AMP i s a c t u a l l y q u a n t i t a t e d w i t h t h i s method. In a d d i t i o n to the d i f f i c u l t i e s i n v o l v e d i n separa-t i n g s m a ll q u a n t i t i e s of ^ H - c y c l i c AMP from l a r g e q u a n t i -t i e s of other t r i t i u m l a b e l e d n u c l e o t i d e s , the assay of adenyl c y c l a s e i n / t i s s u e p r e p a r a t i o n s i s complicated by the presence of ATPases and c y c l i c AMP phosphodiesterases. There i s much evidence which i n d i c a t e s t h a t adenyl c y c l a s e i s an enzyme of the plasma membrane, and as such, i t has been very d i f f i c u l t to s o l u b i l i z e . The a c t i v i t y , t h e r e -f o r e , i s measured i n t i s s u e p r e p a r a t i o n s such as crude homogenates ( 3 3 ) , washed p a r t i c l e s ( 1 0 ) , and plasma mem-branes ( 4 0). The h y d r o l y s i s of ATP by ATPases can be counteracted by a s s a y i n g the adenyl c y c l a s e a c t i v i t y i n the presence of ATP co n c e n t r a t i o n s f a r i n excess of the K m f o r ATPases (37). A l t e r n a t i v e l y , pyruvate kinase and phosphoenolpy-ruvate or c r e a t i n e phosphokinase and phosphocreatine can be added to the assay to regenerate ATP from ADP and phos-phate. We chose t o i n c l u d e the pyruvate kinase - phospho-enolpyruvate r e g e n e r a t i n g system, and problems a s s o c i a t e d w i t h t h i s system are d e a l t w i t h i n the D i s c u s s i o n . Yet another s o l u t i o n to the problem of ATP h y d r o l y -s i s i s to s u b s t i t u t e the ATP analogue, a d e n y l y l i m i d o d i -phosphate (AMP-PNP) f o r the ATP s u b s t r a t e . AMP-PNP i s a 'substrate f o r the adenyl c y c l a s e of plasma membranes but not f o r plasma membrane ATPases or m i t o c h o n d r i a l ATPases w i t h i n r a t l i v e r (40,46). I n the analogue a NH grouping r e p l a c e s the t e r m i n a l bridge oxygen of ATP (46). The imidodiphosphate (P-N-P) l i n k a g e has been found as p a r t of a n a t u r a l product i n a l g a l '•polyphosphates",(27). L a r s e n et a l (27) analyzed the c r y s t a l and molecular s t r u c t u r e of sodium imidodiphosphate i n r e l a t i o n to t h a t of sodium pyropnosphate and found a s t r i k i n g s i m i l a r i t y between the two molecules. I n general the s t r u c t u r e of P-N-P has a g r e a t e r s i m i l a r i t y to P-O-P than does the s t r u c t u r e of P-C-P (27). This might e x p l a i n the a b i l i t y of AMP-PNP to a c t as a su b s t r a t e f o r the adenyl c y c l a s e of r a t l i v e r plasma membranes and the i n a b i l i t y of adenyl-y l methylenediphosphate (AMP-PGP) to do so ( 4 6 ) . There are a number of other b i o l o g i c a l r e a c t i o n s i n which AMP-PNP can mimic ATP f u n c t i o n s (27»46). I n the present study, we have found t h a t AMP-PNP i s a su b s t r a t e f o r salmon t e s -t i s adenyl c y c l a s e . Yount et a l (46) have analyzed some p r o p e r t i e s of AMP-PNP. As the tetrasodium s a l t i t i s s t a b l e f o r months at -20°, and f o r at l e a s t 16 hours i n s o l u t i o n at 25° at n e u t r a l pH. At pH 8 . 5 AMP-PNP binds C a 2 + , Mg 2 +, and Mn 2 + more t i g h t l y than does ATP. The t e r m i n a l phosphate pro-ton of AMP-PNP t i t r a t e s a t a pK a of 7.7"-± 0.1 and the t e r m i n a l phosphate proton of ATP t i t r a t e s a t a pK a of 7.1. We chose to sy n t h e s i z e AMP-PNP from trihutylammonium imidodiphosphate and AMP by a procedure u s i n g 1,1'-carbon-:.y I d i i m i d a z o l e d e s c r i b e d by Hoard and Ott (21) f o r the s y n t h e s i s of d e o x y o l i g o n u c l e o t i d e t r i p h o s p h a t e s . Two other methods of s y n t h e s i z i n g AMP-PNP have been d e s c r i b e d . I t can be sy n t h e s i z e d by the r e a c t i o n of imidodiphosphate w i t h P -adenosine P -dip h e n y l pyrophosphate ( 4 6 ) . R o d b e l l et a l (40) have d e s c r i b e d a procedure i n which AMP-PNP s y n t h e s i s from ATP and PNP i s c a t l a y z e d by amino a c y l t r a n s f e r RNA synthetases. C y c l i c - 3 ' , 5'-AMP phosphodiesterase a c t i v i t y which converts c y c l i c - 3 5 ' - A M P to 5'-AMP has been detected i n salmon t e s t i s homogenates, but has not been c h a r a c t e r i z e d ( 3 3 ) . I n t i s s u e s from other species (mostly mammalian) two d i s t i n c t and separable c y c l i c AMP phosphodiesterases have been c h a r a c t e r i z e d ( 1 ) . The main d i f f e r e n c e between the two enzymes i s t h e i r K m f o r c y c l i c AMP. One has a K m i n the range of 1 to 5 jM and i s considered the h i g h a f -f i n i t y phosphodiesterase; the other o f t e n has a K m of about 100 ^ uM. I n g e n e r a l , the h i g h a f f i n i t y enzyme i s l o c a t e d i n the p a r t i c u l a t e or membrane f r a c t i o n s and the low a f f i n i t y enzyme i s found i n the s o l u b l e f r a c t i o n ( 1 ) . The r o l e of c y c l i c AMP phosphodiesterases i n c o n t r o l -l i n g c e l l u l a r l e v e l s of c y c l i c AMP i s u n c e r t a i n . Because many phosphodiesterase assays have not taken i n t o account the p o s s i b l e e x i s t e n c e of two, or more, c y c l i c n u c l e o t i d e phosphodiesterases, Appleman et a l ( 1 ) f e e l t h a t i n v e s -t i g a t i o n of t h i s problem has been hindered. So f a r there i s l i t t l e evidence f o r a c o n t r o l of c y c l i c AMP l e v e l s by d i r e c t hormonal s t i m u l a t i o n or i n h i b i t i o n of these en-zymes. I n s u l i n , however, does seem to r e g u l a t e phospho-d i e s t e r a s e a c t i v i t y ( 1 ) . I n order to assay adenyl c y c l a s e a c t i v i t y i n salmon t e s t i s homogenates the c y c l i c AMP phosphodiesterase(s) had to be i n h i b i t e d . Theophylline and s a t u r a t i n g amounts o f ' c o l d " c y c l i c AMP (2rmM) were added f o r t h i s purpose. Theophylline alone allowed the accumulation of up t o 30$ as much l a b e l e d c y c l i c AMP as was accumulated i n the pres-ence of e i t h e r 2 mM "cold" c y c l i c AMP or 2 mM "cold" c y c l i c AMP p l u s 7 mM t h e o p h y l l i n e ( 3 3 ) . Theophylline appears to be a competitive i n h i b i t o r of both the high K m and low K m c y c l i c AMP phosphodiester-ases ( 1 ) . I t i s one of the most w i d e l y used phosphodi-esterase i n h i b i t o r s , but there are many others ( 1 , 4 2 ) . 27. Recently a number of c y c l i c AMP analogues have been syn-t h e s i z e d which are much more potent i n h i b i t o r s . Simon et a l (42) have reviewed these, and among the most potent are 8-bromo-cyclic AMP, 8 - m e t h y l t h i o - c y c l i c AMP, 2*-0-a c e t y l - c y c l i c AMP p l u s a number of other 8- and 2 1- d e r i v -atives., of c y c l i c AMP. A d e r i v a t i v e of 4-benzyl-2-imida-z o l i d i n o n e was 1 0 0 0 times more potent than t h e o p h y l l i n e i n i n h i b i t i n g c y c l i c AMP phosphodiesterase a c t i v i t y i n r a t e r y t h r o c y t e s ( 1 ) . I n a d d i t i o n , c y c l i c IMP and the n a t u r a l l y o c c u r r i n g n u c l e o t i d e , c y c l i c GMP, have i n h i b i -ted some c y c l i c AMP phosphodiesterase p r e p a r a t i o n s ( 4 2). There are many problems a s s o c i a t e d w i t h the i n c l u s i o n of theophylline i n adenyl c y c l a s e assays. Appleman et a l ( 1 ) have l i s t e d a number of phenomena which t h e o p h y l l i n e may a f f e c t i . e . " c a l c i u m e f f l u x , ATP d e p l e t i o n , p r o t e i n s y n t h e t i c e f f e c t s , oxygen consumption and heart r a t e and endocrine s e c r e t i o n s " . I t i s obvious, t h e r e f o r e , t h a t there are many i n h i b i t o r s , other than t h e o p h y l l i n e , which should be t e s t e d f o r t h e i r potency as i n h i b i t o r s of s a l -mon t e s t i s c y c l i c AMP ph o s p h o d i e s t e r a s e ( s ) . The mechanism by which adenyl c y c l a s e l i n k s hormonal i n f o r m a t i o n to c e l l u l a r responses i s a problem of major importance. Two recent review a r t i c l e s have i n v e s t i g a t e d the experimental evidence and hypotheses r e l a t e d to the 28. mechanism of hormonal s t i m u l a t i o n of adenyl cy c l a s e (37) and the means by which c y c l i c AMP r e g u l a t e s i n t r a c e l l u l a r f u n c t i o n s (26). Even though there i s very l i t t l e e x p e r i -mental data on hormonal c o n t r o l and c e l l u l a r f u n c t i o n s of t e s t i c u l a r adenyl cy c l a s e i n salmonids there are major aspects of the adenyl cy c l a s e system which appear to t r a n -scend species and t i s s u e d i f f e r e n c e s . Adenyl c y c l a s e i s e s s e n t i a l l y a multi-component en-zyme which i s , most l i k e l y , l o c a t e d i n the plasma membrane. The c a t a l y t i c sub-unit which converts ATP to c y c l i c AMP i s probably a p r o t e i n or l i p o p r o t e i n l o c a t e d on the i n n e r surface of the plasmd membrane. Many p r o p e r t i e s of t h i s component are s i m i l a r i n a wide v a r i e t y of t i s s u e s and sp e c i e s . The hormone r e c e p t o r or d i s c r i m i n a t o r sub-unit i s probably a l s o a p r o t e i n , g l y c o p r o t e i n , g l y c o l i p i d or l i p o p r o t e i n which i s an i n t e g r a l component of the plasma membrane, but t h i s component i s exposed on the outer sur-face of the c e l l . I t i s proposed t h a t f o r each type of hormone there i s a s p e c i f i c type of r e c e p t o r . A que s t i o n which i s s t i l l unresolved i s "what i s the r a t i o of hor-mone re c e p t o r s to c a t a l y t i c s u b - u n i t s ? " This r a t i o w i l l probably vary i n d i f f e r e n t c e l l types. Some c e l l s can respond to more than one k i n d of hormone and i t i s b e l i e v e d t h a t a s i n g l e c a t a l y t i c sub-unit can be a s s o c i a t e d w i t h 2 9 . a number of d i f f e r e n t hormone r e c e p t o r s . ( 3 7 ) . I n g e n e r a l , the i n t e r a c t i o n of a hormone w i t h an ap-p r o p r i a t e r e c e p t o r r e s u l t s i n an a l t e r a t i o n of the s t r u c -t u r a l components of the system which leads to an in c r e a s e i n the c a t a l y t i c a c t i v i t y of adenyl c y c l a s e . Some unre-solved questions are: "what i s the a c t u a l p h y s i c a l change which r e s u l t s i n in c r e a s e d c a t a l y t i c a c t i v i t y ? ; what are the k i n e t i c s of the response?; and how many molecules of hormone e l i c i t a maximal response from a s i n g l e c a t a l y t i c s u b - u n i t ? " There are many hypotheses about the nature of the c o u p l i n g between the hormone r e c e p t o r and the ca t a -l y t i c sub-unit; P e r k i n s (37) has discussed a number of models. The e x i s t e n c e of a t h i r d component of the adenyl c y c l a s e system, a t r a n s d u c i n g s u b - u n i t , has been proposed as the means of r e l a y i n g i n f o r m a t i o n from the hormone re c e p t o r to the c a t a l y t i c s ub-unit. The i n v e s t i g a t i o n of adenyl c y c l a s e has been hampered by experimental d i f f i c u l t i e s . The responsiveness of adenyl c y c l a s e to hormones i s a very l a b i l e p r o p e r t y and u n t i l r e c e n t l y no component of the adenyl c y c l a s e system had been p u r i f i e d (37) . U n t i l a l l the components of adenyl c y c l a s e are p u r i f i e d and r e c o n s t i t u t e d i n t o a f u n c t i o n a l u n i t , the exact mechanism of adenyl c y c l a s e a c t i v a t i o n w i l l probably remain a mystery. 30. The second major aspect of adenyl c y c l a s e f u n c t i o n i s the i n t r a c e l l u l a r r o l e of c y c l i c AMP. So f a r the a c t i -v a t i o n of p r o t e i n kinase i s t h e o n l y w e l l documented mech-anism by which c y c l i c AMP a c t s i n e u k a r y o t i c c e l l s . Mul-t i p l e forms of c y c l i c AMP dependent p r o t e i n k i n a s e s have been found but f u n c t i o n a l l y there e x i s t s only a s i n g l e c y c l i c AMP dependent p r o t e i n kinase ( 2 6 ) . C y c l i c AMP dependent p r o t e i n kinase c o n s i s t s of 2 s u b - u n i t s , a r e g u l a t o r y (R) and c a t a l y t i c (C) s u b - u n i t . The holoenzyme i s i n a c t i v e but b i n d i n g of c y c l i c AMP to R f r e e s C from R and thus a c t i v a t e s C. The enzyme i s probably l o c a t e d i n a l l s u b - c e l l u l a r f r a c t i o n s w i t h the p o s s i b l e e x c e p t i o n of the m i t o c h o n d r i a l f r a c t i o n ( 2 6 ) . The r e a c t i o n which the c y c l i c AMP dependent p r o t e i n kinase c a t a l y z e s i s the -phosphorylation of c e r t a i n p r o t e i n s main-l y on s e r i n e r e s i d u e s but a l s o on some threonine r e s i d u e s . This enzymic a c t i v i t y r e p r e s e n t s only a f r a c t i o n of the p r o t e i n p h o s p h o r y l a t i o n s o c c u r r i n g w i t h i n a c e l l and i s d i s t i n c t from n o n - c y c l i c AMP dependent p r o t e i n phospho-r y l a t i o n 26) . The s p e c i f i c i t y of a c t i o n of c y c l i c AMP i s r e g u l a t e d by the nature of the s u b s t r a t e s which are a v a i l a b l e to the p r o t e i n kinase i n a given c e l l . This model, there-f o r e , i m p l i e s t h a t a s i n g l e p r o t e i n kinase must be able to 31 . recognize a wide v a r i e t y of p r o t e i n s u b s t r a t e s but not j u s t any and a l l p r o t e i n s . I t has been suggested t h a t the a c t i v e s i t e of the p r o t e i n kinase recognizes a s p e c i f -i c , , three dimensional s t r u c t u r e (26). The o v e r a l l p i c t u r e which emerges from adenyl c y c l a s e s t r u c t u r e and f u n c t i o n i s the f o l l o w i n g . The adenyl cy-clase c a t a l y t i c component - cyclic-3',5'-AMP - p r o t e i n k i n a s e system i s u n i v e r s a l l y present i n a l l animal species and t i s s u e s . The d i v e r s i t y a r i s e s from the v a r i a t i o n s i n the hromones and hormone r e c e p t o r s and from the v a r i -a t i o n s i n the p r o t e i n kinase s u b s t r a t e s (26). W i t h i n the salmon t e s t i s the gonadotropic s t i m u l a -t i o n of adenyl c y c l a s e has been demonstrated (33) but i t remains to be determined which c e l l t ype(s) contained the adenyl c y c l a s e t h a t was responding to the hormone(s). I t has a l r e a d y been mentioned t h a t the t a r g e t c e l l s of s a l -mon g o n a d o t r o p i c s ) are unknown. The f u n c t i o n of c y c l i c AMP dependent h i s t o n e and protamine kinases w i t h i n the salmonid t e s t i s was discussed e a r l i e r i n the I n t r o d u c t i o n . No c o r r e l a t i o n between hormonal s t i m u l a t i o n of developing germ c e l l s and a c t i v a t i o n of these p r o t e i n kinases has yet been found but the p o s s i b i l i t y of t h i s type of a l i n k does e x i s t . 32. MATERIALS AND METHODS I . M a t e r i a l s U n i f o r m l y l a b e l e d adenosine-^H-5'-monophosphate, diammonium s a l t (12.6 Ciper mmole) and u n i f o r m l y l a b e l e d a d e n o s i n e - 5 ' - t r i p h o s p h a t e , tetrasodium s a l t (7.93 Ct per mmole) were purchased from New England Nuclear. Aden-osine 5'-triphosphate ( 8 - % ) , l i t h i u m s a l t (18.3 Ci per mmole) was obtained from Schwarz/Mann. The u n i f o r m l y l a b e l e d adenosine-^H-5'-triphosphate, tetrasodium s a l t , and the adenosine 5'-triphosphate (8-^H) l i t h i u m s a l t were obtained i n 50% et h a n o l ; these n u c l e o t i d e s were evap-orated . to dryness at room temperature under a stream of n i t r o g e n . The r e s i d u e s were d i s s o l v e d i n s o l u t i o n s of "cold" ATP to give a f i n a l ATP c o n c e n t r a t i o n of 1.7 mM and a s p e c i f i c a c t i v i t y of 16 uCi'to 53 }xOi per jumole. Omnifluor was purchased from New England Nulcear and NCS was obtained from Amersham/Searle. S c i n t a n a l y z e d toluene was purchased from F i s h e r . Adenosine, cyclic-3',5 1-AMP, 5'-AMP, ADP, and ATP were purchased from P-L Bi o c h e m i c a l s . Pyruvate kinase and t r i s o d i u m 2-phosphoenolpyruvic a c i d were obtained from Calbiochem. C r y s t a l l i z e d , l y o p h i l i z e d bovine serum albumin from Sigma was used f o r de t e r m i n a t i o n of standard curves i n p r o t e i n assays. The detergents and t h e i r s u p p l i e r s were L u b r o l PX, a fattyyfethylene oxide condensate w i t h an average molecu-l a r weight of 600 d a l t o n s , from Canadian I n d u s t r i e s L t d . , Nonidet P40 from S h e l l Chemicals, T r i t o n X-100 or a l k y l phenoxy polyethoxy ethanol from Rohm and Haas, and Tween 80 or polyoxyethylene (20) s o r b i t a n monooleate from F i s h e r . The DEAE c e l l u l o s e was a S c h l e i c h e r and S c h u e l l prod-uct and DOWEX 50 W-X2 was purchased from Bio-Rad Labora-t o r i e s , . T r ibutylamine from Eastman was r e d i s t i l l e d un-der vacuum and s t o r e d i n a dark b o t t l e at room temperature. Reagent grade p y r i d i n e was r e f l u x e d over c a l c i u m hydride and then s t o r e d over c a l c i u m h y d r i d e . Dimethylformamide (DMP) was d r i e d over molecular si e v e f o r at l e a s t 10 days p r i o r to use. A l l other chemicals were of reagent grade and were purchased commercially. Oncorhynchus tshawytscha (Chinook salmon) were cap-tured d u r i n g t h e i r spawning m i g r a t i o n on the F r a s e r R i v e r , B r i t i s h Columbia. The t e s t e s were e x c i s e d , f r o z e n on dry C02» a n d then t r a n s p o r t e d to the l a b o r a t o r y where they were s t o r e d at -80°. The t e s t e s from 2 salmon were em-ployed i n these s t u d i e s . The f i r s t salmon was c o l l e c t e d June 2, 1970; the t e s t e s weighed 9 9 . 5 g and represented 34. 2.2% of the body weight. The second f i s h was c o l l e c t e d June 29, 1970; the t e s t e s weighed 125 g and represented 2.5% of the body weight. The t e s t e s were p a r t i a l l y thawed, cut i n t o s m all p o r t i o n s of 1 to 2 g and then r e f r o z e n at -80° f o r l a t e r use. A l l experiments were completed w i t h -i n 25 months of the c o l l e c t i o n of the t i s s u e samples. I I . Methods A Synthesis of T r i t i u m Labeled A d e n y l y l Imidodiphosphate F i r s t l y , sodium imidodiphosphate (PNP) was prepared from d i p h e n y l chlorophosphate a c c o r d i n g to the procedure of Yount e t a l ( 4 6 ) . M o l e c u l a r weight was determined by a n a l y s i s of a c i d l a b i l e phosphate. P u r i t y of the PNP was checked by ascending chromatography i n 2 s o l v e n t systems. System A was i s o p r o p a n o l : DMF: methyl e t h y l ketone; H2O: concentrated ammonia (20:20:20:39:1, by volume).and sys-tem.B was n-propanol: concentrated ammonia: R^O (6:3:1, by volume). Compounds were detected w i t h a phosphate spray reagent ( 2 4 ) , a f t e r a c i d h y d r o l y s i s of the chroma-togram (46). T r i t i u m l a b e l e d a d e n y l y l imidodiphosphate (%-AMP-PNP) (IV) was s y n t h e s i z e d a c c o r d i n g to a s l i g h t l y modi-f i e d v e r s i o n of the general procedure of Hoard and Ott (21) f o r the s y n t h e s i s of deox y o l i g o n u o l e o t i d e t r i p h o s -phates. The o v e r a l l r e a c t i o n s are summarized i n equations 1 and 2. According to Hoard and Ott ( 2 1 ) , s i d e products of the r e a c t i o n would be adenosine-5' phosphoramidate (ADP-NHg), 5'-AMP, and the symmetrical pyrophosphate, diadenosine-5'-diphosphate. HOP OCH;, I I OH ori I I I 0" ,0" i H i I I I + "OPNPO*(BU 3RH) 4 DNAF 0 0 ?-Ho- ?-CX> y = \ > OPNPOiR'OCH, + HN NH 8 feJ ^ ti n il O 0 " OH OH IV The tributylammonium s a l t of the imidodiphosphate (V) was prepared by the method of Yount et a l (46) ex-cept t h a t water was removed by coevaporation w i t h p y r i -dine i n s t e a d of chloroform and the s a l t was then d i s s o l v e d i n DMP and used immediately. The i m i d a z o l i d a t e ( I I I ) •2. prepared from ^H-AMP (16 p.C/umole) and 1 ,1 • - c a r b o n y l d i -i m i d a z o l e ( I I ) was formed by the procedure of Hoard and 36 Ott ( 2 1 ) . A s o l u t i o n c o n t a i n i n g about 0.5 mmole t r i b u t y l ammonium imidodiphosphate i n 0.5 ml DMF was added w i t h vigorous mixing to 0.05 mmole of the t r i t i u m l a b e l e d i m i d azolidate„» This r e a c t i o n mixture was shaken at room temperature f o r 20 hours. Next, the p r e c i p i t a t e was r e -moved and an equal volume of methanol, f o l l o w e d by two volumes of water were added to the s o l u t i o n . This e n t i r e s o l u t i o n was a p p l i e d to a DEAE c e l l u l o s e column ( 1 . 2 x 4 0 cm) and was chromatographed at room temperature w i t h a l i n e a r g r a d i e n t of ammonium bicarbonate (0 to 0.2 M). The e l u t i o n of n u c l e o t i d e was monitored by f o l l o w i n g the absorbance at 260 77m of the e l u a t e . T r i t i u m l a b e l e d aden y l imidodiphosphate was i d e n t i f i e d by chromatography i n the f o l l o w i n g three systems: System A, System B, and System G - i s o b u t y r i c a c i d : 1 M ammonium hydroxide (5:3, by volume). The peak c o n t a i n i n g H> AMP-PNP was d r i e d at room temperature on a r o t a r y evaporator; the n u c l e o t i d e was d i l u t e d to 1.7 mM and then was sto r e d a t -20°. B Adenyl Cyclase Assay The adenyl c y c l a s e a c t i v i t y i n the t e s t i s of 0. tshawytscha was measured i n t i s s u e homogenates which were prepared by the f o l l o w i n g method. One gram of f r o z e n 37. t e s t i s was minced, then placed i n 5 ml of i c e c o l d 0.01 M Tr i s - H O l (pH 7.5) and homogenized w i t h 10 slow passes of a l o o s e - f i t t i n g , t e f l o n p e s t l e of a P o t t e r - E l v e h j e m type homogenize!*. Urquhart (45) has i n d i c a t e d t h a t t h i s type of gentle homogenization i s s u f f i c i e n t to rupture spermatogonia and spermatocytes but does not a f f e c t the sm a l l e r c e l l s , the.spermatids and spermatozoa. A stage 2 salmon t e s t i s c o n t a i n s mainly primary and secondary spermatocytes and very few spermatids (45); t h e r e f o r e these t i s s u e p r e p a r a t i o n s should have contained very few whole c e l l s . The homogenate was f i l t e r e d through 4 l a y e r s of c heesecloth and the f i l t r a t e was assayed w i t h i n 15 min of p r e p a r a t i o n . During t h i s e n t i r e procedure the homog-enate was kept on i c e . The enzyme a c t i v i t y of the f i l t e r e d homogenate was assayed a c c o r d i n g to a s l i g h t l y m o d ified v e r s i o n of the procedure d e s c r i b e d by Menon and Smith ( 3 3 ) . The assay system contained 35 mM T r i s - H C l (pH 7.5), 14 mM MgSO^, 2 mM c o l d cyclic-3',5'-AMP, 7 mM t h e o p h y l l i n e , 7 mM NaP, 7 mM KC1, 18 mM phosphoenolpyruvate, 3 u n i t s pyruvate k i -nase per a s s a y , 0.4 mM ^H-ATP (20 to 53 yuC/jumole) and homog-enate. ( u s u a l l y 2.5 t o 6.0 yug p r o t e i n per pi of ass a y ) . I n some experiments 0.4 mM ^H-AMP-PNP (16 jaG/imole) was s u b s t i t u t e d f o r 0.4 mM ^K-ATP. I n these cases the assay system remained the same except t h a t the ATP regen-e r a t i n g system, KC1 - phosphoenolpyruvate and pyruvate k i n a s e , was omitted. The t o t a l assay volume was e i t h e r 170 or 100 p.1 and the assay temperature was e i t h e r 37° or 30°. A f t e r an' a p p r o p r i a t e r e a c t i o n time the r e a c t i o n tube was heated i n a 'temp bloc k ' heater at 100° f o r 3 min. The tube was c h i l l e d on i c e and c e n t r i f u g e d at 7500 rpm f o r 10 min i n a S o r v a l l RC-2 c e n t r i f u g e f i t t e d w i t h an SS 34 r o t o r . The supernatant was chromatographed on e i t h e r Whatman 40 SCP or Whatman 3 MM paper by descending chromatography i n the 1 M ammonium acetate (pH 7.5): ethanol (3:7, by volume) system f o r 20 hours at room temperature. The volume of supernatant which was chromatographed was 100 u l or 60 pi depending on whether the t o t a l assay volume was 170 jul or 100 pi, r e s p e c t i v e l y . A spot of 0.05 umole of cyclic-3',5'-AMP was r o u t i n e l y co-chromatographed and 0.05 umole each of ATP, ADP, 5'-AMP, and adenosine were p e r i o d i c a l l y co-chromatographed as standards. The chromatogram, a f t e r d r y i n g , was viewed i n a Chromato Vue under short wave l e n g t h l i g h t (200-280 7^ m) and the u l t r a -v i o l e t absorbing regions were marked. The spot co-chromatographing w i t h the c y c l i c AMP was cut out and placed i n a s c i n t i l l a t i o n v i a l c o n t a i n i n g 20 ml of 39. s c i n t i l l a t i o n f l u i d composed of NCS, water, s c i n t a n a l y z e d to l u e n e , and onmifluor (100 ml: 16 ml:38X)0 ml: 15.2 g ) . The paper was soaked i n the s c i n t i l l a t i o n f l u i d f o r 24 hours before the r a d i o a c t i v i t y was determined i n a Nuclear-Chicago, Mark I l i q u i d s c i n t i l l a t i o n counter. C o n t r o l s f o r the assay were prepared by s u b s t i t u t i n g homogenate which had been b o i l e d f o r 3 min f o r the f r e s h homogenate. The p r o t e i n content of t i s s u e p r e p a r a t i o n s was determined by the b i u r e t method (13) or by the method of Lowry et a l ( 3 1 ) . The f i n a l adenyl c y c l a s e a c t i v i t y was expressed as the p r o d u c t i o n of c y c l i c AMP/min/mg p r o t e i n . 40. RESULTS I . Synthesis of T r i t i u m Labeled A d e n y l y l Imidodiphosphate A M o l e c u l a r Weight and P u r i t y of Sodium Imidodiphosphate The formula weight of the tetrasodium imidodiphosphate (PNP) as determined by i n o r g a n i c ( a c i d l a b i l e ) phosphate was 337, corresponding to tetrasodium imidodiphosphate* t e t r a h y d r a t e . Our PNP was chromatographed beside a sample of Na^PNP'10 H 20 k i n d l y provided by Dr. Ralph Yount. Both p r e p a r a t i o n s had the same m o b i l i t y (R^) i n s o l v e n t systems A and B (Table 1). A s m a l l amount of i n o r g a n i c phosphate was present i n our PNP sample. The m o b i l i t i e s recorded f o r PNP and P^ i n s o l v e n t A compare f a v o r a b l y w i t h those g i v e n by Yount et a l (46.) (see Table 2 of t h i s T h e s i s ) ; the m o b i l i t i e s i n s o l v e n t B, however, are much d i f f e r e n t . B P u r i f i c a t i o n of T r i t i u m Labeled A d e n y l y l Imidodiphosphate The procedure f o r s y n t h e s i s of 3 H - A M P - P N P has been o u t l i n e d under Methods. The r e s u l t s of chromatographic p u r i f i c a t i o n of the ^H-AMP-PNP r e a c t i o n mixture on a DEAE c e l l u l o s e column are shown i n F i g u r e 1. The adenosine content of peak V as determined from the O.D. at 2607^m and an absorbancy index of 1.54x10^ was found to be 11.9 41 . Table 1. C h a r a c t e r i z a t i o n of A d e n y l y l Imidodiphosphate by Paper Chromatography. Compound A B C Peak V ATP ADP PNP (our sample) PNP (Dr. Yount) 0.24 (0.51) 0.31 0.35 0.18 (0.30) 0.18 0.30 0.13 (0.34) 0.16 0.20 0.07 ( 0 . 1 5 ) 0.07 0 . 1 5 0.41 (0.49) 0.37 0.50 Peak V from .the DEAE c e l l u l o s e chromatographic p u r i -f i c a t i o n of the ^ H-AMP-PNP r e a c t i o n mixture was compared w i t h ATP and ADP by descending chromatography i n 3 s o l -vent systems - A, B, and C on Whatman 40 SCP paper. See Methods f o r a d e s c r i p t i o n of the s o l v e n t s . Our PNP sam-ple was compared w i t h a PNP p r e p a r a t i o n provided by Dr. Yount and w i t h i n o r g a n i c phosphate (P-j_) by ascending chro-matography i n s o l v e n t s A and B on Whatman 31 ET paper. Bracketed numbers represent the m o b i l i t i e s of minor com-ponents present i n peak V and our PNP sample. 42. Table 2. C h a r a c t e r i z a t i o n of A d e n y l y l Imidodiphosphate by Paper Chromatography.' Compound R f A B AMP-PNP 0.39 0.34 ATP 0.50 0.37 ADP 0.54 0.42 ADP-NH2 0.69 0.55 PNP 0.20 0.29 P i 0.30, (two 0.45 spots) 0.35 Ascending chromatography w i t h Whatman No. 31 ET paper was used w i t h s o l v e n t systems A and B. (See Methods of t h i s Thesis f o r a d e s c r i p t i o n of the s o l v e n t s . ) 3. This t a b l e was taken from i n f o r m a t i o n given i n Table I of the f o l l o w i n g paper: Yount, R.G., Babcock, D., B a l l a n t y n e , W., and O j a l a , D. 1971. B i o c h e m i s t r y , 10, 2484-2489. 43. Figure 1 . Chromatographic P u r i f i c a t i o n of -'H-AMP-PNP on DEAE C e l l u l o s e . 9 -• 8 -7 -6 -F r a c t i o n Number The r e a c t i o n product of AMP-PNP s y n t h e s i s (see Methods) was a p p l i e d to a 1.2x40 cm DEAE c e l l u l o s e column and e l u t e d w i t h a l i n e a r g r a d i e n t of 0-0.2 M NH4HCO5 (2.5 1 t o t a l volume). F r a c t i o n s of 15 ml were c o l l e c t e d at a r a t e of 6 ml/min. The gradient was run at room temperature. The e l u t i o n of n u c l e o t i d e was f o l l o w e d by mo n i t o r i n g the absorbance of each f r a c t i o n at 260 ^ m ( ); the increase i n s a l t c o n c e n t r a t i o n was fo l l o w e d by measuring the con-d u c t i v i t y of every f i f t h f r a c t i o n (• • ) . F r a c t i o n s 75-91 were pooled and d r i e d (see Methods), and the major c o n s t i t u e n t of peak V was c h a r a c t e r i z e d as AMP-PNP by paper chromatography (see R e s u l t s ) . 44 . yAmoles. There were o r i g i n a l l y 50 yumoles of ^ H-AMP; th e r e -f o r e , 11.9 jumoles of H-AMP-PNP represented a y i e l d of 24%. However, the y i e l d was somewhat l e s s than 24% be-cause of the presence of an un q u a n t i t a t e d i m p u r i t y i n peak V (see below). Peak V was c h a r a c t e r i z e d by chromatography i n 3 s o l -vent systems (Table 1). The m o b i l i t i e s of peak V, ATP, .. and ADP i n s o l v e n t s A and B were compared w i t h the m o b i l -i t i e s , g i v e n by Yount et a l (46) (see Table 2 of t h i s T h e s is) f o r AMP-PNP, ADP-NH2, ATP, and ADP. Although the R.£ v a l u e s i n Table 1 were q u a n t i t a t i v e l y d i f f e r e n t from those g i v e n by Yount et a l , the m o b i l i t i e s of these nu-c l e o t i d e s r e l a t i v e t o each other were q u a l i t a t i v e l y the same i n both cases. This a p p l i e d t o both s o l v e n t s A and B. This comparison supports an i d e n t i f i c a t i o n of the major component of peak V as H-AMP-PNP and an i d e n t i f i -c a t i o n of the minor component of peak V as H-ADP-N^. Yount and co-workers found t h a t the major i m p u r i t y of AMP-PNP was ADP-NH'2» and t h a t i t s presence r e s u l t e d from the h y d r o l y s i s of the t e r m i n a l phosphate of AMP-PNP (46). 45-I I . Adenyl Cyclase A c t i v i t y i n 0. Tshawytscha T e s t i s A The ATP-Regenerating System The e f f e c t i v e n e s s of the pyruvate kinase - phospho-e n o l p y r u v i c a c i d ATP - r e g e n e r a t i o n system was p e r i o d i -c a l l y checked. A 20 ^ ul a l i q u o t of the supernatant from the adenyl c y c l a s e assay was chromatographed on Whatman 40 SCF paper i n the i s o b u t y r i c a c i d : 1 M ammonium hydrox-i d e : 0.1 M EDTA (125:75:2, by volume) s o l v e n t system ( 1 0 ) . A f t e r the chromatogram was thoroughly d r i e d n u c l e o t i d e spots which were detected under s h o r t wave l e n g t h u l t r a v i o l e t l i g h t (200 to 280?j>m) were cut out and r a d i o a c t i v e content was determined by the procedure d e s c r i b e d f o r the adenyl c y c l a s e assay (see Methods). A f t e r an adenyl c y c l a s e assay mixture had been incubated f o r 30 min at 30° 95% of the t r i t i u m l a b e l i n the assay supernatant was recov-ered i n the ATP p l u s ADP spots and 82% of the l a b e l was i n the ATP spot. B E f f e c t of Time, Temperature and Substrate The adenyl c y c l a s e a c t i v i t y of salmon t e s t i s homog-enates was s e n s i t i v e to the l e n g t h and temperature of the enzyme assay ( F i g u r e 2 ) . The formation of c y c l i c AMP from ATP was p r o p o r t i o n a l to the l e n g t h of i n c u b a t i o n at F i g u r e 2 . E f f e c t of Time, Temperature, and Substrate on the Adenyl Cyclase A c t i v i t y of Salmon T e s t i s Homogenates. 4 6 . 30CH Time Min Assay c o n d i t i o n s were o u t l i n e d under Methods. Curves formed by s o l i d l i n e s ( ) i n d i c a t e t h a t ^H-ATP was the s u b s t r a t e , and those formed by broken l i n e s ( ) i n d i -cate t h a t ^H-AMP-PNP was the s u b s t r a t e . The t o t a l assay volume was 1 7 0 ill and the assay temperatures were those given beside each curve. 47. 30 w; formation of c y c l i c AMP from AMP-PNP was p r o p o r t i o n a l to i n c u b a t i o n time at 1 5 ° and 30°. The r a t e of p r o d u c t i o n of c y c l i c AMP from ATP at 37° was approximately twice that at 30°. S i m i l a r l y , the r a t e of pr o d u c t i o n of c y c l i c AMP from AMP-PNP at 30° was about twice the r a t e of pro-d u c t i o n at 1 5 ° . ATP appeared to be a b e t t e r s u b s t r a t e f o r the salmon t e s t i s adenyl c y c l a s e than was AMP-PNP; at 30° c y c l i c AMP was s y n t h e s i z e d from ATP at twice the ra t e i t was sy n t h e s i z e d from AMP-PNP. However, the ^ H-AMP-PNP sample was not pure and the true r a t e of produc-t i o n of c y c l i c AMP from AMP-PNP was probably higher than i n d i c a t e d i n Figu r e 2. G E f f e c t of D i v a l e n t Cations The adenyl c y c l a s e a c t i v i t y of salmon t e s t i s r e q u i r e s the presence of e i t h e r Mg or Mn i n the assay medium (F i g u r e 3 ) . The maximal r a t e of enzyme a c t i v i t y i n the 2 + presence of Mn could be as much as 5 times g r e a t e r than the maximal r a t e obtained w i t h Mg . However, i n another experiment ( r e s u l t s not shown) maximal s t i m u l a t i o n w i t h Mn^ was 2.5 times g r e a t e r than w i t h Mg^ . The op-t i m a l Mg 2 + c o n c e n t r a t i o n was approximately 10 mM and f u r t h e r i n c r e a s e s i n M g 2 + c o n c e n t r a t i o n , up to 50 mM, n e i -t h e r i n c r e a s e d nor decreased the r e a c t i o n r a t e . Maximal 48. F i g u r e 3. The E f f e c t of Changes i n Magnesium or Manganese Ion Concentration on the A c t i v i t y of Adenyl Cyclase i n Salmon T e s t i s Homogenates. 30 The assay contents were those g i v e n under Methods except that the c o n c e n t r a t i o n of MgCl 2 (o o) was v a r i e d , or M gCl 2 was omitted and v a r i o u s c o n c e n t r a t i o n s of MnCl 2 (• •) were s u b s t i t u t e d . The adenyl c y c l a s e a c t i v i t y was measured over a p e r i o d of 30 min at 37°. The t o t a l assay volume was 170 pi, and the s u b s t r a t e was ^H-ATP. a c t i v a t i o n of the enzyme w i t h Mn was achieved a t about 5 mM and t h i s r a t e was maintained from 5 t o 20 mM Mn*1 . F u r t h e r i n c r e a s e s i n Mn c o n c e n t r a t i o n up to 50 mM caused some decrease i n enzyme a c t i v i t y , but the r a t e of c y c l i c AMP p r o d u c t i o n w i t h 50 mM Mn*- was s t i l l more than 3 times the r a t e w i t h 50 mM Mg ( F i g u r e 3 ) . D E f f e c t of F l u o r i d e Ion I n the absence of F~ the adenyl c y c l a s e a c t i v i t y of salmon t e s t i s was very low (about 1 pmole c y c l i c AMP/ min/mg p r o t e i n at 37°). The maximal r a t e of c y c l i c AMP pr o d u c t i o n at 12 mM P" was 7 times the enzymic r a t e i n the absence of any added F~ (Figure 4 ) . F l u o r i d e i b n con-c e n t r a t i o n s above 20 mM brought a marked decrease i n a c t i v i t y . E E f f e c t of Detergents and Some Other Reagents Table 3 g i v e s the r e s u l t s of a study that was under-taken to determine the e f f e c t s of a v a r i e t y of reagents on the adenyl cy c l a s e a c t i v i t y i n homogenates of chinook salmon t e s t i s . The a d d i t i o n of 0.01 M ^-mercaptoethanol or 0.3 M KC1 to the homogenizing medium had no e f f e c t on the adenyl c y c l a s e a c t i v i t y . G l y c e r o l , when present i n the homogenizing medium i n co n c e n t r a t i o n s of 5 to 20% 50. F i g u r e 4. The E f f e c t of Changes i n F l u o r i d e Ion Concen-t r a t i o n on the A c t i v i t y of Adenyl Cyclase i n Salmon T e s t i s Homogenates. 9 3 5 10 15 20 25 50 55 40 45 50 F~ Concentration mM The enzyme was assayed f o r 30 min at 37° by the pro-cedure o u t l i n e d under Methods. The t o t a l assay volume was 170 u l and the s u b s t r a t e was %-ATP. 5 1 . Table 5. E f f e c t of a V a r i e t y of Reagents on the Adenyl Cyclase A c t i v i t y of Salmon T e s t i s Homogenates Reagent and I t s C o n c e n t r a t i o n i n the Homogenizing Medium Adenyl Cyclase A c t i v i t y (pmole c y c l i c AMP/min/ mg p r o t e i n ) + Reagent C o n t r o l 0.01 M |3-Mercaptoethanol 0.01 M p-Mercaptoethanol* 3.24 1.53 3 . 1 5 2.07 1$ L u b r o l PX* 1% Nonidet P40 0.25% Sodium Deoxycholate* 1% T r i t o n X100 1% Tween 80 0.03! 30.0 ^ 0 0 1 .65 2.24 68.5 1.53** 3.17** 2.13 0.3 M K C l 0.3 M KCl 3.47 7.06 3.24** 5.72** 5% G l y c e r o l * 10% G l y c e r o l 20% G l y c e r o l * 1.85 1.52 1 .62 2.11 2.11 2.11 3 mM M g C l 2 + 10 mM NaF 2.24 1.90 For each of the above experiments adenyl c y c l a s e a c t i v i t y was measured simu l t a n e o u s l y i n two homogenates. One homogenate was a c o n t r o l which contained t e s t i s t i s s u e i n e i t h e r 0.25 M sucrose and 0.01 M T r i s - C l (pH 7.5) or 0.25 M sucrose, 0.01 M T r i s - C l (pH 7.5) and 0.01 M £-mer-captoethanol, the l a t t e r , marked by a double a s t e r i s k (**). The o t h e r , experimental homogenate, contained the same i n g r e d i e n t s as the c o n t r o l plus the reagent being t e s t e d . Assays were performed as o u t l i n e d under Methods. The assay temperature was 30° and the volume was 170 ^ ul u n l e s s the experiment i s marked w i t h a s i n g l e a s t e r i s k (*) i n which case the volume was 100 pi. A l l enzyme a c t i v i t i e s i n the t a b l e represent averages of d u p l i c a t e d e t e r m i n a t i o n s . 1 . Both the c o n t r o l and the t e s t homogenate f o r t h i s experiment c o n t a i n e d , i n a d d i t i o n to T r i s - C l and sucrose, 3 mM MgCl 2 and 10 mM NaF. 2. The s p e c i f i c a c t i v i t y was expressed as pmole c y c l i c AMP/min/ml enzyme because the presence of Nonidet P40 i n the homogenate i n t e r f e r e d w i t h p r o t e i n d e t e r m i n a t i o n . 52. s l i g h t l y reduced the enzyme a c t i v i t y . Of the 5 detergents t e s t e d only Tween 80 and Nonidet P40 d i d not destroy the adenyl c y c l a s e a c t i v i t y of the homogenate; they d i d however reduce the a c t i v i t y . Two other n o n - i o n i c d e t e r -gents, L u b r o l PX and T r i t o n X - 100 destroyed t h i s enzyme a c t i v i t y i n salmon t e s t i s . I n g e n e r a l , i o n i c detergents e l i m i n a t e a l l aspects of adenyl c y c l a s e a c t i v i t y (37) and we found t h a t deoxycholate destroyed t h i s enzymic a c t i v i t y i n salmon t e s t i s . A 1 5 min p r e i n c u b a t i o n at 0° to 4° of a salmon t e s -t i s homogenate w i t h 3 mM MgCl2 and 10 mM NaF caused a minimal i n c r e a s e i n the f l u o r i d e s t i m u l a t e d adenyl c y c l a s e a c t i v i t y , j P S t a b i l i t y I t was p r e v i o u s l y suggested t h a t the adenyl c y c l a s e of salmon t e s t i s homogenates i s a very unstable enzyme (3 3 ) . We t h e r e f o r e monitored enzymic a c t i v i t y at v a r i o u s i n t e r v a l s a f t e r homogenization of the t i s s u e . An enzyme sample s t o r e d on i c e maintained the i n i t i a l r a t e of c y c l i c AMP p r o d u c t i o n f o r 6 hours but was completely i n a c t i v e when assayed 24}£ hours a f t e r p r e p a r a t i o n ( F i g u r e 5 ) . Storage of a homogenate at room temperature caused the r a t e of enzymic r e a c t i o n to decrease i n p r o p o r t i o n to 53. F i g u r e 5. S t a b i l i t y of Adenyl Cyclase i n a Salmon T e s t i s Homogenate. Homogenates were s t o r e d e i t h e r on an i c e bath (o o) or at room temperature (• •) and were assayed at v a r i o u s i n t e r v a l s a f t e r p r e p a r a t i o n . The p r o d u c t i o n of c y c l i c AMP was measured over a 30 min per i o d at 30°. The assay was performed as o u t l i n e d under Methods w i t h ^H-ATP as the s u b s t r a t e . The t o t a l assay volume was 170 p i . 54. the age of the t i s s u e p r e p a r a t i o n . A salmon t e s t i s homog-enate which was h e l d at room temperature f o r 6 hours converted ATP to c y c l i c AMP at h a l f the r a t e that a f r e s h homogenate d i d . A f t e r storage f o r 2A% hours at room tem-perature a salmon t e s t i s homogenate had no adenyl c y c l a s e a c t i v i t y . G S u b c e l l u l a r D i s t r i b u t i o n Oncorhynchus tshawytscha t e s t i s homogenates were f r a c t i o n a t e d by d i f f e r e n t i a l c e n t r i f u g a t i o n and the f r a c -t i o n s • were assayed f o r adenyl c y c l a s e a c t i v i t y (Table 4 ) . T e s t i s t i s s u e was homogenized i n two d i f f e r e n t types of s o l u t i o n s , an i s o t o n i c medium and a h y p e r t o n i c medium. The adenyl c y c l a s e a c t i v i t y of t e s t i s homogenized i n i s o -t o n i c medium was a p p a r e n t l y d i s t r i b u t e d throughout a l l the s u b c e l l u l a r f r a c t i o n s w i t h a major p o r t i o n of the ac-t i v i t y appearing i n the low speed or 600g sediment. A-bout 10% of the adenyl c y c l a s e a c t i v i t y was contained i n the s o l u b l e or 105,000g supernatant f r a c t i o n . The enzyme s p e c i f i c a c t i v i t i e s (pmoles c y c l i c AMP/min/mg p r o t e i n ) of the homogenate, the low speed sediment, and a l l the supernatant f r a c t i o n s were approximately equal. The 6300g sediment, however, had a very h i g h s p e c i f i c a c t i v -i t y of 111 pmole c y c l i c AMP/min/mg p r o t e i n i n Experiment 5 5 . Table 4 . S u b c e l l u l a r D i s t r i b u t i o n of Adenyl Cyclase i n Salmon T e s t i s Homogenates. Homogenizing Medium A ( I s o t o n i c ) Homogenizing Medium B (Hypertonic) Experiment I Experiment I I Experiment I I I Experiment IV F r a c t i o n $ S.A. lo S.A. % S.A. S.A. Whole Homo. 100 6.6 100 7.8 100 7.1 100 2.8 600g SD 59 8.6 55 8.8 4 4.5 0 0 6300g SD 20 111 . 22 - 14 21 . 56 14.8 105000g SD 9 - 0 0 31 10. - -105000g SN 14 5.2 8 3.1 0 0 - -600g SN 43 - 39 7.5 87 6.5 100 3.1 6300g SN 23 - 26 6.1 62 5.3 56 2.0 Abb r e v i a t i o n s : % % of t o t a l a c t i v i t y in whole homogenate1 S.A. s p e c i f i c a c t i v i t y i n pmole c y c l i c AMP/min/ mg p r o t e i n Whole Homo.whole homogenate SD sediment SN supernatant j Salmon t e s t i s (5g) was homogenized i n e i t h e r medium A, 0. 25 M sucrose, 0.01 M T r i s - C l (pH 7.5) (25 ml;, f o r ex-periments I and I I , or medium B, 0.25 M sucrose, 0.01 M T r i s - C l (pH 7.5), 0.3 M K C l , and 0.01 M p-mercaptoethanol (25 m l ) , f o r experiments I I I and IV. These homogenates were then f r a c t i o n a t e d by d i f f e r e n t i a l c e n t r i i ' u g a t i o n a c c o r d i n g t o t h e s c h e m e o f ivienon a n a S m i t h ( 3 3 ) . The f r a c t i o n s were assayed f o r p r o d u c t i o n of ^ H - c y c l i c AMP from -^ H-ATP (see Methods). I n experiment I the assay was performed a t 37° and the r e a c t i o n l a s t e d 30 min; i n ex-periment I I the r e a c t i o n time was 35 min, the temperature, 30°; i n experiment I I I r e a c t i o n time was 31 min, the temperature, 30°; and i n experiment IV r e a c t i o n time was 60 min, the temperature 30°. For experiments I , I I , and I I I the assay volume was 170 u l . The assay volume was 100 yul i n experiment IV. I n each experiment the s u b c e l l u -l a r f r a c t i o n s were resuspended i n the same medium i n which the homogenate was prepared. 1. Total activnVy per fracViort w a s ca lcuiaVeol a s pmole cyclic min / fotoJ pro+ein. 56. I (Table 4 ) . The adenyl c y c l a s e a c t i v i t y of t e s t i s homogenized i n the h i g h s a l t c o n c e n t r a t i o n medium was not l o c a t e d i n e i t h e r the low speed sediment, or the h i g h speed su-pernatant. C e n t r i f u g a t i o n of the 6300g supernatant at 105,000g r e s u l t e d i n a 50% l o s s of t o t a l enzyme a c t i v i t y . As w i t h the i s o t o n i c homogenates, the s p e c i f i c a c t i v i t y of the 6300g sediment was h i g h e r than the s p e c i f i c a c t i v -i t i e s of the other f r a c t i o n s . 57 DISCUSSION I ; The Adenyl Cyclase Assay. A ATP-Regeneration System The ATP-regenerating system of pyruvate kinase and phosphoenolpyruvate was capable of m a i n t a i n i n g over 80% of the t r i t i u m l a b e l as ATP a f t e r a 30 min i n c u b a t i o n of the standard adenyl c y c l a s e assay mixture at 30°. The ATP c o n c e n t r a t i o n , t h e r e f o r e , was maintained at about 0.3 mM which i s w e l l above the K m of the salmon t e s t i s enzyme f o r ATP of 0.05 mM ( 3 3 ) . The r e g e n e r a t i n g system does increase the complexity of the adenyl c y c l a s e assay and some si d e e f f e c t s of the system should be considered. The e f f e c t s of any a l t e r a t i o n i n the standard enzyme assay, such as the a d d i t i o n of d etergents, should be an-a l y z e d to determine whether adenyl c y c l a s e a c t i v i t y or pyruvate kinase a c t i v i t y or both have been a f f e c t e d . Pyruvate k i n a s e , l i k e adenyl c y c l a s e , r e q u i r e s Mg 2 + f o r i t s a c t i v i t y ; t h e r e f o r e , omission of Mg from the assay i n h i b i t s both enzymes (10). I t has been suggested t h a t an u n i n h i b i t e d c y c l i c AMP phosphodiesterase a c t i v i t y plus a r e l a t i v e l y h i g h c o n c e n t r a t i o n of c o l d c y c l i c AMP p lus an ATP-regenerating system could combine to e f f e c t i v e l y convert c y c l i c AMP to ATP thereby d i l u t i n g the r a d i o a c -t i v l y l a b e l e d ATP ( 3 7 ) . B E f f e c t of Time and Temperature The p r o d u c t i o n of c y c l i c AMP from ATP by a salmon t e s t i s homogenate was a time dependent r e a c t i o n which was l i n e a r f o r over 60 min at 30° but was not l i n e a r f o r even 20 min a t 37°. This l a t t e r r e s u l t was repeated i n 3 separate experiments ( r e s u l t s not shown). I t seems p o s s i b l e t h a t t h i s enzyme could be unstable at 37° con-s i d e r i n g t h a t the body temperature of the chinook salmon o 4 i s u s u a l l y about 10 . The p r o d u c t i o n of c y c l i c AMP from ATP at 37° when c a t a l y z e d by a guinea p i g v e n t r i c l e prep-aration.; i s a l i n e a r r e a c t i o n f o r at l e a s t 30 min ( 1 0 ) . Even though the r a t e of p r o d u c t i o n of c y c l i c AMP from ATP i s lower at 30° than at 37°, i t seems p r e f e r a b l e to assay the salmon t e s t i s enzyme at 30° because of the l i n -e a r i t y of the r e a c t i o n a t t h i s temperature. 4. The temperature of the waters of the P a c i f i c Ocean i n which these f i s h spend much of t h e i r l i f e and the temperature of the E r a s e r R i v e r d u r i n g the month of June when the f i s h f o r these experiments were captured are both approximately 10°. 5 9 . G E f f e c t of M g 2 + and Mn 2 + The d i v a l e n t c a t i o n requirement of salmon t e s t i s adenyl c y c l a s e was f i l l e d by e i t h e r Mg 2 + or Mn 2 + although the V m a x obtained i n the presence of Mn 2 + was much g r e a t e r than w i t h Mg 2 +. The optimal c o n c e n t r a t i o n of Mn 2 + was 5 mM and t h a t of Mg 2 + was 1 0 mM. Adenyl c y c l a s e s from mammalian sources have a l s o shown a gr e a t e r V w i t h & max Mn 2 + and maximal s t i m u l a t i o n of the enzyme w i t h lower concentrations of Mn than Mg ( 1 1,37). The reason f o r the g r e a t e r a c t i v a t i o n of the enzyme w i t h Mn*- i s unknown but the g r e a t e r a f f i n i t y of Mn 2 + f o r ATP i s not the an-swer, si n c e h i g h e r c o n c e n t r a t i o n s of Mg would e v e n t u a l l y achieve the same V as obtained w i t h Mn 2 + (37). I t has been demonstrated t h a t ATP con c e n t r a t i o n s i n excess of the Mg* c o n c e n t r a t i o n i n the assay medium are i n h i b i t o r y ( 3 7 ) . The true s u b s t r a t e f o r adenyl c y c l a s e i s thought to be the ATP-Mg complex and f r e e ATP i s thought to i n h i b i t trie r e a c t i o n . Concentrations of Mg* f a r i n excess of the ATP c o n c e n t r a t i o n b r i n g about maximal en-zymic s t i m u l a t i o n both i n the salmon t e s t i s enzyme and i n many mammalian enzymes ( 1 0,37). Two hypotheses have been proposed to e x p l a i n the r o l e of t h i s e x t r a Mg 2 +. Drummond and Duncan (10) have proposed t h a t the Mg 2 + 6 0 . b i n d s a t a second s i t e ( p o s s i b l y a l l o s t e r i c ) on the en-zyme and i n c r e a s e s t h e r e a c t i v i t y o f the c a t a l y t i c s i t e w i t h the s u b s t r a t e so t h a t the maximal r e a c t i o n v e l o c i t y i s g r e a t l y i n c r e a s e d . A n o t h e r h y p o t h e s i s i s t h a t f r e e ATP b i n d s t o the c a t a l y t i c s i t e w i t h a much s t r o n g e r a f -f i n i t y t h a n does the ATP-Mg 2 + complex and t h e r e b y f r e e ATP i n h i b i t s t h e r e a c t i o n . V e r y h i g h c o n c e n t r a t i o n s o f M g 2 + a r e t h e r e f o r e r e q u i r e d t o compete the f r e e ATP out of the c a t a l y t i c s i t e ( 3 7 ) . H i g h c o n c e n t r a t i o n s o f Mn have caused d e c r e a s e s i n a d e n y l c y c l a s e a c t i v i t y ( 1 1 , 3 3 , 3 7 ) . The e x t e n t o f t h i s i n h i b i t i o n w h i c h we o b s e r v e d was n o t as marked as the i n h i b i t i o n w h i c h was p r e v i o u s l y r e p o r t e d f o r a salmon t e s -t i s homogenate ( 3 3 ) , n o r was i t as g r e a t as t h a t o b s e r v e d i n a g u i n e a p i g v e n t r i c l e p r e p a r a t i o n ( 1 1 ) . There has been a s u g g e s t i o n t h a t t h e d e c r e a s e d a d e n y l c y c l a s e a c -t i v i t y o f b o v i n e c e r e b r a l c o r t e x a t h i g h M n 2 + c o n c e n t r a -p-f o + t i o n s i s r e l a t e d t o a c o m p e t i t i o n o f Mnc w i t h bound Ca^ . I t i s n o t known i f C a 2 + does p l a y a r o l e i n the c a t a l y t i c a c t i v i t y o f a d e n y l c y c l a s e . C o n c e n t r a t i o n s o f Ca above 1 mM a r e u s u a l l y i n h i b i t o r y t o a d e n y l c y c l a s e ( 3 7 ) . Menon and S m i t h (33) r e p o r t e d t n a t C a 2 + had no e f f e c t on salmon t e s t i s a d e n y l c y c l a s e ; however, no c o n c e n t r a t i o n s were g i v e n . 6 1 . I n h i b i t i o n of hormonally induced adenyl c y c l a s e a c t i v i t y by Mn 2 + has been report e d i n s e v e r a l d i f f e r e n t systems (37). Although the V _ of salmon t e s t i s adenyl max cyc l a s e i s l a r g e r i n the presence of Mn* than Mg , we have p e r s i s t e d i n u s i n g Mg 2 + r a t h e r than Mn 2 + i n the en-zyme assay because of the two problems, j u s t mentioned, which are a s s o c i a t e d w i t h Mn 2 +. D E f f e c t of F The f l u o r i d e i o n s t i m u l a t e s adenyl c y c l a s e i n almost a l l broken c e l l p r e p a r a t i o n s but does not s t i m u l a t e the enzyme i n i n t a c t c e l l s even though NaP, most l i k e l y enters the c e l l r e a d i l y (37). The mechanism by which F" a c t i -vates adenyl c y c l a s e i s unknown. The complex, MgF 2, i s known to e x i s t and i t has been proposed t h a t the a c t i v e form of P~ i s a magnesium-fluoride complex (10). Drummond and Duncan observed t h a t i n c r e a s e s i n the Mg 2 + concentra-t i o n reversed the i n h i b i t i o n of guinea p i g v e n t r i c l e adenyl c y c l a s e which occurred at high f l u o r i d e concentra-t i o n s . T h i s , and other o b s e r v a t i o n s , l e d them to suggest t h a t P~ s t i m u l a t e s the enzyme through the fo r m a t i o n of a fluoride-magnesium complex which i n c r e a s e s the r e a c t i v i t y of the c a t a l y t i c s i t e to a g r e a t e r extent than magnesium alone could. A number of anions other than f l u o r i d e were 62. t e s t e d f o r t h e i r e f f e c t on adenyl c y c l a s e a c t i v i t y and i t was found t h a t enzymic s t i m u l a t i o n was h i g h l y s p e c i f i c f o r the f l u o r i d e anion (10) . Basal a c t i v i t y of salmon t e s t i s adenyl c y c l a s e as measured i n the absence of NaF was not zero, as p r e v i o u s l y r e p o r t e d ( 3 3 ) , but was very low. A b a s a l adenyl c y c l a s e a c t i v i t y has been recorded i n a number of w h o l e - c e l l and b r o k e n - c e l l p r e p a r a t i o n s ( 3 7 ) . The r e l a t i o n s h i p of b a s a l a c t i v i t i e s which have been recorded i n v i t r o to the phys-i o l o g i c a l f u n c t i o n of adenyl c y c l a s e i s not known (37). I n summary, a comparison of the optimal c o n d i t i o n s f o r a s s a y i n g salmon t e s t i s adenyl c y c l a s e w i t h the optimal c o n d i t i o n s f o r assaying mammalian adenyl c y c l a s e s from a v a r i e t y of t i s s u e s i n d i c a t e many s i m i l a r i t i e s between the f i s h enzyme and the mammalian enzymes, e s p e c i a l l y w i t h regard to Mg'1 , Mn^ , and F~ s t i m u l a t i o n . 63. I I . S o l u b i l i z a t i o n of Salmon T e s t i s Adenyl Cyclase A S u b - C e l l u l a r D i s t r i b u t i o n The main reason f o r s u b j e c t i n g salmon t e s t i s homog-enates to d i f f e r e n t i a l c e n t r i f u g a t i o n was to determine the amount of adenyl c y c l a s e a c t i v i t y i n the 105,000g ( h i g h speed) supernatant. A previous study i n d i c a t e d t h a t 21% of the salmon t e s t i s enzyme was s o l u b l e ( 3 3 ) , but i n t h i s study only about 10% of the a c t i v i t y was recovered i n the h i g h speed supernatant. I n a d d i t i o n , we found t h a t about 55% of the a c t i v i t y , rather' than 14% (33) sed-imented.. at 600g. Although no attempt was made to i d e n t i f y the contents of the 600g sediment, t h i s f r a c t i o n probably contained whole c e l l s , plasma membranes and n u c l e i ( 32). The r e s u l t s of many s t u d i e s , i n v o l v i n g a v a r i e t y of e x p e r i -mental techniques and d i f f e r e n t t i s s u e s have i n d i c a t e d t h a t adenyl c y c l a s e i s l o c a t e d i n the plasma membrane (10,37). There i s some evidence which i n d i c a t e s t h a t adenyl c y c l a s e may be l o c a t e d i n other c e l l u l a r membranous s t r u c -t u r e s ( 3 7 ) . I n r e l a t i o n t o the h i g h s p e c i f i c a c t i v i t i e s which we observed i n the 63C0g sediment, i t i s i n t e r e s t i n g to note t h a t the hi g h e s t s p e c i f i c a c t i v i t i e s of adenyl c y c l a s e i n s u b c e l l u l a r f r a c t i o n s of r a t t e s t i s were i n the m i t o c h o n d r i a l f r a c t i o n ( 1 2 ) . B E f f e c t of Detergents Adenyl c y c l a s e i s g e n e r a l l y c h a r a c t e r i z e d as a mem-brane enzyme ( 3 7 ) , thus the f i n d i n g t h a t most of t h i s enzyme a c t i v i t y i n salmon t e s t i s was l o c a t e d i n p a r t i c u -l a t e f r a c t i o n s (Table 4) was not s u r p r i s i n g . I t i s prob-ab l y safe to assume tha t the c a t a l y t i c adenyl c y c l a s e a c t i v i t y of salmon t e s t i s i s a membrane-bound enzyme. At t h i s p o i n t i t should be mentioned t h a t some r e -searchers have c l a s s i f i e d membrane p r o t e i n s i n t o 2 cate-g o r i e s - p e r i p h e r a l and i n t e g r a l ( 5 0 ) . Among the c r i t e r i a f o r i d e n t i f i c a t i o n of p e r i p h e r a l p r o t e i n s i s the a b i l i t y to d i s s o c i a t e these p r o t e i n s from the membrane by m i l d treatment such as in c r e a s e d i o n i c s t r e n g t h (50). Since homogenization of salmon t e s t i s i n a medium c o n t a i n i n g 0.3 M KC1 d i d not render adenyl c y c l a s e a c t i v i t y s o l u b l e a f t e r c e n t r i f u g a t i o n at 105,000g (Table 4) i t might be considered t h a t t h i s enzyme i s not ' p e r i p h e r a l ' . Membrane p r o t e i n s , other than those c l a s s i f i e d as p e r i p h e r a l , have proven to be extremely d i f f i c u l t to s o l -u b i l i z e (48). Many bonds, i n c l u d i n g hydrophobic i n t e r -a c t i o n s , hydrogen bonds, h y d r o p h i l i c i n t e r a c t i o n s and e l e c t r o s t a t i c i n t e r a c t i o n s are thought to be important i n m a i n t a i n i n g the i n t e g r i t y of the membrane (49). Very s t r o n g hydrophobic i n t e r a c t i o n s are b e l i e v e d to be of key importance i n b i n d i n g membrane p r o t e i n s i n t o the l i p i d m a t r i x of the membrane (49,50). To s o l u b i l i z e mem-brane p r o t e i n s these bonds must be broken without denatur i n g the p r o t e i n s . Four c a t e g o r i e s of reagents which have been of use i n s o l u b i l i z i n g membrane p r o t e i n s are dete r g e n t s , organic s o l v e n t s , l i p o l y t i c enzymes, and cha o t r o p i c agents (48). As a p r e l i m i n a r y to the s o l u b i l i z a t i o n of salmon t e s t i s adenyl c y c l a s e the e f f e c t s of s e v e r a l detergents on the enzyme a c t i v i t y were examined. The b a s i s f o r choo i n g 4 n e u t r a l detergents - L u b r o l PX, Nonidet P40, T r i t o n X100, and Tween 80 was the knowledge that n e u t r a l d e t e r -gents are capable of s o l u b i l i z i n g membrane p r o t e i n s i n the n a t i v e s t a t e ( 4 8 ) . T r i t o n X100 has been used success f u l l y to s o l u b i l i z e some membrane enzymes i n c l u d i n g some mammalian adenyl c y c l a s e s ( 3 7 ) . The adenyl c y c l a s e s were however, i n s o l u b l e i n the absence of the detergent ( 3 7 ) . Gat heart adenyl c y c l a s e has been s o l u b i l i z e d by homog-e n i z a t i o n of the t i s s u e i n the presence of L u b r o l PX (37) This enzyme p r e p a r a t i o n i s hor m o n e - i n s e n s i t i v e , NaF-sensi-t i v e , and remains s o l u b l e a f t e r the removal of the deter-gent ( 3 7 ) . These experiments suggested t h a t T r i t o n X100 and L u b r o l PX might be v a l u a b l e a i d s i n s o l u b i l i z i n g 66. salmon t e s t i s adenyl c y c l a s e , but our r e s u l t s showed th a t these 2 detergents destroyed the enzyme a c t i v i t y i n crude homogenates (Table 3). A p o i n t of some i n t e r e s t i s that i n c r e a s e s i n c y c l i c phosphodiesterase a c t i v i t y have been a s s o c i a t e d w i t h the presence of T r i t o n X100 ( 3 7 ) . Salmon t e s t i s homogenized i n the presence of 1% Nonidet P40 or 1% Tween 80 r e t a i n e d approximately 45% and 75%, respec-t i v e l y , of the adenyl c y c l a s e a c t i v i t y measured i n the absence of these detergents (Table 3 ) . I t i s not known, however, whether these detergents i n any way s o l u b i l i z e d t h i s enzymic a c t i v i t y . Future s t u d i e s on the s o l u b i l i z a -t i o n of t h i s enzyme should, t h e r e f o r e , b e g i n by more f u l l y a s s e s s i n g the e f f e c t s of Nonidet P40 and Tween 80 on salmon t e s t i s adenyl c y c l a s e . Since i t i s probable t h a t the c a t a l y t i c adenyl c y c l a s e a c t i v i t y of salmon t e s t i s can be a t t r i b u t e d to a membrane bound p r o t e i n , the m i l i e u of t h i s p r o t e i n i s an important f a c t o r to c o n s i d e r when attempting to s o l u b i l i z e i t . A p o r t i o n of the enzyme i s very l i k e l y i n c l o s e a s s o c i a t i o n w i t h the a p o l a r r e g i o n of p h o s p h o l i p i d molecules (49,50). A requirement f o r p h o s p h o l i p i d s i n the a c t i v i t y of many membrane-bound enzymes has now been shown ( 4 8 ) . Although there i s no evidence to i n d i c a t e t h a t c a t a l y t i c or NaF-s e n s i t i v e adenyl c y c l a s e a c t i v i t y i s dependent on phos-67. p h o l i p i d s , there i s reason to suggest t h a t l i p i d s may be i n v o l v e d i n the hormone r e c e p t o r sub-unit and i n the c o u p l i n g mechanism of adenyl c y c l a s e ( 3 7). Thus, a s o l u b l e , i n t a c t , hormone-sensitive adenyl c y c l a s e system w i l l probably i n c l u d e s p e c i f i c l i p i d molecules. 68. I l l . J r l t i u m ^ L a b e l e d A d e n y l y l Imidodiphosphate A Synthesis The s y n t h e s i s of -^H-AMP-PNP (based on the procedure of Hoard and Ott ( 2 1 ) ) from ^ H - A M P and tributlyammonium imidodiphosphate u s i n g 1 , 1 ' - c a r b o n y l d i i m i d a z o l e was not e n t i r e l y s u c c e s s f u l , s i n c e there was some i m p u r i t y i n the f i n a l product. This i m p u r i t y was e l u t e d i n the same peak as the ^H-AMP-PNP from the DEAE c e l l u l o s e column. Two ways to in c r e a s e the p u r i t y of the ^ H-AMP-PNP would be (a) to i s o l a t e n u c l e o t i d e from only the homogeneous por-t i o n of peak V ( F i g u r e 1) and (b) to f u r t h e r p u r i f y t h i s product by paper chromatography. Hoard and Ott (21) i n d i c a t e d t h a t 2•-deoxyadenosine 5'-triphosphate (dATP) which was s y n t h e s i z e d from 2'-deoxyadenosine 5'-monophosphate and pyrophosphate was not completely r e s o l v e d by DEAE c e l l u l o s e chromatography. Paper chromatography of the peak c o n t a i n i n g dATP i n d i c a -ted the presence of an u n i d e n t i f i e d i m p u r i t y having the parent n u c l e o t i d e spectrum. The o v e r a l l y i e l d of nucleo-t i d e i n the DEAE c e l l u l o s e chromatographic peak c o n t a i n i n g the dATP was 73%; however, because of the presence of the i m p u r i t y , dATP was i s o l a t e d from only the homogeneous 69. p o r t i o n of the peak and the y i e l d was reduced to 36%. Our y i e l d of %-AMP-PNP i n i t s impure form was 24%. The s y n t h e s i s of AMP-PNP by the r e a c t i o n of imidodiphos-phate w i t h P^-adenosine P 2 - d i p h e n y l pyrophosphate gave y i e l d s of 20 to 35% (46). The enzymatic s y n t h e s i s of AMP-PNP from ATP and PNP us i n g amino a c i d tRNA synthetases gave y i e l d s of 60 T O 75% (40). B AMP-PNP as a Substrate f o r Salmon T e s t i s Adenyl Cyclase The adenyl c y c l a s e of salmon t e s t i s was capable of c a t a l y z i n g the pr o d u c t i o n of c y c l i c AMP from AMP-PNP. This process appeared to proceed at a slower r a t e than w i t h the ATP su b s t r a t e but the '^H-AMP-PNP p r e p a r a t i o n contained some i m p u r i t i e s which were not q u a n t i t a t e d . R o d b e l l et a l (40) rec o r d the use of an AMP-PNP sample which was contaminated w i t h 1 5 % ADP-NH2. I t i s p o s s i b l e t h a t e s s e n t i a l l y pure AMP-PNP would be as good an adenyl c y c l a s e s u b s t r a t e as ATP because i t was found t h a t ATP and AMP-PNP behaved i d e n t i c a l l y as the su b s t r a t e f o r the adenyl cyclase of r a t l i v e r plasma membranes (40). The major advantage of AMP-PNP over ATP i n the adenyl c y c l a s e assay i s that AMP-PNP i s a poor s u b s t r a t e f o r ATPases (40) and t h e r e f o r e the ATP-regenerating system i s not needed. AMP-PNP would be a u s e f u l adenyl c y c l a s e s u b s t r a t e f o r 70. k i n e t i c s t u d i e s of the enzyme and i n assays which r e q u i r e very low s u b s t r a t e c o n c e n t r a t i o n s ( 4 0 ) . 7 1 . CONCLUSIONS The F~ s t i m u l a t e d adenyl c y c l a s e a c t i v i t y i n homog-enates o f maturing chinook salmon t e s t i s was examined w i t h r e s p e c t t o : (a) time and temperature dependence, (b) Mg and M n 2 + requirement, ( c ) e f f e c t o f d i f f e r e n t P~ c o n c e n t r a -t i o n s , , (d) e f f e c t o f d e t e r g e n t s , (e) s t a b i l i t y , ( f ) sub-s t r a t e s p e c i f i c i t y and (g) s u b c e l l u l a r d i s t r i b u t i o n . The o p t i m a l temperature t o assay adenyl c y c l a s e i n salmon t e s t i s homogenates was 30°. T h i s was the h i g h e s t temperature examined a t which the enzymic r e a c t i o n was l i n e a r f o r over 30 min. Changes i n the r a t e o f p r o d u c t i o n o f c y c l i c AMP from ATP w i t h time a t 37° i n d i c a t e d t h a t the enzyme may not be s t a b l e a t 3 7 ° . The d i v a l e n t c a t i o n requirement of the enzyme was f i l l e d by e i t h e r Mg 2 + or Mn 2 +; o p t i m a l c o n c e n t r a t i o n s o f the 2 c a t i o n s were 1 0 to 1 5 mM and 5 to 1 5 mM, r e s p e c t i v e l y . There i s no e x p l a n a t i o n f o r the g r e a t e r maximal enzymic s t i m u l a t i o n by Mn + than by M g 2 + which was observed not on l y i n the a c t i v i t y o f salmon t e s t i s homogenates but a l s o i n the adenyl c y c l a s e a c t i v i t y from a v a r i e t y o f other sources ( 1 1 ,37) The b a s a l a c t i v i t y o f salmon t e s t i s a d e n yl c y c l a s e was s t i m u l a t e d 7 - f o l d by 12 mM P". T h i s i s comparable to the 72. maximal F ~ - s t i m u l a t e d i n c r e a s e i n adenyl c y c l a s e a c t i v i t y observed i n v a r i o u s t i s s u e p r e p a r a t i o n s from other species ( 3 7 ) . P r e l i m i n a r y s t u d i e s on detergent treatment of salmon t e s t i s homogenates i n d i c a t e d that Tween 80 and Nonidet P40, but not T r i t o n X100 nor L u b r o l PX might be u s e f u l as a i d s to s o l u b i l i z i n g the c a t a l y t i c adenyl c y c l a s e a c t i v i t y i n t h i s t i s s u e . I t was p r e v i o u s l y s t a t e d t h a t the adenyl c y c l a s e a c t i v i t y i n salmon t e s t i s homogenates was very u n s t a b l e ( 3 3 ) , but r e s u l t s g i v e n i n t h i s Thesis i n d i c a t e t h a t the enzyme i s s t a b l e f o r up to 6 hours at 0° to 4°. The en-zyme, however, was t o t a l l y i n a c t i v e a f t e r storage f o r 1 day at 0° to 4°, and was very unstable when s t o r e d at room temperature (24°). The s u b s t r a t e s p e c i f i c i t y of the salmon t e s t i s enzyme was extended to i n c l u d e the ATP analog, AMP-PNP. The analog was s y n t h e s i z e d from adenosine-5' phosphorimidazolate and tributylammonium imidodiphosphate by the general method of Hoard and Ott ( 2 1 ) . The AMP-PNP p r e p a r a t i o n was contaminated w i t h an i m p u r i t y , probably ADP-NH2* which was not q u a n t i t a t e d and thus the y i e l d of AMP-PNP was estimated t o be somewhat l e s s than 24%. Pure AMP-PNP would probably be a good s u b s t r a t e f o r the salmon t e s t i s 73. enzyme i n assaysT-requiring low s u b s t r a t e c o n c e n t r a t i o n and an absence of the ATP-regenerating system. Adenyl c y c l a s e a c t i v i t y was found i n a l l the s u b c e l l -u l a r f r a c t i o n s of salmon t e s t i s homogenates. The l o c a l i z a -t i o n of about 55% of the adenyl c y c l a s e a c t i v i t y i n the 600g sediment and oriby about 10% of the a c t i v i t y i n the 105»000g supernatant was c o n t r a r y to a former r e s u l t ( 3 3). The s i g n i f i c a n c e of the h i g h s p e c i f i c a c t i v i t y i n the 6300g sediment i s unknown. F i n a l l y , some ideas f o r f u t u r e experimentation w i t h the adenyl c y c l a s e a c t i v i t y i n the developing salmonid t e s t i s are proposed. Experiments d i r e c t e d towards d e f i n i n g hormonal c o n t r o l s of t e s t i c u l a r a c t i v i t y may y i e l d i n t e r e s t -i n g r e s u l t s . S t i m u l a t i o n of the adenyl c y c l a s e a c t i v i t y i n salmon t e s t i s s l i c e s w i t h salmon p i t u i t a r y e x t r a c t s has a l r e a d y been demonstrated ( 3 3 ) . A s e p a r a t i o n of the d i f f e r e n t types of germ c e l l s w i t h i n the developing salmonid t e s t i s i s p o s s i b l e ( 3 0 ) . I f adenyl c y c l a s e ac-t i v i t y can be demonstrated i n any of these c e l l s , a study of the e f f e c t s of salmon p i t u i t a r y e x t r a c t s (8) on t h i s a c t i v i t y would be u s e f u l i n e l u c i d a t i n g hormonal c o n t r o l of spermatogenesis. The e f f e c t of some s t e r o i d hormones i s mediated by c y c l i c AMP (3 7 ) . Testosterone s t i m u l a t e s the adenyl c y c l a s e 74. i n r a t seminal v e s i c l e s and c y c l i c AMP has been shown to s t i m u l a t e s e v e r a l key g l y c o l y t i c and hexose monophosphate shunt enzymes i n seminal v e s i c l e s of r a t s (44) . These f i n d i n g s l e a d one to speculate whether the s t i m u l a t o r y e f f e c t of androgens on spermatogenesis w i t h i n the t e s t i s of hypophysectomized t e l e o s t s (29) might be mediated by c y c l i c AMP. Therefore, not only g o n a d o t r o p i c s ) but a l s o the t e s t i c u l a r androgens of salmonids should be t e s t e d f o r t h e i r e f f e c t on salmon t e s t i s adenyl c y c l a s e . The c a t a l y t i c a c t i v i t y of salmon t e s t i s adenyl c y c l a s e i s f a i r l y low compared w i t h a c t i v i t i e s i n other t i s s u e s from other species ( 1 0 ) . 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