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Some studies on adenylate cyclase in brain Ma, Yvonne Suk-Fong 1972

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c( SOME STUDIES ON ADENYLATE CYCLASE IN BRAIN by YVONNE SUK-FONG MA B. Sc., (Hons.), University of British Columbia, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Pharmacology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1972 In present ing th i s thes is in part i al f u l f i lmen t o f the requirements fo r an advanced degree at the Un ive rs i t y of B r i t i s h Columbia, I agree that the L ib ra ry sha l l make it f r ee l y ava i l ab le for reference and study. I fu r ther agree that permission for extensive copying of th is thes is for s cho l a r l y purposes may be granted by the Head of my Department or by h is representa t i ves . It is understood that copying or pub l i c a t i on o f th i s thes is fo r f i nanc i a l gain sha l l not be allowed without my wr i t ten permiss ion. Depa rtment.of The Un ive rs i t y of B r i t i s h Columbia Vancouver 8, Canada Date t^L*-*-^-* (i) ABSTRACT The Gilman's cyclic AMP binding assay was used to examine the possibility of adopting this method for adenylate cyclase determinations. Cyclic AMP determinations were not invalidated by the reagents used in the adenylate cyclase reaction. Cyclic AMP measured by the binding assay was directly proportional to adenylate cyclase activity. Although variability in recovery of cyclic AMP was obtained, it could be reduced by performing triplicate assays. Thus, the cyclic AMP binding assay, with some reservations, would appear applicable for measuring adenylate cyclase activity. Adenylate cyclase in rat brain was studied by using the cyclic AMP binding method for determination of product formed. Rat brain cortex was fractionated by the method of Whittaker. The highest adenylate cyclase activity was found in the fraction containing the highest acetylcholin-esterase activity, and this fraction was shown by electronmicroscopic studies to be rich in synaptosomes. A modified sucrose gradient was used for isolating satisfactory synaptosomal fractions (the layer between 1.0 M and 1.1 M sucrose). Properties of synaptosomal adenylate cyclase were examined. The enzyme was dependent on the concentrations of ATP 2+ 2+ and Mg or Mn ion. The enzyme was stimulated by fluoride and inhibited by calcium ion. Synaptosomal adenylate cyclase was not sensitive to catecholamines or adenosine. No hormonal stimulation was obtained in the presence of GTP. In experiments where the effects of endogenous catecholamines were reduced by the addition of oC and (h (ii) adrenergic blocking agents or by prior treatment of the animals with reserpine, hormonal stimulation of adenylate cyclase in particulate prep-arations could not be demonstrated. (iii) T ABLE OF CONTENTS Page INTRODUCTION 1 EXPERIMENTAL PROCEDURE 19 I. Materials 19 II. Methods 20 A. Cyclic AMP Binding Assay 20 B. Adenylate Cyclase Assay 26 C. Acetylcholinesterase Assay 30 D. Subcellular Fractionation of Rat Brain Cortex 30 E. Electron Microscopy 37 F. Protein Determination 37 RESULTS 39 I. Studies on the Cyclic AMP Binding Assay 39 A. Preliminary Examination and Modification of Gilman's Method 39 B. Level of Cyclic AMP Measured in Heart and Liver Extracts 42 C. Recovery of Known Amounts of Cyclic AMP Added 42 II. Adenylate Cyclase Determination by the Cyclic AMP Binding Assay 42 A. Effects of Adenylate Cyclase Incubation Mixtures on the Binding Assay 42 B. Recovery of Known Amounts of Cyclic AMP in the Adenylate Cyclase Assay 45 (iv) TABLE OF CONTENTS (Continued) Page C. Correlation between Level of Cyclic AMP Measured and Enzyme Activity 50 D. Comparison with [ l 4 c ] - A T P Adenylate Cyclase Assay 52 III. Brain Adenylate Cyclase Studied with Cyclic AMP Binding Assay 59 A. Subcellular Distribution of Adenylate Cyclase in Rat Cerebral Cortex 59 B. Purification of Synaptosomal Fraction 65 C. Properties of Synaptosomal Adenylate Cyclase 69 D. Attempts to Demonstrate Hormonal Sensitivity in Brain Adenylate Cyclase 74 DISCUSSION 82 BIBLIOGRAPHY 87 (v) LIST OF TABLES No. Title Page I. Level of cyclic AMP measured in tissue extract 43 II. Recovery of cyclic AMP by the cyclic AMP binding method 44 III. Recovery of cyclic AMP in the adenylate cyclase incubation by the binding assay 48 IV. Adenylate cyclase and acetylcholinesterase activities in rat brain subcellular fractions prepared by the method of Whittaker 60 V. Adenylate cyclase and acetylcholinesterase activities in sub synaptosomal fractions prepared by a second sucrose density gradient 66 VI. Adenylate cyclase and acetylcholinesterase activities in fractions prepared by a Fi c o l l -sucrose density gradient 68 VII. Adenylate cyclase and acetylcholinesterase activities in fractions prepared by a modified sucrose density gradient 70 VIII. Effect of epinephrine on synaptosomal adenylate cyclase 76 DC. Effects of various agents on synaptosomal adenylate cyclase 77 X. Effect of norepinephrine on adenylate cyclase from rat brain homogenized in the presence of oC and adrenergic blocking agents 79 XI. Effects of hormones on reserpinized rats 80 XII. Effect of GTP on hormonal stimulation 81 (vi) LIST OF FIGURES No. Title Page 1. Schematic representation of hormone action on adenylate cyclase complex 4 2. An outline for the preparation of cyclic AMP binding protein 22 3. A sketch of the standard curve for the cyclic AMP binding assay 27 4. A flow diagram for the preparation of primary subcellular brain fractions 32 5. Submitochondrial fractionation in a sucrose density gradient 33 6. Preparation of synaptosomes with a Ficoll-sucrose density gradient 35 7. Preparation of synapto somes in a modified sucrose density gradient 38 8. A standard curve for the cyclic AMP binding method 40 9. The relationship between the amount of cyclic AMP measured by the binding assay and the aliquot of adenylate cyclase control sample used 46 10. Recovery of cyclic AMP in the adenylate cyclase incubation by the binding assay after BaZn preci-pitation or Dowex chromatography 51 11. Amount of cyclic AMP measured by the cyclic AMP binding assay with respect to the concen-tration of brain protein used 53 12. Amount of cyclic AMP measured by the cyclic AMP binding assay as a function of incubation time in the adenylate cyclase reaction 54 (vii) LIST OF FIGURES (Continued) No. Title Page rl 4 1 13. Comparison between the L C J-ATP adenylate cyclase assay and the coupling of cyclic AMP binding assay to adenylate cyclase determination at different brain protein concentrations 56 14. Comparison between the C*^c]-ATP adenylate cyclase assay and the coupling of cyclic AMP binding assay to adenylate cyclase determination at different times of incubation 57 15. Electronmicrographs of Sj-C submitochondrial fraction 62 16. Effect of ATP concentration on synaptosomal adenylate cyclase 72 17. Effects of ions on synaptosomal adenylate cyclase 73 ABBREVIATIONS ATP ADP AMP cyclic AMP GTP cyclic GMP ATCH EDTA Tris DEAE PPO POPOP adeno sine 51 -tripho sphate adenosine 5'-diphosphate adenosine monophosphate adenosine S'.B'-cyclic monophosphate guano sine 5'-tripho sphate guano sine 3', 5'-cyclic monophosphate acetyl thio choline ethylene diamine tetraacetate tris (hydroxymethyl) aminomethane N, N - di ethyl amino ethyl ethe r 2, 5-diphenylonazole 1, 4-bis [2-(5-phenylonazolyl)]-benzene (ix) ACKNOWLEDGEMENTS I am very fortunate to have Dr. G. I. Drummond as my supervisor for this project. He is an excellent teacher. Through his patience and guidance, I have acquired much valuable experience. I would like to express my deepest gratitude for his encouragement and instruction during the course of this work. I am also grateful to Dr. M. C. Sutter and Dr. D. Godin for many useful discussions. I wish to thank Dr. P. Sulakhe, Dr. P. Gray, Dr. D. Severson, Dr. S. W. French and Miss S. Hemmings for their technical assistance and advice. I wish to express my appreciation to my parents, the Pharmacology Department and the Medical Research Council of Canada for financial support. 1 INTRODUCTION All living organisms have a constant internal environment despite continuous external influences and complex, dynamic cellular activities. The discovery and exploration of the mechanisms of biological regulation which control and integrate all vital physiological and metabolic processes have provided a fascinating theme in science. Two regulatory systems, the nervous and the endocrine, have evolved to play a role in the animal kingdom. Nervous control is rapidly established, signalled by the number of electrical impulses passing along the nerve-target junction. The endocrine system provides sustained effects. Specific hormones are produced to control activities at specific organs. The biological significance of the two regulatory systems has been widely studied in different tissues and in different organisms. Regulation by Endocrine Systems Studies on the biochemical events of hormone action began in 1957 (1, 2) when Sutherland and his coworkers discovered that the hepatic glycogenolytic effect of epinephrine and glucagon was intracellularly mediated by a heat stable factor, which they isolated and characterized as adenosine S'.S'-cyclic monophosphate (cyclic AMP). Since then, the ubiquity of cyclic AMP in living organisms and its involvement as a second messenger to many regulatory hormones has been intensely investigated. These findings have established that cyclic AMP has many physiological and metabolic roles in such diverse cell functions as synthesis 2 of proteins, glycogenolysis, lipolysis, steroidogenesis, active transport, morphogenesis, etc. The participation of cyclic AMP in neural transmission and metabolism has also been implicated. Hormonal control of cellular activity seems to be mediated by a change in the intracellular concentration of cyclic AMP (3 - 8). The level of cyclic AMP in cells is regulated by two enzymes. Adenylate cyclase (3, 9) catalyzes the formation of cyclic AMP from adenosine triphosphate (ATP). Adenosine 31, 5'-cyclic phosphate 3'phospho-hydrolase (phosphodiesterase) (10) catalyzes the hydrolysis of cyclic AMP to adenosine monophosphate (AMP). Hormones may exert their effects either by stimulation or inhibition of adenylate cyclase or of cyclic AMP pho sphodiesterase. Adenylate cyclase is widely distributed in nature. It is not only found in animal tissues (9, 11) but also in microorganisms (12, 13) and possibly in plants (14, 15). The enzyme is generally associated with cell membranes and has the characteristics of a lipoprotein (9). Hormonal stimulation of the enzyme has been demonstrated in particulate fractions from liver (9), kidney (16), adipose tissue (17, 18), heart (19) and skeletal muscle (20, 21). Solubilized preparations, while catalytic ally active, are usually unresponsive to hormonal stimulation. The mechanism of hormonal activation is frequently assumed to involve an allosteric interaction between the hormone and a receptor. The receptor could possibly be either the enzyme itself or a site on the membrane with which it is associated. Of great physiological significance is the specificity of 3 adenylate cyclase from different tissues to different hormones. For example, adenylate cyclase from liver was found to be stimulated by glucagon and epinephrine (3, 22 - 24), but the enzyme from kidney and toad bladder was found to be activated by neurohypophyseal hormones (25 - 28). More exciting is the finding that each hormone-sensitive receptor from the same enzyme preparation is different. In adipose tissue, adenylate cyclase could be stimulated simultaneously by seven hormones (17, 29 - 31). When epinephrine stimulation was blocked by a ^ adrenergic blocking agent, the glucagon activation remained. Mechanism for selective stimulation of adenylate cyclase has been dis-cussed by Hechter (32) and Rodbell (33, 34). Adenylate cyclase was sug-gested as an enzyme complex consisting of several discriminators, a transducer and an amplifier unit as shown schematically in Figure 1. Each hormone interacts with a separate and distinct discriminator. The effects of specific hormonal binding to discriminator are translated in the transducer resulting in activation of adenylate cyclase in the amplifier unit. A highly sensitive and efficient control system can thus be envisaged. Another interesting feature of adenylate cyclase demon-strated in broken cell preparations in all adult mammalian tissues is its sensitivity to stimulation by fluoride ion (9). Fluoride ion has been an important agent in understanding the mechanism of activation of the enzyme. Adenosine 31, 5'-cyclic monophosphate phosphodiesterase has a similar cellular distribution as adenylate cyclase. Unlike adenylate 4 D I S C R I M I N A T O R S H O R M O N E - ? * A M P L I F I E R T R A N S D U C E R A T P c y c l i c A M P E X T R A C E L L U L A R 4 C E L L M E M B R A N E > I N T R A C E L L U L A R Figure 1: Schematic Representation of Hormone Action on Adenylate Cyclase Complex 5 cyclase, it occurs in soluble as well as in particulate forms (10, 35, 36) "and is not sensitive to hormones. Little is known about the regulation of cyclic AMP phosphodiesterase activity. The enzyme in most mammalian tissues is stimulated by imidazole and inhibited by methylxanthines (3), puromycin (37), benzylisoquinoline alkaloid papaverine (38), phenothiazine tranquilizers (39), and reserpine (39). Not all phosphodiesterases are specific for cyclic AMP. Some have been reported to hydrolyze other cyclic nucleotides as well as cyclic AMP, especially cyclic GMP (40). In terms of the second messenger hypothesis, the production of cyclic AMP in response to a hormone would be required to initiate a metabolic or physiological event in the cell. Cyclic AMP can modulate the activity of a number of enzymes (41 - 44). Cyclic AMP was first shown to participate in a complex sequence of reactions resulting in activation of glycogen phosphorylase, the rate limiting enzyme of glycogenolysis. Cori (45, 46) and Sutherland (47) have demonstrated that glycogen phosphorylase in skeletal muscle and in liver occurred in two interconvertible forms. The conversion of inactive phosphorylase b to the active form a was brought about by a phosphorylation catalyzed by phosphorylase b kinase whose activity itself was subject to metabolic control. Ingeniously, Krebs and his associates (48) found that cyclic AMP was an allosteric cofactor in a reaction in which a protein kinase, termed by them phosphorylase b kinase kinase, catalyzed the phosphory-lation of inactive phosphorylase b kinase and converted the enzyme to its active form. Adenosine triphosphate served as phosphate donor in this reaction which was completely dependent on the presence of cyclic AMP; the apparent K m of cyclic AMP was 1 x 10" M. The cyclic AMP depen-dent protein kinase also catalyzed, at least in vitro, the conversion of active glycogen synthetase I to inactive glycogen synthetase D (49) and activated adipose tissue lipase (50, 51). In addition, caesin, protamine and histone were phosphorylated by the same protein kinase (52). Protein kinases have been isolated from many animal tissues (53) and are also present in Escherichia coli (54). The enzyme was found to contain two subunits (55, 56). Cyclic AMP bound to a regulatory subunit resulted in activation of the catalytic subunit. Based on these findings, Greengard and his collaborators have proposed that cyclic AMP exerts its intracellular effects by activation of specific protein kinases (54). Regulation by Neural Mechanisms The regulatory mechanisms of the nervous system have been studied extensively. The findings are less conclusive than the endocrine system. One important drawback is the fact that neurotransmitter, or neurotransmitters, in the mammalian central nervous system have as yet to be identified. Because cyclic AMP is of considerable physiological significance as a mediator of hormone action in the endocrine system, a similar regulatory role of the cyclic nucleotide in the nervous system has been postulated (32). The mammalian nervous system is centrally controlled by activi-ties in the brain and in the spinal cord with messages transmitted to and from all parts of the body via the peripheral nerves. Although nervous tissue is a heterogenous mass of nerve and glial cells, the nerve cell is distinctive and carries all the electrical messages. The nerve cell con-sists of a cell body and elongated cytoplasmic axonal and dendritic processes where electrical impulses can travel long distances. A striking anatomical feature at the interneuronal or neuromuscular junction is the synapse. The synapse is composed of three parts: pre-synaptic nerve terminal, the synaptic cleft, and the postsynaptic mem-brane. The presynaptic nerve terminal appears swollen and is packed with vesicles. It makes intimate contact with the thickened membrane of the postsynaptic neuron or muscle through a synaptic cleft. On mild homogenization of nerve tissue, synapses separate from the nerve fibres and form closed membranous structures called synaptosomes (57). Eccles (58) and Katz (59) have provided strong evidence that the synapse is the site of interneuronal or neuromuscular transmission. Neural transmission is mediated by a chemical transmitter or neurohormone which is stored in the synaptic vesicles and released quantally from the presynaptic nerve ending in response to electrical activity. The neurotransmitter then acts on the postsynaptic membrane to stimulate or inhibit ion fluxes resulting in changes of membrane potential. Peripheral nerves are classified as cholinergic or adrenergic. Cholinergic nerves utilize acetylcholine as the chemical transmitter, while adrenergic nerves utilize catecholamines as neurohormones. The identity of neurotransmitters in the central nervous system is not settled, although there are many attractive candidates. Potential neuro-hormones are norepinephrine, dopamine, histamine, serotonin, acetyl-8 cho l i ne , g lu tamate and -am inobu t y r a t e . T h e s e subs tances a r e c o n -cen t r a t ed in a v a r i e t y of ne r ve end ings in the b r a i n (57, 60). Whi le the p r o c e s s of t r a n s m i t t e r r e l e a s e and the m e c h a n i s m of synapt i c t r a n s -m i s s i o n r e q u i r e e luc ida t i on , the p o s s i b l e ro l e of c y c l i c A M P in ne r ve funct ion and m e t a b o l i s m has s t imu l a t ed m a n y i n ve s t i g a t i ons . A d e n o s i n e 3 1 , 5 ' - cyc l i c monophosphate i s p r e s e n t in the ne r vous s y s t e m . Concen t r a t i ons as h igh as 5, 000 m o l e s pe r m g of synapt i c p r o t e i n o r 5, 000 to 10, 000 m o l e c u l e s p e r synapt i c v e s i c l e have been r e p o r t e d r e c e n t l y by Johnson (61) i n ne r ve endings of ra t c e r e b r a l Cor tex . Changes i n the l e v e l o f b r a i n c y c l i c A M P were shown to be a s s o c i a t e d w i th n e u r a l a c t i v i t i e s . S e i z u r e o r e l e c t r o c o n v u l s i v e shock l eads to e l eva t i on of b r a i n c y c l i c A M P (62, 63). T h e ne r vous s y s t e m a l so conta ins the e n z y m e s regu la t ing the i n t r a c e l l u l a r concen t r a t i on of c y c l i c A M P . A m o n g m a m m a l i a n t i s s u e s , the b r a i n has the m o s t ac t i ve adeny la te c y c l a s e (9) and p h o s p h o d i e s t e r a s e (10, 36), a l though r e l a t i v e e n z y m e spec i f i c a c t i v i t i e s d i f fe r in d i f fe rent s p e c i e s and in d i f fe rent b r a i n a r e a s (64, 65). In the g r e y ma t t e r of the c e r e b r a l co r t ex where there a r e concen t r a t ed s to res of ne r ve c e l l bod ies and s y n a p s e s , adeny la te c y c l a s e ac t i v i t y is at l eas t 22 t i m e s h i g h e r , on a p r o t e i n b a s i s , than that of the l i v e r . Subce l l u l a r f r a c t i o n a -t ion s tud ies by De R o b e r t i s and h is c o w o r k e r s (35, 65) r e v e a l e d that m o s t of ra t b r a i n adenylate c y c l a s e is pa r t i cu l a t e , 6 0 % of the total e n z y m e ac t i v i t y in the homogenate was found in the m i t o c h o n d r i a l f r a c t i on (10, OOOxg pe l l e t ) . S u b m i t o c h o n d r i a l f r a c t i ona t i on in a d i s con t inuous sucrose density gradient showed that most of the adenylate cyclase was actually present in the synaptosomal fraction. These findings are important because they indicate that, at the subcellular level, there is a correlation between the distribution of the cyclic AMP forming system and neurohormones. Loh et al (66) have studied the effects of neurohormones on brain adenylate cyclase in vivo. By direct administration of monoamines into the central nervous system, norepinephrine and histamine caused pro-longed dose-dependent activation of brain adenylate cyclase. Weiss and Costa (67) have examined the properties of adenylate cyclase from rat pineal gland. They found that the enzyme was stimulated in vitro by fluoride ion and catecholamines (L-epinephrine, L-norepinephrine and isoproterenol). The catecholamine effect on rat pineal adenylate cyclase was blocked by 3^ adrenergic blocking agents. Brain adenylate cyclase in broken cell preparations was also highly sensitive to fluoride ion. However, hormonal stimulation of the enzyme was generally small, and stimulation was difficult to reproduce. In many studies, adenylate cyclase from brain homogenates failed to respond consistently and decisively to hormonal treatment (63, 68 - 70). Only a few investigators have been able to demonstrate significant activation of the brain enzyme by cate-cholamines. Klainer et al (71) in 1962 showed that epinephrine caused a 10 to 80% increase in cyclic AMP formation with washed particulate cere-bellar preparations from dog, cat, sheep, pig, cow and steer; 25 to 125% increases in cyclic AMP were obtained with preparations from the cerebral cortex. In 1971, Drummond and his associates (72) reported that epinephrine caused a modest but significant and reproducible stimu-lation in 3, OOOxg washed pellet from rat cerebral cortex. McCune (73) also claimed that adenylate cyclase from rat brain was consistently (10 to 40%) activated by norepinephrine and (15%) stimulated by dopamine. Recently Greengard et_ al (74) demonstrated that norepinephrine or dopamine produced a 70% activation of adenylate cyclase from bovine superior cervical ganglia. In contrast to broken cell systems, brain adenylate cyclase is significantly stimulated by neurohormones in slice preparations from several species. By measuring the level of cyclic AMP in brain slices after a few minutes exposure to biogenic amines, Kakuichi and Rail (75 - 77) found that norepinephrine and histamine were effective in enhancing the accumulation of cyclic AMP. Their observations were later confirmed by Shimizu (78, 79) and Krishna (80). These workers r 14 1 used brain slices prelabelled with L C J-adenine and measured the amount of [^C 3-cyclic AMP formed when slices were incubated with various agents. An 18-fold increase in the cyclic AMP was obtained after 3 to 6 min exposure of rabbit or guinea pig cerebellar slices to norepinephrine, a 10-fold increase occurred in the presence of histamine, and a 3-fold increase was produced by serotonin. When rabbit or guinea pig cerebral slices were incubated, norepinephrine caused a 70% increase in cyclic AMP in 6 min in both cases, and histamine stimulated 25-fold and 10-fold respectively. Cyclic AMP level in human cerebral slices 11 also responded to biogenic amines (81, 82). Although stimulation by catecholamines varied greatly among species and brain areas, the hor-monal effect on cyclic AMP levels in brain slices was markedly enhanced in the presence of theophylline, a methylxanthine inhibitor of cyclic AMP phosphodiesterase. Studies with incubated brain slices became more exciting when Rail (83 - 85) and Shimizu (86) found that exposure of slices from guinea pig cerebral cortex to 40 mm KC1 for 4 min led to a 10-fold increase in the intracellular level of cyclic AMP. In addition, adenosine, electrical stimulation and depolarizing agents were also capable of stimulating the production of cyclic AMP in guinea pig cortical slices. In contrast to the stimulatory effect of catecholamines, the actions of adenosine and depolarizing agents were antagonized by theophylline. Incubation of cerebral or cerebellar slices with other possible neurotransmitters such as acetylcholine, dopamine, glutamate and "Jf -aminobutyrate caused little or no change in the level of cyclic AMP. The effects of catecholamines and electrical stimulation in elevating the level of cyclic AMP in brain slices may be physiologically important. Intracellular levels of cyclic AMP in reaggregated brain cell cultures from embryonic mouse brain are also increased 4 to 6-fold by norepinephrine (87). With isolated rabbit and bovine superior cervical ganglia, norepinephrine, dopamine or electrical stimulation produced a several-fold increase in cyclic AMP content (74, 88). Gilman (89) has also observed a stimulatory effect of prostaglandin (PGE, ) on cyclic AMP 12 accumulation in neuronal tumor cells. Some information on the specificity of brain adenylate cyclase was obtained from experiments performed by measuring cyclic AMP levels in brain slices. The enzyme was shown to have specific receptors for interaction with hormones. Schmidt et al (90) could not detect norepinephrine stimulation in brain slices obtained from rats in their first 3 days postpartum. They claimed that the adenylate cyclase receptor for catecholamine had not developed. The receptor for the response of the brain slices to histamine has been pharmacologically distinguished from the adrenergic receptor responsible for the effects of catecholamines. While norepinephrine response in rat brain was blocked by ec and 3 adrenergic blocking agents and psychotropic drugs, the histamine effect was maintained unless an antihistaminic agent was present (91). The effect of simultaneous addition of maximal amounts of norepinephrine and histamine was approximately equal to the sum of the effects of the agents tested individually. Adenosine 3*, 5'-cyclic monophosphate may be involved in the regulation of brain function by mediating the actions of catecholamines. Evidence was provided from studies on the changes in the level of cyclic AMP in response to neurohormones and electrical stimulation. The actions of cyclic AMP in the central nervous system were also confirmed from investigations made by direct application of theophylline, an inhibitor of cyclic AMP degradation, or cyclic AMP, usually the dibutyryl derivative of cyclic AMP which is not susceptible to phosphodiesterase hydrolysis. 13 Adenosine 3', 5'-cyclic monophosphate is possibly associated with synaptic transmission. Greengard et al (74) have shown that dopamine caused an accumulation of cyclic AMP in isolated bovine superior cervical ganglion whose interneurons contain dopamine. An oC adrenergic antagonist, phentolamine, abolished the increase in cyclic AMP produced by dopamine. Electrical stimulation of the preganglionic nerve fibres also resulted in an increase of cyclic AMP which was blocked by phen-tolamine. Hence, they proposed that cyclic AMP mediates the synaptic activity of dopamine when preganglionic stimulation caused a release of dopamine from the interneurons. Bloom and his coworkers (92 - 94) have demonstrated that cyclic AMP mediates the action of norepinephrine in Purkinje cells. They found that in the cerebellar cortex there were norepinephrine containing afferents which make direct axo-dendritic and axo-somatic synapses with Purkinje cells, and this projection served to inhibit the Purkinje cell. With extracellular recording of spontaneous activity, applied cyclic AMP and its dibutyryl derivative mimicked the inhibitory action of norepinephrine. They also indicated that norepinephrine inhibition of Purkinje cells is the same as cyclic AMP inhibition which is brought about by hyperpolarization of the neuronal membrane resulting in an increase of transmembrane resistance. Adenosine 3',5'-cyclic monophosphate was also suggested to facilitate the effect of epinephrine in promoting neuromuscular transmission. First, Birks and Macintosh (95) had shown that epinephrine resulted in increased acetylcholine release by the superior cervical ganglion. 14 In 1958, Krnjevic and Miledi (96), using the phrenic nerve diaphragm preparation, demonstrated an increased frequency of miniature end-plate potentials and increased amplitude of the end-plate potential in response to epinephrine. Then Breckenridge (97) observed that theophylline potentiated the effects of epinephrine on the sciatic-gastronemius preparation of the cat. This potentiation was blocked by propranolol, a (3 adrenergic blocking agent, and did not occur in a curarized preparation. Later, Goldberg (98) also reported that dibutyryl cyclic AMP or theophylline, like epinephrine, increased the amplitude of the end-plate potential in isolated rat diaphragm, and increased the frequency of miniature end-plate potentials. The action of cyclic AMP in releasing acetylcholine from motor neurons was discussed by Breckenridge et al (99). They suggested that cyclic AMP acts by accelerating the rapid axoplasmic flow of synaptic vesicles or neurosecretory particles along neurotubules. Their suggestion was based on their finding that adenylate cyclase accumulates proximal to an occlusion in the chicken sciatic nerve, and morphological studies have indicated that adenylate cyclase is associated with an intracellular organelle which is moved by axoplasmic flow. Work has also been reported which suggests that cyclic AMP mediates the action of norepinephrine on the production of N-acetyl sero-tonin and melatonin in the pineal gland. Norepinephrine and dibutyryl cyclic AMP were found to be potent stimulators of melatonin synthesis (100, 101). Klein and his colleagues (10Z, 103) have shown that dibutyryl cyclic AMP and theophylline, like norepinephrine, stimulated the conversion oi erotonin to [ C ] -melatonin and elevated the concentration of C^4C 3-N-acetyl serotonin in the incubation media of pineal glands incubated with 0.1 mM [*^C ]-serotonin. The regulatory action of norepinephrine and dibutyryl cyclic AMP in melatonin synthesis was found to stimulate N-acetyl transferase, the enzyme which N-acetylates serotonin. By direct analogy to the role of cyclic AMP in endocrine controlled metabolism, cyclic AMP may also be mediating the effects of catecholamines in nerve metabolism. Cyclic AMP dependent glycogen synthetase (104) and cyclic AMP dependent protein kinase (53, 105) have been isolated from brain tissue. In addition, brain cyclic AMP level was found to increase during anoxia following decapitation (62). All these investigations suggest that cyclic AMP has a significant role in nerve function. It may be an important mediator of catecholamine action and it may also mediate the effects of adenosine and electrical stimulation. Why is hormonal activation of adenylate cyclase in broken cell preparations difficult to demonstrate consistently? It is possible that in vitro conditions required for hormonal activation of the brain enzyme have not been established. A better understanding of the conditions required to detect hormonal sensitivity could elucidate the nature of hormone specificity and of the actions of cyclic AMP in the nervous system. Such an investigation represents part of the work described in this thesis. 16 Methods for Determination of Cyclic AMP and Adenylate Cyclase Activity Cyclic AMP has attracted much attention in biological research. Many investigations have been carried out to explore the actions of the cyclic nucleotide in cellular regulation. To cope with the studies on cyclic AMP, simple and sensitive methods are required for measuring adenylate cyclase activity and cyclic AMP levels in tissues. In recent years, several facile methods have been developed for cyclic AMP deter-mination. A cyclic AMP binding assay has been described by Gilman (106). The method is highly sensitive and specific for cyclic AMP. It can detect as little as 0. 05 to 0. 10 ^ moles of cyclic AMP, and extensive work-up of the tissue samples is frequently not necessary. The principle of the cyclic AMP binding assay is based on the competition of tritiated and unlabelled cyclic AMP for a specific protein binding site. The binding protein is a partially purified cyclic AMP dependent protein kinase. Affinity of the binding protein for cyclic AMP is enhanced by the addition of a heat stable proteinaceous inhibitor of cyclic AMP dependent protein kinase. Maximal binding is achieved at pH 4. The binding reaches equilibrium after one hour of incubation at 0° at a saturating concentration of tritiated cyclic AMP in the presence or absence of unlabelled nucleotide. The cyclic AMP-protein complex is then adsorbed on a cellulose ester millipore filter. The amount of tritiated cyclic AMP in the protein complex is determined by scintillation spectrometry. Lower counts are recorded in samples with higher concentrations of unlabelled cyclic AMP added, because binding of tritiated cyclic AMP to the binding protein is 17 competitively diluted by unlabelled cyclic nucleotide. Unknown amounts of cyclic AMP can thus be determined by comparing the radioactivity measured with that obtained by standard amounts of unlabelled cyclic AMP. The methods for adenylate cyclase determination are based on the catalytic ability of the enzyme to convert labelled ATP to cyclic AMP. In the early studies of Sutherland, cyclic AMP formed was measured by a complex and tedious procedure involving a series of reactions leading to activation of glycogen phosphorylase and breakdown of glycogen. In 1968, radioactive ATP ( C 1 4 c ] - or [oC-32P 3 -ATP) was employed by Krishna, Weiss and Brodie (107). Labelled cyclic AMP formed was isolated by Dowex chromatography. The eluates containing cyclic AMP were sub-jected to barium hydroxide and zinc sulfate precipitation to remove the last traces of contaminating nucleotides and inorganic phosphate. The radioactive cyclic AMP was then quantitatively determined by liquid scintillation spectrometry. In 1970, a simplier adenylate cyclase assay was developed by Drummond et al (19). Descending paper chromatography was used to separate the cyclic AMP formed during adenylate cyclase incubation. The cyclic AMP containing areas of the chromatograms were cut out and counted. This method is simple but has some limitations. The radioactive substrate is very expensive, and the assay could not be reliably used with labelled substrate above 0. 3 mM because of high background counts in the cyclic AMP area. With the availability of the simple and inexpensive cyclic AMP binding assay, it seemed appropriate to determine if this method of 18 cyclic AMP determination could be applied to measure the product formed during adenylate cyclase determinations. Unlabelled ATP could be used as substrate. Substrate concentration in the incubation could be higher than 0.3 mM. Tritiated cyclic AMP used in the cyclic AMP binding compounds. Many samples could be assayed in one experiment. Tripli-cate or quadruplicate assays could be performed. Small amounts of cyclic AMP formed during the adenylate cyclase incubation could be easily detected. Statement of the Problem The purpose of the present work was twofold: 1. To explore the possibility that determination of cyclic AMP by the specific binding assay could be used routinely for adenylate cyclase determination, particularly in brain preparations or more broadly as a general method of adenylate cyclase activity determination. 2. Hopefully with this assay, or other assays in current use, to examine the properties of neural adenylate cyclase. A primary objective was to explore possible procedures for demonstrating hormonal stimula-tion of the enzyme in broken cell fractions and in intracellular particulate preparations such as synaptosomes. method is much less expensive than [ C]-ATP or [oL-32p]-ATP. The or [ 3 2P] labelled EXPERIMENTAL PROCEDURE I. Materials Uniformly labelled L 1 4C 1-ATP (tetrasodium salt, 418 to 462 mCi per mmole) and C H J-cyclic AMP (ammonium salt, 24. 1 Ci per mmole) were obtained from New England Nuclear. Ethanol was removed from the commercial solutions under vacuum. C C J-Adenosine triphosphate was diluted with unlabelled ATP and water to give a specific activity of 18 to 22 microcuries per micromole. The solution was stored at -18°. t H ] -Adeno sine 3', 5'-cyclic monophosphate solution was diluted three hundred times with water to give a final specific activity of 80 microcuries per micromole. Unlabelled ATP (disodium salt) and cyclic AMP (phosphoric acid) were purchased from Sigma Chemical Co., St. Louis. Pyruvate kinase (rabbit muscle) and 2-phosphoenolpyruvate (trisodium salt) were obtained from Calbiochem. Various preparations of pyruvate kinase when assayed by the method of Bucher and Pfleiderer (108) catalyzed the phosphorylation of adenosine diphosphate with specific activities ranging from 64 to 219 micromoles per minute per milligram of protein. Hormones and drugs were obtained from the following sources: epinephrine (L-adrenaline bitartrate), K & K Laboratories, Plainsview, New York; norepinephrine (L-arternol-D-bitartrate), Mann Laboratories, New York; Isopropylnorepinephrine hydrochloride (isoproterenol hydro-chloride), Winthrop Laboratories, New York; D, L-propranolol, Ayerst Laboratories, Montreal; histamine dihydrochloride, Fischer Scientific 20 Co., Vancouver; serotonin creatinine sulfate (5-hydroxytryptamine creatinine sulfate), K &t K Laboratories, Plainsview, New York; adenosine, Nutritional Biochemicals Corporation, Cleveland, Ohio; phentolamine methane sulfonate (regitin-methansulfonate), Ciba Co. Ltd., Dorval, Quebec; theophylline. Western Laboratories Ltd. , Vancouver; pentobarbital (nembutal), British Drug House Canada Ltd., Toronto; serpasil (reserpine), Ciba Co. Ltd., Dorval^Quebec. Crystalline sucrose was purchased from Allied Chemical, Morristown, New Jersey. Ficoll was obtained from Sigma Chemical Co., St. Louis. DEAE cellulose (cellulose N, N-diethylaminoethyl ether) was purchased from Eastern Organic Chemicals, Rochester, New York. Dowex AG 50WX4, 200 to 400 mesh (H + form) was obtained from Bfo-Rad Laboratories, Richmond, California. Prior to use, the resins were washed with 1 N sodium hydroxide, then with water followed by 1 N hydrochloric acid. The resins were then washed extensively with water to remove fines. DEAE cellulose was stored in 5 mM potassium phosphate (K^PO^), pH 7. Dowex AG 50WX4 was suspended in water 50% (v/v). II. Methods A. Cyclic AMP Binding Assay - modified Method of Gilman 1. Cyclic AMP binding protein preparation Procedures were patterned after those of Walsh et al (48) and Miyamoto et al (109) for the preparation of protein kinase. The binding protein was purified up to the DEAE cellulose step. Fresh bovine 21 heart was obtained from the slaughter house and immediately stOTed in ice. Fat was removed and ventricles were sliced and stored at -80°. Frozen beef ventricles, 250 g, were thawed and chopped into small pieces. The tissue was homogenized for 2 min in a Waring Blendor in 3 vol of 4 mM EDTA, pH 7, and was processed at 4° as outlined in Figure 2. After centrifugation at 27, OOOxg for 30 min, the supernatant was adjusted to pH 4. 8 by dropwise addition of 1 N acetic acid and was kept in ice for 10 min before the precipitate formed was removed by sedimentation at 27, OOOxg for 30 min. The supernatant obtained was adjusted to pH 7 with 1 M K-jPO^, pH 7. 2. Ammonium sulfate crystals were slowly added (32.5 g (NH^^SO^ per 100 ml supernatant), and the mixture was stirred for 30 min. The precipitate formed was collected by centrifugation at 16, OOOxg for 20 min. The pellet was dissolved in 40 ml of 5 mM K 3 P 0 4 and 2 mM EDTA, pH 7. The solution was dialyzed for 14 hr against 20 vol of 5 mM K 3 F 0 4 and 2 mM EDTA, pH 7 with 3 changes of buffer. After the precipitate formed was removed by centri-fugation at 27, OOOxg for 30 min, the supernatant was applied onto a DEAE cellulose column 30 x 5 cm previously equilibrated with 5 mM K 3 P 0 4 and 2 mM EDTA, pH 7. The column was then eluted with 0.008 M K 3 P 0 4 and 2 mM EDTA, pH 7. After the first peak of protein had come off, the column was eluted with 0. 30 M KjPO^ and 2 mM EDTA, pH 7. The fractions containing the second peak of protein were pooled and concentrated by (Nr^^SC^ precipitation (42 g (NH^^SO^ per 100 ml of eluate). The precipitate was collected at 16, OOOxg for 20 min. 22 30% (w/v) homogenate in 4 mM EDTA, pH 7 27, 000 x g 30 min Pellet Supernatant I Pellet pH 4. 8 with 1 N HAc 27, 000 x g 30 min 1 Supernatant pH 7 with 1 M K,PO, 3 4 (NH 4) 2S0 4 precipitation, 32. 5 g/100 ml 16, 000 x g 20 min I Supernatant 1 Pellet dissolved in 5 mM K PC>4, pH 7 dialyzed 14 hr 27, 000 x g 30 min Supernatant DEAE cellulose column I , 1 Pellet 0. 008 M K 3P0 4, pH 7 Protein Peak 1 0. 3 M KoPO,, pH 7 Protein Peak 2 I (NH^J^SO^ precipitation 42 g/100 ml 16, 000 x g 20 min r — J Supernatant Pellet I dissolved in 5 mM K,P0 4, pH 7 I dialyzed 14 hr stored Figure 2: An Outline for the Preparation of Cyclic AMP Binding Protein The pellet was dissolved in 5 ml of 5 mM K^PC^ and 2 mM EDTA, pH 7. The solution was dialyzed against 20 vol of the same buffer for 14 hr with 3 changes of buffer. The precipitate formed was removed by sedimentation at 27, OOOxg for 30 min. The supernatant was the cyclic AMP binding protein solution. It was diluted with 5 mM K^PO^ and 2 mM EDTA, pH 7. In an early preparation, cyclic AMP binding protein was stored in a solu-tion of protein concentration 1. 3 mg per ml at -18°. The protein was found to lose binding activity upon repeated freezing and thawing. In later preparations, cyclic AMP binding protein was dispensed in 2 ml glass ampules (65 ywg in 0.3 ml, or 130 jug in 0.6 ml) and stored at -80°. Immediately before use, the contents of each ampule were diluted with an equal volume of water. 2. Protein kinase inhibitor protein preparation The preparation of inhibitor protein was modelled after that of Appleman et al (110). Frozen rabbit skeletal muscle was obtained from Pel Freeze Biologicals, Inc., Arkansas. Minced rabbit muscle, 450 g, was homogenized in 10 mM Tris HCl, pH 7. 5, and was boiled for 10 min. After removal of particulate materials by filtration under vacuum, the filtrate was precipitated with 1/9 vol of 50% trichloroacetic acid. The precipitate was collected at 15, OOOxg and dissolved in water, and the pH was adjusted to 7 with 1 N sodium hydroxide. This fraction was dialyzed against water at room temperature overnight, and the precipitate which formed was discarded. The final protein con-centration in the supernatant was 2.6 mg per ml. 24 3. Tissue extracts Fresh pieces of rat liver and rat heart were frozen in liquid nitrogen. Weighed tissue samples (80 to 100 mg wet weight) were homogenized in 5 ml of 5% trichloroacetic acid in a No. C Potter Elvehjem homogenizer with 30 passes. After centrifugation at 8, OOOxg for 10 min at 4°, 0. 5 ml 1 N hydrochloric acid was added to the supernatant, which was then extracted 5 times with 10 ml of ether. Last traces of ether were removed from the extract with a stream of nitrogen. The extract was dried in a Virtis lyophilizer and stored at -80°. For the cyclic AMP binding assay, the dried residue was diluted with 50 mM sodium acetate/acetic acid, pH 4. 4. Cyclic AMP standard Unlabelled cyclic AMP (phosphoric acid) was dissolved in water to give a stock solution of concentration 2 nmoles per ml and stored at -18°. Just before the cyclic AMP binding assay, a solution of 200 f> moles per ml was prepared by diluting the stock solution 10 times with water. Two to ten yo moles (10 to 50 /x\ of solution) of cyclic AMP were used routinely in constructing the standard curve. 5. Cyclic AMP binding assay mix The assay mix for the cyclic AMP binding assay contained 3 components: 1. Fifty ml of 100 mM sodium acetate/acetic acid, pH 4. 2. Twenty ml of protein kinase inhibitor protein (2. 6 mg protein per ml). 25 3. Thirty ml of [ rl]-cyclic AMP (67 ^ moles per ml or 1 x 10 5 DPM per ml). 6. Cyclic AMP binding assay (106) The standard binding reaction was conducted in ice in a final vol of 200 jul: lOOyjl of assay mix containing 2 ^ moles [ 3H ]-cyclic AMP, 10 to 70 yul of sample or cyclic AMP standard, and 30^1 cyclic AMP binding protein (3.2 jug protein per 30/<1). Sufficient binding protein was added to bind less than 30% of the L 3H 3-cyclic AMP. Reactions were initiated by addition of binding protein. Two blank reaction tubes contained water instead of binding protein. Equilibrium o was reached in one hr at 4 . The mixtures were diluted with 1 ml of cold 20 mM K^PO^, pH 6, after 1 hr and 15 min of incubation. Four to five min later, they were passed through 24 mm millipore filters (cellulose ester, HAWP 02400, HA 0.45 JJ, 25 each, white plain, 24 mm), previously rinsed with the same buffer. The filters were then rinsed with 8 ml of cold 20 mM K-^PO^, pH 6, and were placed into clean glass counting vials and dried at 150° F for one hr. Five ml of scintillation fluid containing 4 g PPO and 50 mg POPOP in litre of toluene was added to each vial. The radioactivity was determined at room temperature in a Nuclear Chicago Isocap/300 model fluid counter. The efficiency for [ H J measured was approximately 40%. Efficiency for each sample was read from a quench curve obtained by plotting channel ratio against efficiency given by quenched [ H ] standards. The CPM recorded for each sample were corrected to DPM. The blank count (usually less than 26 400 DPM) was subtracted from the total DPM. A standard curve was drawn on log-log graph paper with DPM against total ^moles of cyclic AMP ([ H ] - cyclic AMP and unlabelled cyclic AMP standard) present in each tube. As illustrated in Figure 3, a reduction in DPM was observed with increasing amounts of unlabelled cyclic nucleotide added per tube. A straight line relationship was obtained on a logarithmic plot between 2 to 12 moles of total cyclic AMP per tube. Amounts of cyclic AMP in an unknown sample with a given DPM could thus be read from the standard curve. B. Adenylate Cyclase Assay 1. Brain enzyme preparation Guinea pigs (500 to 600 g) or rats (200 to 250 g) were anesthetized with pentobarbital (35 mg per kg) intraperitoneally, and bled by severing the jugular vein. Whole cortex or brain was removed and was washed in 10 mM Tris HC1, pH 8. The wet tissue was weighed and homogenized in 10% (w/v) with Tris-buffer in a Potter Elvehjem homogenizer with 10 passes. The homogenate was diluted to 4% (w/v) with Tris-buffer and centrifuged at 3, OOOxg for 10 min. The pellet was washed with Tris-buffer in a vol equal to the 4% homogenate and recentrifuged at 3, OOOxg for 10 min. The washed pellet was then suspended in Tris-buffer to form a 10% suspension (w/v). The suspension was stored in ice and assayed immediately. The protein concentration used for each adenylate cyclase assay was between 6 to 150 >ug per tube. Figure 3: A Sketch of the Standard Curve for the Cyclic AMP Binding Assay 28 r 1 4 1 2. i C J-ATP adenylate cyclase assay The procedure was the same as described by Drummond et al ( 72.). The assay mixture contained the following in a vol of 150 40 mM Tris HC1, pH 7.5; 20 mM phosphoenolpyruvate (PEP), approximately 2 international units of pyruvate kinase and 6 mM KC1 were employed for ATP regeneration; 8 mM theophylline and 2 mM unlabelled cyclic AMP to minimize hydrolysis of labelled product by cyclic AMP phosphodiesterase; 18 mM MgS0 4 and 0. 3 mM C 1 4 C 3-ATP (20 ^uCi per yimole) were used as substrate complex. Enzyme was added last. A control tube contained water instead of enzyme. Incubations were carried out for 5 min at 37°, and reactions were terminated by placing the tubes in a boiling water bath for 3 min. After the precipitated protein was removed by centrifugation at 8, OOOxg for 10 min, 100 1 of the supernatant was spotted on Whatman No. 3 chromatography paper. Nucleotides were separated by descending chromatography in a solvent system consisting of 1 M NH^ Ac and ethanol (15:35 v/v) at room tempera-ture for 18 to 22 hr. After drying, the cyclic AMP spot was identified under ultraviolet light. The area was cut out and placed in 18 ml of scintillation fluid (4 g PPO, 50 mg POPOP dissolved in 1 litre of toluene). The radioactivity was measured in a Nuclear Chicago Scintillation r 14 1 Spectrometer. The counting efficiency obtained for L C J was approxi-mately 6 5%. The amount of radioactive cyclic AMP formed was cal-r 14 i culated from the specific activity of the L C J-ATP substrate after correction for radioactivity present in the cyclic AMP area of control incubations. Specific activity of adenylate cyclase was expressed as 29 picomoles of cyclic AMP formed per minute per milligram of protein. 3. Coupling of cyclic AMP binding assay to adenylate cyclase assay The principle was identical to the L*^ C 3-ATP adenylate cyclase assay. However, unlabelled ATP (1 mM final concen-tration) was used as substrate, and unlabelled cyclic AMP was omitted. Assays were performed in triplicate. Cyclic AMP formed during the incubation was determined by the cyclic AMP binding assay. After ter-mination of the adenylate cyclase reactions by boiling, the clear super-natants obtained after centrifuging were stored at -18° until assay for cyclic AMP. The extracts generally required 5 to 20 times dilution with 50 mM NaAc/HAc, pH 4. Usually 50 ju 1 of the diluted extracts were added to the binding protein mixture, and duplicates were assayed. Any chemicals used in the adenylate cyclase incubation were examined for their effects on cyclic AMP binding. In recovery studies, known amounts of cyclic AMP were added to adenylate cyclase tubes before or after boiling. Experi-ments were also performed where Ba(OH) and ZnSO precipitation and Dowex chromatography (107) were utilized for removing other nucleotides, such as ATP, ADP and AMP, before cyclic AMP determination. In these experiments, adenylate cyclase incubation was carried out in a final vol of 450 After termination of the reaction, 1 5 0 1 of 0. 25 M ZnSO^ was added to each tube followed by 150yul of 0.25 M Ba(OH) 2. The thick, white precipitate was removed by centrifugation at 8, OOOxg for 10 min. 30 An aliquot (700 ju\) of the supernatant was diluted with 50 mM NaAc, pH 4, before assay for cyclic AMP. Where column chromatography was employed, boiled and centrifuged reaction mixtures (500 ju 1) were mixed with 10 yul of [ H]-cyclic AMP having a specific activity of 2, 000 counts per minute in 0.01 picomole. The solution was chromatographed on Dowex (Dowex AG 50WX4, 200 to 400 mesh, H + form). Columns were prepared by pipetting 2 ml of a 50% (v/v) suspension of the resin into a small glass tube (outer diameter 7 mm). The column was eluted with water, and the third and fourth ml eluates were collected in calibrated centrifuge tubes. The amount of L Hj-cyclic AMP recovered in these eluates was measured; recoveries ranged from 82 to 92%. Cyclic AMP present in the eluates was then determined by the binding method. . C. Acetylcholinesterase Assay Acetylcholinesterase activity was measured by the method of Ellman et al (111). The rate of hydrolysis of acetylthiocholine was deter-mined at 25° in a mixture containing 0. 1 M K^PO^, pH 7; 0. 3 mM acetyl-thiocholine, 0. 3 mM dithiobis-nitrobenzoate and protein. Increase in absorbance resulting from hydrolysis was followed at 412 nm. The specific activity of acetylcholinesterase was expressed in nanomoles of acetylthiocholine hydrolyzed per minute per milligram of protein. D. Subcellular Fractionation of Rat Brain Cortex 1. Preparation of primary fractions All procedures were carried out at 4° according to 31 the method of Whittaker (112). Rats (200 to 250 g) were anesthetized with pentobarbital (35 mg per kg) and bled through the jugular vein. The cortex was removed and washed in 20 ml of cold 0. 32 M sucrose, pH 7. 2, containing 50 >iM Tris HC1, 10 M MgS0 4 and 10 JJ> M EDTA. After weighing, the cortex was homogenized in 40% (w/v) of 0. 32 M sucrose-buffer in a Potter Elvehjem homogenizer with a loosely fitting teflon pestle (ten passes by hand followed by ten passes with an electric motor). The smooth homogenate was made to 10% (w/v) with 0. 32 M sucrose-buffer, and was filtered through 4 layers of cheesecloth. The filtrate was subject to a series of differential centrifugations as described in Figure 4 for the purpose of isolating the nuclear fraction (900xg pellet), the crude mitochondrial fraction (11, 500xg pellet) and the microsomal fraction ( l l , 500xg supernatant). 2. Preparation of submitochondrial fractions The crude mitochondrial fraction (11, 500xg pellet) was fractionated in a discontinuous sucrose density gradient as described by Whittaker (112). The sucrose gradient was composed of 0.8 M, 1.0 M and 1. 2 M sucrose (8 ml of each) in a 30 ml (1 in x 3 in) cellulose tube for the Spinco Rotor SW 25. 1. The gradient had been left in the cold room for at least 2 hr before use. The crude mitochondrial pellet (11, 500xg pellet) was obtained as indicated in Figure 4 and was dispersed in 5 ml 0.32 M sucrose, pH 7.2. Two to five ml of this suspension containing approximately 30 mg of protein was layered onto the sucrose gradient as shown in Figure 5A. Centrifugation was conducted at 0° at 50, OOOxg in a 32 Cerebral Cortex Homogenized in 6 vol of 0. 32 M sucrose, pH 7 10% (w/v) in 0. 32 M sucrose homogenate filtered through 4 layers of cheesecloth 900 x g 10 min I Pellet (Nuclear fraction) washed 2X and recentrifuged, combined all supernatants Supernatant 11, 500 x g 20 min Pellet (Mitochondrial fraction) washed IX and recentrifuged, " 1 Supernatant (Microsome and soluble protein) Figure 4: A Flow Diagram for the Preparation of Primary Subcellular Brain Fractions 33 Figure 5A Figure 5B '//////// 0 . 8 M 1.0 M 1.2 M 0 . 3 2 M 0 . 8 M 1.0 M Si Si - B s , - C Before Centrifugation 1.2 M V W ( ! ^ ^ V ^ V S l - D After Centrifugation Figure 5: Submitochondrial Fractionation in a Sucrose Density Gradient Sample was the crude mitochondrial pellet (11, 500xg pellet) suspended in 0. 32 M sucrose, pH 7. After 2 hour centrifu-gation at 50, OOOxg, fractions Sj-A, S^B, S^C and pellet S^-D were separated out. Fraction S^-A was the myelin layer, fraction S^-B was the nerve membrane fragment layer, fraction S^-C was the synaptosomal layer and pellet S^-D was the mitochondrial layer. 34 Model L Ultracentrifuge with Spinco Rotor SW 25. 1. After 2 hr, 4 distinct layers, fractions S^-A, Sj-B, S^-C and pellet S^-D, were evident as illustrated in Figure 5B. Each layer was removed quickly and care-fully with a pasteur pipette. Fraction S^-A (the layer between 0. 32 M and 0. 8 M sucrose) was diluted to 50% (v/v) with 0.15 M sucrose. Frac-tions Sj-B (the layer between 0. 8 M and 1.0 M sucrose) and S^-C (the layer between 1.0 M and 1.2M sucrose) were suspended slowly in 0.32 M sucrose 50% (v/v). The three fractions were pelleted at 100, OOOxg for 30 min in a Model L Ultracentrifuge with Spinco Rotor 40. Pellets S^-A, Sj-B, S^-C and S^-D were dispersed in 0. 32 M sucrose by light homo-genization in a small glass homogenizer with a loosely fitting pestle. As concluded by Whittaker, fraction S^-A was the myelin fraction, S^-B fraction was mainly nerve membrane fragments, S^-C fraction contained synaptosomes and S^-D fraction was packed with mitochondria. The crude mitochondrial fraction (11, 500xg pellet) was also fractionated in a discontinuous Ficoll-sucrose density gradient. The gradient was composed of 10 ml 7. 5% Ficoll (w/v) in 0. 32 M sucrose and 10 ml 13% Ficoll (w/v) in 0. 32 M sucrose according to Cotman et al (113). The crude mitochondrial fraction was layered onto and sedimented in a Ficoll-sucrose density gradient as described for the sucrose gradient. After 2 hr centrifugation, 3 fractions, F-A, F-B and pellet F-C, were separated out as indicated in Figure 6B. From enzymatic and electron-micrographic studies, Cotman _et al had shown that fraction F-A (the layer between 0. 32 M sucrose and 7. 5% Ficoll) was the myelin fraction, F-B Figure 6A Figure 6B 0 . 3 2 M 7.5 % i i H i i i i i i i i i i i i i i i i i : 1 3 . 0 % F - A F - B F- C efore Centrifugation After Centrifugation Figure 6: Preparation of Synaptosomes with a Ficoll-sucrose Density Gradient The Ficoll-sucrose gradient was composed of 10 ml 7.5% Ficoll (w/v) in 0.32 M sucrose and 10 ml 13% Ficoll (w/v) in 0.32 M sucrose. Sample was the crude mitochondrial pellet (11, 500xg pellet) suspended in 0.32 M sucrose, pH 7. After 2 hour centri-fugation at 50, OOOxg, fractions F-A, F-B and pellet F-C were separated out. Fraction F-A was the myelin fraction, fraction F-B was the synaptosomal layer and pellet F-C was the mito-chondrial layer. 36 fraction (the layer between 7. 5 % and 13% Ficoll) was mainly nerve ending particles, and pellet F-C was the mitochondrial pellet. Fractions F-A and F-B were diluted to 50% (v/v) in 0.15 M and 0. 32 M sucrose respect-ively. The two suspensions were then pelleted at 100, OOOxg for 30 min in a Model L Ultracentrifuge. 3. Preparation of synaptosomes Fractions containing nerve ending particles were obtained after the crude mitochondrial fraction (11, 500xg pellet) was fractionated in a discontinuous sucrose gradient or in a Ficoll-sucrose density gradient. These synaptosomal fractions, S^-B and S^-C fractions from the sucrose gradient and F-B fraction from the Ficoll-sucrose gradient, were highly contaminated and were purified by refractionation in a second sucrose density gradient consisting of 0. 8 M sucrose, 1. 0 M sucrose and 1.2 M sucrose (8 ml of each). Similar to the first sucrose density gradient, 3 subfractions ( S2-A, S2-B, S2-C) and a pellet (S2-D) were separated out. Fraction S 2-C was a better synaptosomal preparation than fraction S^-C or fraction F-B. Enriched and enzymatically active synaptosomal _ fractions were also prepared from the crude mitochondrial pellet fractionated in a narrower sucrose density gradient. The sucrose gradient was composed of 5 ml 0.8 M sucrose, 8 ml 1.0 M sucrose, 8 ml 1.1 M sucrose and 5 ml 1.2 M sucrose. Fractionation of the crude mito-chondrial pellet in this modified gradient was carried out as described by Whittaker (112). After 2 hr of centrifugation at 50, OOOxg, 4 distinct fractions 37 (S-A, S-B, S-C, S-D) and a pellet (S-E) were apparent, as shown in Figure 7. Fraction S-C, the layer between 1.0 M and 1. 1 M sucrose, was removed and pelleted at 100, OOOxg for 30 min. This fraction was taken as the working synaptosomal fraction. E. Electron Microscopy Electronmicrographs were prepared according to the method of French et al_(ll4). Fraction S^-C (obtained by fractionation of the crude mitochondrial pellet in a sucrose density gradient) was pelleted in a polyethylene microfuge tube and was fixed for one hr in ice cold 2. 5% glutaraldehyde in 0. 1 M sodium cacodylate, pH 7. 3. After storing one week in buffer at 4°, the pellet was postfixed in 1% osmium tetroxide in cold veronal acetate buffer for one hr. Then the pellet was dehydrated in graded alcohols and embedded in a mixture of Araldite and Epon. Sliver sections were cut with a diamond knife in an LKB Ultratome III and placed unsupported on 200 mesh grids. They were stained with alkaline lead citrate and examined with a Phillips 300 electron microscope at 60 KV, F. Protein Determination All protein determinations were made by the method of Lowry et al (115). 38 Figure 7A Figure 7B 0 . 8 M 1 . 0 M 1.1 M 1.2 M 0 . 3 2 M 0 . 8 M 1.0 M 1.1 M S - A S - B S - C S - D S - E Before Centrifugation After Centrifugation Figure 7: Preparation of Synaptosomes in a Modified Sucrose Density Gradient The sucrose density gradient was composed of 5 ml 0. 8 M sucrose, 8 ml 1. 0 M sucrose, 8 ml 1. 1 M sucrose and 5 ml 1. 2 M sucrose. Sample was the crude mitochondrial pellet (11, 500 xg pellet) suspend-ed in 0.32 M sucrose, pH 7. After 2 hour centrifugation at 50, OOOxg, fractions S-A, S-B, S-C, S-D and pellet S-E were separated out. Fraction S-A was the myelin layer, S-B fraction was the nerve mem-brane fragment layer, fraction S-C was the enriched synaptosomal layer, fraction S-D was the synaptosomal and mitochondrial layer and pellet S-E was the mitochondrial layer. 39 RESULTS I. Studies on the Cyclic AMP Binding Assay A. Preliminary Examination and Modification of Gilman's Method Experiments were conducted to determine whether radio-activity could be reliably determined by counting directly from the filter discs. Millipore filters were not dissolved in ethyl cellusolve but were dried at 150° F for one hr, and the radioactivity on the filters was counted in 5 ml toluene, PPO and POPOP scintillation fluid. Although the counting efficiency on paper was only 63% of that measured when the filter was dissolved in ethyl cellusolve and counted in aquasol, the slopes of the standard curves obtained by the two methods were identical. This finding indicated that the cyclic AMP-protein complex was not affected by high temperature, and the same amount of cyclic AMP was measured whether radioactivity was determined on paper or in solution. The radio-activity on millipore filters was also found to be stable at room tempera-ture for several days. Counts per min were recorded from samples before and after the vials had sat at room temperature for 48 hr and were found to be identical. This finding was useful because radioactivity could be determined by a scintillation counter which operates at ambient temperature. The Nuclear Chicago Isocap/300 was used. This instrument counts tritium from paper at room temperature with 40% efficiency. Figure 8 shows a standard curve obtained by the cyclic AMP binding method with radioactivity determined by counting from the intact millipore filter at room temperature. Similar to the result obtained 40 Total p moles of tritiated and unlabelled cyclic AMP was plotted against DPM. The results are averages of four separate experi-ments. 41 by Gilman, the standard curve was a straight line on a log-log scale when DPM (after correction for the blank) was plotted against total /o moles of tritiated and unlabelled cyclic AMP standard per tube. Routinely, we have adopted the procedure of determining radioactivity from the intact filter disc. Eliminating the dissolution of the filters greatly facilitates the experimental procedure. The procedure we have adopted is as follows. The binding reaction was carried out at pH 4 and 0° in a final vol of 200 yul. The incubation mixture contained 50 mM NaAc/HAc, pH 4, 2 f>moles C H 3-cyclic AMP (specific activity of 50, 000 disintegrations per minute per picomole), 52 yug protein kinase inhibitor protein, and 3.2 j-Kg of binding protein (which was saturated by 0.4 fo moles t H 3-cyclic AMP. Standards (2 to 10 moles of unlabelled cyclic AMP) were always run. After one hr and 15 min of in-cubation at 0°, the mixtures were diluted with 1 ml of cold 20 mM K,PO , 4 pH 6, and filtered through millipore filters (0.45 jj. pore size and 24 mm in diameter). The filters were dried at 150° F and counted in 5 ml scintillation fluid (4 g PPO and 50 mg POPOP in 1 litre of toluene) at room temperature. The DPM was calculated from CPM recorded divided by the efficiency and corrected for background counts. The standard curve was plotted on log-log graph paper with DPM against total p moles of tritiated and unlabelled cyclic AMP standard per tube. 42 B. Level of Cyclic AMP Measured in Heart and Liver Extracts In preliminary experiments, the cyclic AMP binding assay was used to measure the concentration of cyclic AMP in tissue extracts. The object of the experiment was to show that the modified method gave the same level of cyclic AMP as measured by Gilman and by other assays. Frozen rat ventricles and livers were extracted by the same procedure as described by Gilman. The cyclic AMP level in rat heart ventricle determined by the binding assay was 0. 3 7 7 m o l e s per mg wet weight; the amount of cyclic AMP measured in rat liver extract was 1. 3 4 m o l e s per mg wet weight. Table I shows the level of cyclic AMP measured in rat heart ventricles and rat liver extracts with different samples. The data were similar and comparable to the results obtained by Gilman and by others (116 - 118). C. Recovery of Known Amounts of Cyclic AMP Added Recovery of known amounts of cyclic AMP added to a frog liver extract was studied. As shown in Table II, using various concen-trations of unlabelled cyclic AMP, the percentage recovery determined by the binding assay was 95 - 10%. The data indicated that the binding assay was working as well as described by Gilman. II. Adenylate Cyclase Determination by the Cyclic AMP Binding Assay A. Effects of Adenylate Cyclase Incubation Mixtures on the Binding Assay It was important to determine whether the various . reagents present in the adenylate cyclase mixture would interfere with the binding Table I Level of Cyclic AMP Measured in Tissue Extract /o moles cAMP/mg wet weight 0. 375 0. 301 0. 361 0.461 1. 35 1. 34 Tissue Rat heart ventricle Rat liver The cyclic AMP binding assay was used to measure the levels of cyclic AMP in four different samples of rat ventricle and two different samples of rat liver. Table II Recovery of Cyclic AMP by the Cyclic AMP Binding Method Sample and Condition Cyclic AMP Measured Actual % Recovery Total y>m cAMP/tube Actual prn cAMP/tube Sample in 0. 1 ml 50 mM NaAc, pH 4 Aliquot used tube Sample/tube Total jo mole cyclic AMP /tube p moles cAMP/mg wet wt level of cAMP/ tube Frog Liver A. 116 mg 10 /A\ 11.6 mg 5. 1 0.46 20 JJ\ 22. 2 mg 9.3 0.42 0.44 B. 72 mg + 100 5 jul 3. 6 mg + 5 ^ om cAMP 6. 2 6.6 95 pmoles cAMP 10 yul 7. 2 mg + 10 (om. cAMP 10. 2 13.2 76 20 ^1 10.8 mg 4- 20 ^ m cAMP 28.0 26. 3 106 C. 76 mg + 200 5 yul 3. 8 mg + 10 p m cAMP 9.85 11.7 84 pmoles cAMP 10 ^ 1 7. 6 mg + 20 cAMP 26.75 23. 3 114 Recovery of added cyclic AMP was studied with three samples of a frog liver extract (A, B and C). A was used as control, lOO/s moles cyclic AMP was added to B, and 200 /<* moles cyclic AMP was added to C, Different aliquots of samples (5 to 20 ^  1 per tube) were assayed for cyclic AMP by the binding method. The actual level of cyclic AMP per assay tube was the sum of the expected amount of cyclic AMP in the tissue and the amount of unlabelled cyclic AMP present in the aliquot used. 45 of cyclic AMP to the binding protein. The incubation mixture contained Tris HC1, pH 7. 5, ATP, theophylline, KC1, pyruvate kinase, PEP and MgSO^, fluoride or hormones. These reagents were tested for their effects on cyclic AMP binding assay. Individual or combined reagents, in concentrations normally used for the adenylate cyclase assay, were added to the cyclic AMP binding incubation at saturating concentrations of [ 3 H ] -cyclic AMP. The results indicated that the binding of t 3 H ] - cyclic AMP to protein was not stimulated or inhibited by the presence of ATP or other adenylate cyclase reagents. The level of cyclic AMP determined in. the adenylate cyclase control tubes (containing all reagents except enzyme protein) was generally less than 5 ^ omoles. The levels of cyclic AMP per adenylate cyclase incubation were the same although different aliquots of control sample sample were used in the cyclic AMP binding assay. As indicated in Figure 9, the amount of cyclic AMP measured per binding assay was directly proportional to the aliquot of sample used per tube. Levels of cyclic AMP in adenylate cyclase incubation mixtures con-taining the enzyme were usually 1 0 to 50 times higher than in the control tube. The mixtures were generally diluted 5 to 20 times with 50 mM NaAc/HAc, pH 4, before assay for cyclic AMP. The effect of reagents on the cyclic AMP binding was therefore regarded as negligible. B. Recovery of Known Amounts of Cyclic AMP Added in the Adenylate Cyclase Assay Experiments were performed to determine the recovery of known amounts of unlabelled cyclic AMP added in the adenylate cyclase 46 2 0 6 0 A L I Q U O T ( u l ) ure 9: The Relationship Between the Amount of Cyclic AMP Measured by the Binding Assay and the Aliquot of Adenylate Cyclase Control Sample Used Cyclic AMP levels were measured in duplicate by the cyclic AMP binding assay, using various aliquots of adenylate cyclase mixture (containing all reaction reagents except protein). 47 incubation by the cyclic AMP binding assay. The adenylate cyclase incu-bation mixture contained the following: Tris HC1, pH 7.5, ATP, KCl, theophylline, pyruvate kinase, PEP, MgSO^, with or without enzyme protein. Ten to 400 moles of cyclic AMP were added before or after the reaction was terminated by boiling. Control tubes had no cyclic AMP added. The cyclic AMP levels in the mixture were quantitatively deter-mined by the cyclic AMP binding assay. The amount of added cyclic AMP recovered was the difference between the total cyclic AMP measured and the level of cyclic AMP in the control. The percentage recovery varied widely from 60 to 156% in different assay tubes and in different experiments. However, the average result was 110 - 6% (from 33 assays) in the absence of brain enzyme protein in the adenylate cyclase incubation mix, and 110 - 7% (from 25 assays) in the presence of brain protein in the adenylate cyclase mixture. Data obtained from a study of cyclic AMP recovery are shown on Table III. In this study, percentage recoveries calculated from the amount recovered and the actual amount added varied from 75 to 130%. The discrepancies indicated that something in the adenylate cyclase incuba-tion might be interfering with the cyclic AMP binding assay. Interference by ATP, ADP, AMP and inorganic phosphate was re-investigated. Nucleotides, ADP, AMP and inorganic phosphate were formed from ATP during adenylate cyclase incubation. These nucleotides were precipitated in the presence of ZnSO^ and Ba(OH)^. Cyclic AMP remained in solution. Cyclic AMP could then be separated from the other nucleotides before determination by the binding assay. Cyclic AMP formed 48 Table III Recovery of Cyclic AMP in the Adenylate Cyclase Incubation by the Binding Assay Condition Cyclic AMP moles/as say Cyclic AMP Recovered/as say % Recovery Experiment 1 (no enzyme protein) 1. Control -F~ 1.71- blank level 2. Control +F~ 2. 10 3. 10 /jmoles cAMP 10. 35 8. 64 86.4 -F" 4. 10 /omoles cAMP 10.95 8.85 88. 5 +F~ Experiment 2 (83 yug enzyme protein) 5. Control -F~ 26. 20 basal level 6. Control +F~ 62.90-7. 10 /omoles cAMP 37. 50 9.50 95. 0 -F" 8. 10 ^moles cAMP 72. 50 7. 50 75. 0 +F" 9. 50 /omoles cAMP 83. 25 55. 25 110.0 -F" 10. 50 ^moles cAMP 117. 70 52. 70 105. 0 + F" 11. 100 ^moles cAMP 140.00 112.00 112. 0 - F" 12. 100 ^moles cAMP 195. 00 130.00 130. 0 + F" 49 Table IU Recovery of Cyclic AMP in the Adenylate Cyclase Incubation In Experiment 1, the adenylate cyclase reaction mixture contained all the reagents except brain protein. In Experiment 2, the mixture con-tained all the reagents and 83 ^ ug protein from washed pellet of guinea pig cerebral cortex. Incubations were carried out with or without 8 mM fluoride at 37° for 5 min. Reactions were then terminated by boiling. Ten fomoles cyclic AMP standard were added to Tubes 3 and 4 in Ex-periment 1, and Tubes 7 and 8 in Experiment 2. Fifty /o moles of cyclic AMP were added to Tubes 9 and 1 0 in Experiment 2 and 100 p moles of cyclic AMP were added to Tubes 11 and 12. Tubes 1, 2, 5 and 6 were controls. Incubation mixtures were assayed for cyclic AMP. The amount of known cyclic AMP recovered in each tube was calculated. 50 in the adenylate cyclase reaction was also isolated by ion exchange chromatography using a Dowex AG 50WX4 column. Studies on the recovery of known amounts of cyclic AMP were therefore performed with BaZn precipitation and with Dowex column chromatography. It was hoped that these purification steps would reduce the scatter in the recovery data for cyclic AMP. The results on cyclic AMP recovery obtained with BaZn precipitation and with Dowex chromatbgraphy are presented in Figure 10. It can be seen that the total amount of cyclic AMP measured was not exactly proportional to the amount of cyclic AMP added. Scatter in the percentage recovery of known amounts of cyclic AMP added was present after all the other nucleotides were removed by BaZn precipitation or Dowex chromatography. The results indicated that ATP, ADP, AMP and Pi were not interfering with the cyclic AMP binding. It seems that the BaZn or the Dowex purification step is not necessary when the cyclic AMP binding assay is used for adenylate cyclase determination. Since the scatter in the recovery data for cyclic AMP could be reduced by doing triplicate or quadruplicate assays, it seems to arise more from technical procedures than from interferences by materials in the adenylate cyclase assay. C. Correlation Between Level of Cyclic AMP Measured and Enzyme Activity It was necessary to show that the levels of cyclic AMP measured by the binding assay corresponded to the enzyme activities when different concentrations of enzyme protein were used, when the reactions were incubated at various times, and when fluoride ion was present or 51 ti B a Z n D o w e x 5 0 W x 4 1 O X • A A UJ GO 10 — 3 t— • • Ul in < u >• u • Ul 6 • < mml >• § ADEN •mm Q A A OO A A O O O CL < w If) 2 — • • © • • "o E 1 <c O ! O 1 o 0 1 0 0 0 100 0 1 0 0 A , A 2 B i B 2 c 2 [ P R O T E I N ] ( u g ) Figure 10: Recovery of Cyclic AMP in the Adenylate Cyclase Incubation by the Binding Assay after BaZn Precipitation or Dowex Chromatography 51a Figure 10: Recovery of Cyclic AMP in the Adenylate Cyclase Incubation by the Binding Assay after BaZn Precipitation or Dowex Chromatography Experiments were performed with duplicate adenylate cyclase assays and duplicate cyclic AMP binding assays. Washed pellet from rat cerebral cortex was used. The final vol for the adenylate cyclase incubation was 450 yul and contained the following: 40 mM Tris HC1, pH 7.5, 6 mM KC1, 8 mM theophylline, 18 mM MgS0 4, 1 mM ATP, PEP, pyruvate kinase and protein from washed pellet of rat cerebral cortex. Tubes in Aj_, B^ and C^ were boiled immediately after the addition of brain protein. Tubes in A^, B^ and C~> were incubated for 5 min at 37°. No cyclic AMP was added in adenylate cyclase tubes O, 200 ^omoles cyclic AMP were added to tubes •, and 400 pinoles cyclic AMP were added to tubes In A, adenylate cyclase incubation mixtures were subject to BaZn precipitation. The supernatants were assayed for cyclic AMP by the binding method. In B, adenylate cyclase incubation mixtures were assayed directly for cyclic AMP. In C, 10 >ul of £ 3 H ] -cyclic AMP (2, 000 CPM) was added to each adenylate cyclase incubation. The mixture was chromatographed in a Dowex AG 50 WX4 column as described in Methods, The radioactivity in the cyclic AMP fraction (the third and fourth ml eluate) was measured in order to determine the amount of C H ]-cyclic AMP recovered in the eluate. This fraction was assayed for cyclic AMP by the binding method. 52 absent. Adenylate cyclase incubation was carried out in duplicate in a final vol of 150 ^1. The mixture contained 40 mM Tris HC1, pH 7.5, 6 mM KC1, 8 mM theophylline, 1 mM ATP, 18 mM MgS0 4, PEP and pyruvate kinase. Washed particles from rat cerebral cortex were used. Mixtures were incubated for 5 min at 4 different brain protein concen-trations. Reactions were also carried out with 70 /ig of protein at 4 different times of incubation. After reactions were terminated by boiling, the mixtures were centrifuged and assayed for cyclic AMP. Figures 11 and 12 show the results obtained from two experiments. In A, 8 mM fluoride was added in the adenylate cyclase incubation. The yield of cyclic AMP per min as determined by the binding method was directly proportional to the concentration of brain enzyme used. The p moles of cyclic AMP per mg brain protain determined was also directly pro-portional to the time of adenylate cyclase incubation. In B, reactions were performed in the presence (B^) or in the absence (B^) of 8 mM fluoride. As illustrated in Figures 11 and 12, pmoles of cyclic AMP measured per min adenylate cyclase incubation was 2.5 times higher when the reaction mixture contained fluoride. In the presence or absence of fluoride, cyclic AMP measured by the binding method was directly proportional to the time of adenylate cyclase incubation and the concen-tration of brain protein used. D. Comparison with [ C 1 -ATP Adenylate Cyclase Assay Brain adenylate cyclase activity was measured by the C ^ 4C j-ATP adenylate cyclase assay. Washed particles (3, OOOxg pellet) from guinea pig cerebral cortex (protein concentration ranged from 10 to 53 [ P R O T E I N ] ( u g ) Figure 11: Amount of Cyclic AMP Measured by the Cyclic AMP Binding Assay with Respect to the Concentration of Brain Protein Used Adenylate cyclase activities were measured in duplicate by coupling with cyclic AMP binding assay in various concentrations of washed pellet prepared from rat cerebral cortex. A showed the enzyme activities obtained from one preparation in the presence of 8 mM F~. Bj and B-, showed the enzyme activities determined from another preparation in the presence and absence of 8 mM F". 54 T I M E O F I N C U B A T I O N ( m i n ) A T 3 7 ° Figure 12: Amount of Cyclic AMP Measured by the Cyclic AMP Binding Assay as a Function of Incubation Time in the Adenylate Cyclase Reaction Adenylate cyclase assays were measured by coupling with the cyclic AMP binding assay at different times of incubation with washed pellet of rat cerebral cortex. A showed the enzyme activities (average of quadruplicate adenylate cyclase assays) obtained from one prepara-tion in the presence of 8 mM F". B and showed the enzyme activities (average of duplicate assays) determined from another preparation in the presence and absence of 8 mM F~. 55 150 yUg) were used. Incubation mixtures contained 0.3 mM L C J-ATP, 40 mM Tris HC1, pH 7.5, 2 mM unlabelled cyclic AMP, 18 mM MgSO^, 8 mM theophylline, 6 mM KC1, 8 mM fluoride, and the PEP-pyruvate o kinase ATP regenerating system. Reactions were carried but at 37 for 1 to 10 min after the brain protein was added. Following the termination of the reactions by boiling, the tubes were centrifuged. The supernatant (100 jul) was chromatographed on paper for 18 to 22 hr in a solvent system containing 1 M NH^Ac and ethanol (15: 35, v/v). After drying, areas corresponding to cyclic AMP were cut out and radioactivity was determined. Specific activity of adenylate cyclase was expressed as 14 picomoles of [ C ]-cyclic AMP formed per minute per milligram of brain protein. The specific activities obtained at four different brain protein concentrations and five different times of incubation are indicated by A in Figures 13 and 14. Brain adenylate cyclase activity was also measured by coupling with the cyclic AMP binding assay, using a different preparation of washed particles from guinea pig cerebral cortex. Adenylate cyclase incubations were carried out under the same conditions as described for r 14 T L C J-ATP adenylate cyclase assay. However, 1 mM unlabelled ATP was used as substrate and unlabelled cyclic AMP was omitted. Following incubations at 37°, reaction mixtures were boiled and centrifuged. The supernatants were diluted with 50 mM NaAc/HAc, pH 4, and were assayed in duplicate for cyclic AMP contents by the binding method. The specific activities of adenylate cyclase were determined using four different 56 10 i o < I/) < c E a. < V M _© "o E — — / c A M P A s s a y - o A ^ O ^ ^ " ^ C - A T P A s s a y 1 I i f 1 1 2 0 6 0 1 0 0 [ P R O T E I N ] ( u g ) Figure 13: Comparison Between the C C 1-ATP Adenylate Cyclase Assay and the Coupling of Cyclic AMP Binding Assay to Adenylate Cyclase Determination at Different Brain Protein Concentrations Specific activity of adenylate cyclase was plotted against concen-tration of the brain protein. Adenylate cyclase activity was measured in washed pellet of guinea pig cerebral cortex. In A adenylate cyclase activity from one preparation was determined by using f ^ C ]-ATP as substrate in the presence of 8 mM F~. In B, adenylate cyclase activity from another preparation was analyzed by coupling with cyclic AMP binding assay in the presence of 8 mM F". 57 n l O r— X z o a. 0) E < w M JO "o E T I M E O F I N C U B A T I O N ( m i n ) Figure 14: Comparison Between the [ ]-ATP Adenylate Cyclase Assay and the Coupling of Cyclic AMP Binding Assay to Adenylate Cyclase Determination at Different Times of Incubation Specific activity of adenylate cyclase was plotted against time of adenylate cyclase incubation. Adenylate cyclase activity was measured in washed pellet of guinea pig cerebral cortex. In A, adenylate cyclase activity from one preparation was _ 14 _ determined by using [ C J-ATP as substrate in the presence of 8 mM F~. In B, adenylate cyclase activity from another preparation was analyzed by coupling with cyclic AMP binding assay in the presence of 8 mM F~. 58 concentrations of brain protein and four different times of adenylate cyclase incubation. The results are presented in B of Figures 13 and 14. The specific activities of adenylate cyclase measured by the C ^ C J - A T P assay in A were not comparable to those given by the binding method in B, because ATP concentrations used in the two methods were different, and washed particles from guinea pig cortex used in A was a different preparation than the one used in B. Adenylate cyclase activity was found to vary from one rat brain to another and from one preparation to another. In addition, fluoride activation varied from two to five-fold in the same preparation. However, the specific activities of adenylate cyclase determined by the C C]-ATP as say and the binding assay showed a linear relationship with increasing brain protein concentration and with increasing time of incubation. Studies have been described to show the possibility that the cyclic AMP binding assay could be used to measure adenylate cyclase activity. The findings have indicated that the reagents in the adenylate cyclase reaction mixture did not have any significant effect on [ Hj-cyclic AMP binding, and the level of cyclic AMP measured by the binding method was directly proportional to the time of adenylate cyclase incubation and the enzyme concentration used. Results also showed that there was scatter in the recovery of cyclic AMP added to the adenylate cyclase mixture, and the scatter was not due to interference by other nucleotides present. Scatter might be due to technical problems because it could be reduced with triplicate assays. From these findings, it seems possible 59 that the cyclic AMP binding method could be applied to measure the product of adenylate cyclase reaction. However, this procedure may not be entirely satisfactory for adenylate cyclase assay routinely. The method could actually involve more work and require more time than the paper r l 4 i chromatographic L C J-ATP adenylate cyclase assay. III. Brain Adenylate Cyclase Studied with Cyclic AMP Binding Assay Variability in recovery of cyclic AMP by the binding assay was believed to be caused by technical problems. However, despite this scatter, the cyclic AMP binding assay was used to study the brain adenyl-ate cyclase. The neural enzyme was interesting because hormonal stimu-lation of the enzyme in broken cell brain preparations has not been demonstrated convincingly. Work was therefore primarily focused on finding conditions in which significant hormonal effects could be detected. A. Subcellular Distribution of Adenylate Cyclase in Rat Cerebral Cortex Distribution of adenylate cyclase in rat brain subcellular fractions was first studied in order to show that the binding assay could give the same results as those given by De Robertis (35) and to obtain a highly active and pure enzyme fraction for hormonal studies. Rat brain cortex was fractionated by the classical method of Whittaker. The subcellular distribution of adenylate cyclase activity was measured by coupling with the binding assay. Data obtained from a representative experiment are shown in Table IV. The distribution of the enzyme was very similar to the results obtained by De Robertis and other Table IV Adenylate Cyclase and Acetylcholinesterase Activities in Rat Brain Subcellular Fractions Prepared by the Method of Whittaker Main Protein Adenylate Cycl ase Acetylcholinesterase Fraction Component Yield % Specific Activity /om cAMP Total Activity /o m cAMP Yield % S pecific Activity m ATCH Total Activity am ATCH Yield % min x mg min min x mg min (1) Whole Homogenate 100 272 64200 100 87. 5 20700 100 (2) 900 x g sup 66 . 336 52500 80 97.8 15100 72 (3) 900 x g pellet N 32 252 18900 30 96.8 5760 28 (4) 11, 500 x g sup Mic 23 222 12200 19 97.0 5340 26 (5) 11, 500 x g pellet Mit 36 319 27400 43 117.0 10100 49 (6) Sucrose Gradient S^A (0. 32 M - 0. 8 M) Myelin 4.5 163 1740 2.7 53.8 575 2.8 SL-B (0.8 M - 1.0 M) NMF 2.9 381 2630 4. 1 147.0 1015 4.9 S L-C (1. 0 M - 1. 2 M) S^D (1. 2 M) S 10.5 281 7050 9.4 157.0 3900 19.0 Mit 15.5 152 5600 8.7 70.0 2570 12. 0 33.4 17020 24.9 8060 38. 7 o 61 workers (35, 65). Forty-three percent of the total enzyme activity in the whole homogenate was found in the mitochondrial fraction (11, 500xg pellet). Submitochondrial fractionation in a discontinuous sucrose density gradient showed that the S^-B fraction (the layer between 0.8 M and 1.0 M sucrose) contained the highest specific activity of adenylate cyclase. However, enzyme activity in the mitochondrial fraction was mostly present in the S^-C fraction (the layer between 1.0 M and 1.2 M sucrose). Whittaker and De Robertis have shown that the S^-C fraction contained synaptosomes. Acetylcholinesterase activities in subcellular fractions of rat cerebral cortex were measured. Acetylcholinesterase is generally used as a marker enzyme for synaptosome and synaptosomal membrane. Results, as indicated in Table IV, showed a close correlation between the distribution of acetylcholinesterase activity and adenylate cyclase activity in the rat cerebral cortical fractions. Hence, adenylate cyclase was highly associated with synaptosomes and synaptic membranes. The submitochondrial fraction obtained in the layer between 1.0 M and 1.2 M sucrose was also confirmed by electronmicroscopic studies to be the synaptosomal fraction. Twenty percent of the S^-C pellet was packed with synaptosomes or nerve ending particles. The rest of the pellet was highly contaminated with membranous structures such as myelin, synaptic membrane fragments and mitochondria. Figure 1 5A shows a few beautiful synaptosomes found in the concentrated synaptosomal area. Synaptic vesicles, synaptic cleft and the thickened postsynaptic membrane were clearly indicated. Figure 1 5B gives a view of the heterogeneity 62 Figure 15: Electronmicrographs of S^-C Submitochondrial Fraction The crude mitochondrial pellet was fractionated for 2 hours in a discontinuous sucrose density gradient. Fraction S^-C was the layer obtained between 1.0 M and 1. 2 M sucrose. This fraction was studied under the electron microscope as des-cribed in Methods. In the electronmicrographs, S indicates synaptosome; A, mitochondria; M, myelin; and F, membrane fragments. Figure 1 5A: Magnification is 26, 600 times. Figure 15B: Magnification is 17, 000 times. 63 Figure 15A Figure 15A: Electronmicrograph of - C Submitochondrial Fraction Figure 15B: Electronmicrograph of S^-C Submitochondrial Fraction 65 in the S^-C synaptosomal fraction. Besides synaptosomes, there were myelin, membrane fragments and distorted mitochondria. B. Purification of Synaptosomal Fraction The synaptosomal fraction is interesting because it contains most of the adenylate cyclase present in rat brains. However, the synaptosomal fraction prepared by the method of Whittaker was found to be highly contaminated with myelin and mitochondria. Attempts were therefore made to obtain an enriched and enzymatically active synapto-somal fraction so that the synaptosomal adenylate cyclase could be studied. 1. Sub synaptosomal fractionation on a second sucrose density gradient Synaptosomal fractions, S^-B and S^-C, were first prepared by the method of Whittaker. These fractions were refractionated in a second sucrose density gradient. Four sub synaptosomal fractions, S^-A, S£-B, S^-C and S^-D, were obtained. Fraction S„,-C was expected to be less contaminated with mitochondria than S^-C fraction. Results, as indicated in Table V, have shown that there was a small increase in the specific activity of acetylcholinesterase in S^-C fraction, but the specific activity of adenylate cyclase was less than that in S^-C fraction. Loss in adenylate cyclase activity could have resulted from the long process of preparation because brain adenylate cyclase is known to be a very labile enzyme. Table V Adenylate Cyclase and Acetylcholinesterase Activities in Sub synaptosomal Fractions Prepared by a Second Sucrose Density Gradient Main Protein Adenylate Cyclase Acetylcholine ster as e Fraction Yield Specific Activity Total Activity Yield Specific Activity Total Activity Yield Component % yom cAMP /om cAMP % ri m ATCH r^m ATCH % min x mg min min x mg min (1) Whole homogenate 100 272 64200 100 87. 5 20700 100 (2) 11, 500 x g pellet Mit 36 319 27400 43 117. 0 10100 49 (3) Sucrose gradient S^B (0.8M- 1. 0 M) NMF 2.9 381 2630 4. 1 147 1015 4.9 S^C (1. 0 M - 1. 2 M) S 10. 5 281 7050 9.4 157 3900 19.0 2 (4) Sucrose gradient 13.4 9680 13. 5 4915 23.9 S -A (0. 32 M - 0. 8 M) Myelin 0. 3 191 130 0. 2 163 111 0. 5 S2-B (0. 8 M - 1. 0 M) NMF 0. 7 264 433 0.7 305 500 2.4 S 2-C (1.0M - 1.2 M) S 6. 1 206 2970 4. 1 162. 5 2350 11.4 S2-D (1. 2 M) Mit 3.0 126 910 1.4 104.0 740 3. 6 10.1 4443 6.4 3601 17.9 67 2. Synaptosomal fraction prepared on a Ficoll-sucrose density gradient Synaptosomal fraction was also prepared by the method of Cotman and Matthews (113). Isotonic Ficoll-sucrose density gradients proved to be a preferable medium to hypertonic sucrose gradients for the isolation of synaptosomes, and the synaptosomal fraction was also less contaminated with membrane fragments (119, 120). The crude mitochondrial fraction (11, 500xg pellet) from rat cerebral cortex was therefore fractionated in a discontinuous isotonic Ficoll-sucrose density gradient which was composed of 10 ml 7.5% F i c o l l -sucrose and 10 ml 13% Ficoll-sucrose, After 2 hr centrifugation at 50, OOOxg, 3 fractions, F-A, F-B and F-C, were obtained. Fraction F-B, the layer between 7.5% and 13% Ficoll-sucrose, should be the synapto-somal fraction. Distribution of adenylate cyclase and acetylcholines-terase activities in the subcellular fractions were examined. Results from two experiments, using a Ficoll-sucrose gradient, are tabulated in Table VI. Specific activities of adenylate cyclase in the Ficoll fractions were lower than the crude mitochondrial pellet. Ficoll might be inter-fering with the adenylate cyclase assay. Although the specific activity of adenylate cyclase in F-B fraction was highest among the three sub-mitochondrial fractions, the total enzyme activity was slightly less than the F-C pellet. Many synaptosomes might have sedimented with the mitochondria. In contrast, the acetylcholinesterase in F-B fraction had the highest total activity among the three Ficoll fractions. A sucrose density gradient was used to refractioriate the F-B fraction. Similar to Table VI Adenylate Cyclase and Acetylcholinesterase Activities in Fractions Prepared by a Ficoll-sucrose Density Gradient Fraction Main Component Protein Adenylate Cyclase Acetylcholinesterase Yield % Specific Activity /om cAMP Total Activity /om cAMP Yield % Specific Activity H m ATCH Total Activity n m ATCH Yield % min x mg min min x mg min Experiment 1 (1) Whole homogenate 100 201 27200 100 110 14800 100 (2) 11, 500 x g pellet Mit 28 245 9200 34 150 5650 38 (3) Ficoll-sucrose gradient F-A (0. 32 M - 7. 5%) Myelin 2.9 137 530 2. 0 190 723 4.9 F-B (7. 5% - 13%) S 10. 3 158 2251 8.3 170 2370 16.0 F-C (13%) Mit 11.9 155 2500 9.2 120 1920 13 25. 1 5281 19.5 5513 33.9 Experiment 2 (1) Whole homogenate 100 219 58000 100 117 31600 100 (2) 11, 500 x g pellet Mit 31 250 20800 36 159 13200 42 (3) Ficoll-sucrose gradient F-A (0. 32 M - 7. 5%) Myelin 3. 2 201 1710 3. 0 158 1340 4. 3 F-B (7. 5% - 13%) S 10. 5 253 7200 12.4 176 4850 16.0 F-C (13%) Mit 13.4 210 7770 13. 2 106 3920 12. 0 27. 1 • 16680 28.6 10220 32. 3 69 the results in Table V, the fraction with the highest total acetycholines-terase and adenylate cyclase activities was the layer between 1.0 M and 1.2 M sucrose. The Ficoll-sucrose method is also a long procedure. Inactivation of adenylate cyclase during the preparation was inevitable. Hence, this method was not pursued further. 3. Synaptosomal fraction prepared by a modified sucrose density gradient A single modified sucrose gradient was developed for preparation of an enriched synaptosomal fraction with high adenylate cyclase activity. Since rat brain cortical synaptosomes have been shown to have a buoyant density in the range of 0.9 M to 1. 2 M sucrose, a 1.1 M sucrose solution was added in between the 1.0 M and 1.2 M sucrose gradients with the hope that the synaptosomal fraction between 1.0 M and 1. 1 M sucrose would be less contaminated with myelin and mitochondria. Table VII shows that the highest total acetylcholinesterase activity was found in the synaptosomal fraction S-C (the layer between 1.0 M and 1. 1 M sucrose). Adenylate cyclase activity was also highest in the S-C fraction. The S-C fraction was therefore taken as the satisfactory synaptosomal fraction. C. Properties of Synaptosomal Adenylate Cyclase Since an enriched synaptosomal fraction (S-C fraction) was obtained from the modified sucrose gradient, we were interested in examining the properties of adenylate cyclase in this fraction. Many investigations have demonstrated that adenylate cyclase from most Table VII Adenylate Cyclase and Acetylcholinesterase Activities in Fractions Prepared by a Modified Sucrose Density Gradient Main Protein Adenylate Cyclase Acetylcholinesterase Fraction Component Yield % Specific Activity I& m cAMP Total Activity fom. cAMP Yield % Specific Activity am ATCH Total Activity rim ATCH Yield % min x mg min min x mg min (1) Whole homogenate 100 190 41300 100 90 17000 100 (2) 11, 500 x g pellet Mit 31 222 15000 36 110 4460 26 (3) Sucrose gradient S-A (032 M - 0. 8 M) Myelin 5.5 76 910 2. 2 50 355 2. 1 S-B (0. 8 M - 1.0 M) NMF 2.2 305 1460 3. 5 180 700 4. 1 S-C (1. 0 M - 1. 1 M) S 6. 0 301 3240 7.9 190 1200 7. 1 S-D (1. 1 M - 1. 2 M) S 4.4 261 2500 6.0 160 1090 6.4 S-E (1.2 M) Mit 11.4 93 2300 5. 6 80 945 5.0 - 28. 5 10410 30.2 4390 24. 7 o 71 mammalian tissues is dependent on ATP and metal ions for its activity. The effects of these agents on synaptosomal enzymes were studied. 1. Effect of ATP concentration In Figure 16, the specific activity of adenylate cyclase is shown as a function of ATP concentration in the incubation mixture. In the absence of fluoride but in the presence of 18 mM MgSO^, the maximum velocity of the enzyme reaction was reached at 0.6 mM ATP. This is in agreement with Drummond et al (72) who found the K m of the brain enzyme for ATP to be 0. 1 mM. 2+ 2. Mg requirement Curve A in Figure 17 shows the relationship between specific activity of adenylate cyclase in the synaptosomal fraction and magnesium ion concentration in the presence of 1 mM ATP. Maximum 24-enzyme reaction velocity was reached at 6 mM Mg . The effect of 24-Mg on synaptosomal adenylate cyclase is similar to the results obtained with heart (72), liver (72), skeletal muscle (21) and brain (72) enzymes. 3. Effect of Mn 2 + As shown in Curve B of Figure 17, much higher 24-stimulation of adenylate cyclase activity by Mn was obtained than with 24-similar concentrations of Mg . Manganese ion, 3 mM, gave a maximum enzyme specific activity as high as 480 picomoles of cyclic AMP per 24-minute per milligram protein. This concentration of Mn also activates the cardiac (72) and the skeletal muscle (21) enzymes maximally. 72 l O X z £ 3 -o u a. » E c E a < o o E 1 -Figure 16: Effect of ATP Concentration on Synaptosomal Adenylate Cyclase Specific activity of adenylate cyclase was plotted against ATP concentration. Adenylate cyclase activity in one synaptosomal preparation from rat cerebral cortex was measured in triplicate in 18 mM Mg 2 +. Each adenylate cyclase tube was assayed in duplicate for cyclic AMP. 73 I O X z o a. 5 -E c £ a. < w (A J!) "o E 12 [ l O N ] m M Figure 17: Effects of Ions on Synaptosomal Adenylate Cyclase Specific activity of adenylate cyclase was plotted against ion concentration. Adenylate cyclase activity in one synaptosomal preparation from rat cerebral cortex was measured in tripli-cate in 1 mM ATP. Each adenylate cyclase tube ^ ?- s assayed in duplicate for cyclic AMP level. A was the Mg concentra-24- concentration curve, C was the F" .24-tion curve, B was the Mn concentration curve in the presence of 18 mM Mg^ + and D o i 24-showed the effect of 1 mM Ca in the presence of 18 mM Mg 74 4. Effect of fluoride ion Perkins and Moore (121) have shown that fluoride causes a two-fold increase in brain adenylate cyclase activity from the basal level. The basal level is defined as that activity achieved in the presence of 18 mM Mg and 1 mM ATP. The effect of F~ on the synap-tosomal enzyme was examined. Stimulation of the enzyme could be detected with as little as 2 mM fluoride. Curve C of Figure 17 is the fluoride ion activation curve. A three-fold increase in the basal activity was obtained at 6 mM F~. Rodbell et al (122) has reported that GTP inhibited fluoride-stimulated hepatic adenylate cyclase activity. However, Severson (123) could not demonstrate an effect of GTP on fluoride-activa-tion of skeletal muscle enzyme. Similar to the results of Severson, fluoride-stimulation of synaptosomal adenylate cyclase was not affected by 100 >iM GTP. 2+ 5. Effect of Ca The basal activity of adenylate cyclase in the 2+ presence of 1 mM ATP and 18 mM Mg was reduced to half when 1 mM Ca(Ac)^ was added. Calcium inhibition was also noted in the solubilized brain enzyme prepared by Swislocki (70). D. Attempts to Demonstrate Hormonal Sensitivity in Brain Adenylate Cyclase 1. Effects of hormones on synaptosomal adenylate cyclase The effects of hormones on synaptosomal adenylate 75 cyclase were investigated. Synaptosomal adenylate cyclase was used because it has the highest specific activity among all brain subcellular fractions. Synaptosomal fraction (S-C) was prepared from rat cerebral cortex using a modified sucrose gradient. As shown in Table VIII, various concentrations of epinephrine did not stimulate synaptosomal adenylate cyclase. Instead, inhibition of the enzyme by epinephrine was 2+ often noticed in the presence of 1 mM ATP and 18 mM Mg . A variety of hormones, at concentrations of 67 >*M, were also tested in the synap-tosomal fraction isolated from rat whole brain. As indicated in Table IX, none of the hormones tested activated the synaptosomal adenylate cyclase. In fact, there was a slight inhibition of enzyme activity produced by the hormone. Adenosine and a (h adrenergic blocking agent, D, L-propra-nolol, also had no significant effect on the enzyme. 2. Effect of oC and (3 adrenergic blocking agents added before homogenization Brain adenylate cyclase could be maximally stimu-lated by endogenous neurohormones released during cell rupture. To prevent the enzyme receptor from binding endogenous catecholamines released during homogenization, large doses of oc and (i adrenergic blocking agents were used. Phentolamine (1 mM) and D, L-propranolol (1 mM) were added before rat brain cortex was homogenized in 10 mM Tris HC1, pH 8. The 3, OOOxg washed pellet was then suspended in Tris-buffer containing no blocking agents. Adenylate cyclase activity was determined in the absence and presence of L-norepinephrine. The Table VIII Effect of Epinephrine on Synaptosomal Adenylate Cyclase Specific Activity of Adenylate Cyclase Condition p moles cAMP/min/mg protein Control 254 t 12 Epinephrine added 0. 67 / i M 195 t 3 6. 70 JJM 203 - 12 67.0 juM 1 5 0 - 5 0. 67 m M 176-7 Results were obtained from one preparation of synaptosomes from rat cerebral cortex. The specific activity of adenylate cyclase is the average of three adenylate cyclase assays determined by coupling with the cyclic AMP binding method. 77 Table IX Effects of Various Agents on Synaptosomal Adenylate Cyclase Condition Specific Activity of Adenylate Cyclase p moles cAMP/min/mg protein Control 187 t 11 Hormones: 67 yuM L-Epinephrine 147 - 14 L--Nor epinephrine 133 - 5 Histamine 159 - 12 Serotonin 155 - 11 Adenosine 152-13 Isoproterenol 133- 7 Fluoride 8 mM 462 - 19 (i -blocking agent: D, L-Propranolol 67 yuM 165 - 8 78 results are tabulated in Table X. Norepinephrine still had no effect on brain adenylate cyclase. 3. In reserpinized rat brain cortex Since large quantities of catecholamines are stored in brain tissues and released when brain cells are disrupted, they could interfere with studies when hormones are applied exogenously. To deplete the stores of catecholamine in the nerve endings, rats were given reser-pine (1 mg per kg) once daily for three consecutive days. On the fourth day, S-C synaptosomal fraction was prepared from the cerebral cortex of reserpinized rats, using a modified sucrose density gradient. Adenyl-ate cyclase activity was tested for hormonal sensitivity. As indicated in Table XI, no hormonal response could be detected. 4. The effect of GTP on hormonal response Hormonal stimulation in broken cell preparations of brain adenylate cyclase might be too small to be detected. Guanosine triphosphate (100 /iM) has been found to enhance the epinephrine stimu-lation of adenylate cyclase in skeletal muscle (123) and glucagon stimula-tion of hepatic enzyme (122). Guanosine triphosphate, 100 JU'SA, was therefore added to synaptosomal adenylate cyclase and its effect to intensify hormonal sensitivity was studied. As shown in Table XII, GTP did not increase the enzyme sensitivity to 67yuM epinephrine. 79 Table X Effect of Norepinephrine on Adenylate Cyclase from Rat Brain Homogenized in the Presence of oc and (h Adrenergic Blocking Agents Condition Specific Activity of Adenylate Cyclase p moles cAMP/min/mg protein Control (homogenized in Tris buffer) 159 - 16 4-Control + Norepinephrine 17Z - 15 (67 /Jvl) Experiment (homogenized in oc + (i Blockers) 129 - 12 Experiment + Norepinephrine (67 yuM) 124 ± 7 (670 /iM) 98 t 25 (6.7mM) 96 - 2 One rat cerebral cortex was used. One half was taken as control. The other half was homogenized in 1 mM phento-lamine and 1 mM D, L-propranolol mixed with 10 mM Tris HC1, pH 8. Washed pellet (3, OOOxg pellet) was pre-pared and assayed for adenylate cyclase activity. Different concentrations of norepinephrine were tried. Results are averages of three adenylate cyclase assays. 80a Table XI Effects of Hormones on Reserpinized Rats Two rat cerebral cortices were used as control. Control rats were not given any injections. Reserpinized rats were injected with reserpine (1 mg per kg) once daily for 3 days. They were experimented on the fourth day. Synaptosomal fractions were prepared by the modified sucrose density gradient. Results are averages of triplicate adenylate cyclase assays on synap-tosomal fractions. 80 Table XI Effects of Hormones on Re serpi nized Rats Condition Concentration Specific Activity of Adenylate Cyclase /Dmoles cAMP/min/mg protein Normal Rats (2) Reserpinized Rats (4) Expt. 1 Expt. 2 Control (basal level) 505 - 28 109 - 19 130 - 3 Hormones: L-epinephrine 15 M 349 - 61 80 t 14 127 t 3 50 M 469 - 30 n o t 4 100 M 363 t 25 71 t 20 123 t 3 L-norepinephrine 15 M 324 £ 30 71 ±. 20 125 t u 50 M 355 t 59 113 t 18 120 t 5 100 M 313 - 37 100 - 6 123 t 3 Histamine 15 M 412 t 42 109 - 9 123 ± 6 50 M 334 t 35 99 - 13 115 t 7 100 M 404 t 30 104 ± 13 120 t 10 Serotonin 15 M 331 - 36 90 t 6 131 - 5 50 M 312 t 41 88 t 10 121 t 3 100 M 311 t 10 107 t 19 105 ± 6 Table XII Effect of GTP on Hormonal Stimulation Specific Activity of Adenylate Condition Cyclase /omoles cAMP/min/mg protein Control 296 t 10 (basal level) Epinephrine 202 - 12 (67 yuM) GTP 335 - 8 (100 JJM) GTP (100 >uM) (67 JAM) +- Epinephrine 309 - 17 Experiments were performed with one synaptosomal prep-aration from a rat cerebral cortex. Results are averages of duplicate adenylate cyclase assays. 82 DISCUSSION The findings have shown that the cyclic AMP binding assay can be applied to determine the cyclic AMP formed during adenylate cyclase in-cubations. The reagents in the adenylate cyclase reaction mixture did not interfere with the cyclic AMP binding. The level of cyclic AMP measured by the binding assay increased linearly with increasing time of adenylate cyclase incubation and with increasing enzyme concentration used. However, some scatter in the recovery data for cyclic AMP was obtained by this method. We felt that technical problems could be the cause. Adenosine triphosphate and other nucleotides present in the incu-bation mixture did not affect the cyclic AMP binding; removal of these nucleotides by barium-zinc precipitation or Dowex chromatography did not reduce the scatter in the recovery data for cyclic AMP. The binding assay is a very sensitive method, and it measures the level of cyclic AMP in the range of 2 to 10 /O moles per binding assay tube. The adenyl-ate cyclase incubation mixtures were usually diluted 5 to 30 times before assay for cyclic AMP. A small error in the binding assay would be magnified in the final adenylate cyclase determination. The scatter of results could be reduced with triplicate or -quadruplicate assays. Recently it was found that scatter in triplicate assays was reduced simply by limiting the number of binding assay tubes processed at one time. Al-though the method is inexpensive and easy to perform, the binding assay may not be useful for adenylate cyclase determination routinely. 83 The main reason is that this method involves longer time than the r 14 l paper chromatographic L CJ-ATP adenylate cyclase assay. Appro-priate dilutions for the adenylate cyclase incubation were necessary in order that unknown cyclic AMP levels fall on the range of the. standard curve. The coupling of the cyclic AMP binding assay to adenylate cyclase determination was used to study the adenylate cyclase in rat cerebral cortex. An advantage of the assay was that large experiments could be performed. Subcellular distribution of the adenylate cyclase measured by this method was found to be similar to the results obtained by De Robertis. Rat brain subcellular fractions were prepared by the method of Whittaker. The highest adenylate cyclase activity was found in the fraction containing the highest acetylcholinesterase activity. This fraction was characterized by electronmicroscopic studies and shown to contain many synaptosomes. Adenylate cyclase is probably associated with the synaptosomes which store neurohormones. Studies on hormonal stimulation of synaptosomal adenylate cyclase were carried out. A synaptosome retains all the structural features of the synapse. It therefore provides an ideal system for exploring the role of adenylate cyclase in synaptic transmission. If cyclic AMP is involved in mediating the action of neurotransmitter, synaptosomal adenylate cyclase should be extremely sensitive to neurohormones. 84 Synaptosomal fractions prepared by the method of Whittaker have been studied under the electron microscope. The fraction was highly contaminated with membranous structures from glial cells and nerve fibres. Myelin and mitochondria were abundant. For reliable enzyme studies, the synaptosomal fraction had to be purified. The synaptosomal fraction obtained by the method of Whittaker was refractionated in a second sucrose density gradient. The long procedure generally resulted in loss of protein and inactivation of adenylate cyclase. Synaptosomal fractions were also prepared using an isotonic Ficoll-sucrose gradient. Results of enzymatic studies have indicated that separation of synapto-somes in the Ficoll gradient was not as satisfactory as in the sucrose gradient. Synaptosomal fractions were then prepared by a single modified sucrose density gradient. The fraction between 1.0 M and 1. 1 M sucrose was taken as the best synaptosomal fraction because the greatest amount of acetylcholinesterase and adenylate cyclase activities were found in this fraction. Unfortunately, no electronmicroscopic studies were made to examine the purity of this fraction. It is most likely to be a hetero-genous fraction but many synaptosomes should be present to account for the high acetylcholinesterase activity. Properties of adenylate cyclase were studied in the synaptosomal 2+ 2+ fraction. The ATP, Mg , Mn , and F~ activation curves look similar to those obtained with the washed pellets of heart (72), brain (72) and skeletal muscle (21) enzymes. Maximum enzyme reaction velocity was 2+ * obtained at 0. 6 mM ATP in the presence of 18 mM Mg . Manganese 85 ion activated the enzyme to a greater extent than magnesium ion. The 24-basal activity in 1 mM ATP and 18 mM Mg was increased two to five-fold when 8 mM fluoride was added. Most disappointing was the fact that hormonal sensitivity was still not demonstrated. Epinephrine, norepinephrine, histamine, serotonin and isoproterenol were tried. Although adenosine causes a tremendous increase in cyclic AMP level in brain slices, it was ineffective in stimulating synaptosomal adenylate cyclase. The results on synaptosomal adenylate cyclase are the same as those reported recently by Isaac et al (69). Surprisingly, all hormones used caused a slight inhibition of adenylate cyclase activity in the 2+ presence of 1 mM ATP and 18 mM Mg . The reason for the inhibition is not known. Although GTP at 100 >uM concentration was found to amplify the hormonal stimulation of adenylate cyclase in liver and skeletal muscle (122, 123), GTP has no effect in producing a readily measurable epinephrine activation in the synaptosomal adenylate cyclase. It is possible that the adenylate cyclase is maximally stimulated by endogenous neurohormones released when nerve cells are disrupted during homogenization. However, propranolol was found to have little effect on the basal activity of the synaptosomal enzyme. A large dose of oc. and (3 blocking agents (1 mM phentolamine and 1 mM D, L-propra-nolol) was added to Tris-buffer before homogenization of rat brain cortex. Under these conditions, norepinephrine, as high as 6.7 mM, was unable to produce a stimulation in adenylate cyclase. Rats were reserpinized to deplete the catecholamine stores. Synaptosomal 86 adenylate cyclase activity in reserpinized rats was found in two experi-ments to be lower than that in normal rats. The finding was not conclusive because brain adenylate cyclase activity varies from different brains and in different preparations. Hormonal stimulation of adenylate cyclase was not obtained in reserpinized rats. Swislocki (70) has recently solu-bilized rat brain adenylate cyclase with 0. 1 M Lubrol-PX and purified the enzyme on DE-52 and Bio-Gel A-15 columns. The purified enzyme was sensitive to F~ but not to norepinephrine stimulation. Although hormonal activation of brain adenylate cyclase was not demonstrated in the limited conditions studied, the physiological import-ance of neural adenylate cyclase cannot be neglected. Adenylate cyclase is present in nervous tissue and has high activity in the brain particularly in the synaptosomal fraction. The intracellular concentration of cyclic AMP has been shown to be increased by electrical stimulation and by neurohormones. The conditions required for effecting hormonal stimula-tion on brain adenylate cyclase remain to be demonstrated. There is the possibility that hormone receptors may have been disrupted or destroyed during homogenization. Loss of an essential phospholipid or a change in a membrane protein conformation could result in a loss of hormonal responsiveness in the broken cell preparations. Nerve tissue is a particularly sensitive and complex system. Through its well organized but complicated functions, man is given the ability to think, to explore and to enjoy life. 87 BIBLIOGRAPHY 1. Sutherland, E. W. and Rail, T. W. (1957). J. Biol. Chem. Soc, 79, 3608. 2. Sutherland, E. W. and Rail, T. W. (1958). J. Biol. Chem., 232, 1077. 3. Rail, T. W. and Sutherland, E. W. (1958). J. Biol. Chem., 232, 1065. 4. Sutherland, E. W. and Rail, T. W. (I960). Pharm. Rev., 12, 265. 5. Robison, G. A., Butcher, R. W. and Sutherland, E. W. (1968). Ann. Rev. Biochem. , 37, 149. 6. Jost, J. -P. and Rickenberg, H. V. (1971). Ann. Rev. Biochem., 40, 741. 7. Robison, G. A., Butcher, R. W. and Sutherland, E. W. (1971). Cyclic AMP, Academic Press, New York. 8. Robison, G. A., Nahas, G. G. and Triner, L. (Editors), (1971), Cyclic AMP and Cell Function, Annals of the New York Academy of Sciences, Vol. 185, New York. 9. Sutherland, E. W., Rail, T. W. and Menon T. (1962). J. Biol. Chem., 237, 1220. 10. Butcher, R. W. and Sutherland, E. W. (1962). J. Biol. Chem., 237, 1244. 11. Rail, T. W. and Sutherland, E. W. (1962). J. Biol. Chem., 237, 1228. 12. Hirata, M. and Hayaishi, O. (1965). Biochem. Biophys. Res. Commun., 21, 361. 13. Tao, M. and Lipmann, F. (1969). Proc. Nat. Acad. Sci. (USA), 63, 86. 14. Duffus, C. M. and Duffus, J. H. (1969). Experientia, 25, 581. 15. Pollard, C. J. (1970). Biochim. Biophys. Acta, 201,,511. 16. Dousa, T. and Rychlik, I. (1968). Life Sci. , 7, Part II, 1039. 88 17. Vaughan, M. and Murad, F. (1969). Biochem., _8, 3092. 18. Skala, J., Hahn, P. and Braun, T. (1970), Life Sci., _9, Part I, 1201. 19. Drummond, G. I. and Duncan, L.J. (1970). J. Biol. Chem., 245, 976. 20. Rabinowitz, M. , Desalles, L., Meisler, J. and Lorand, L. (1965). Biochem. Biophys. Acta, 97, 29. 21. Severson, D. L. , Drummond, G. I. and Sulakhe, P. V. (1972). J. Biol. Chem., 247, 2949. 22. Murad, F., Chi, Y. -M., Rail, T. W. and Sutherland, E. W. (1962). J. Biol. Chem., 237, 1233. 23. Bitensky, M. W., Russell, V. and Robertson, W. (1968). Biochem. Biophys. Res. Commun., 31, 706. 24. Hepp, K. D., Edel, R. .and Wieland, O. (1970). Eur. J. Biochem., 17, 171. 25. Chase, L. R. and Aurbach, G. D. (1968). Science, 159, 545. 26. Hynie, S. and Sharp, G. W. G. (1971). Biochem. Biophys. Acta, 230, 40. 27. Dousa, T., Hechter, O., Schwartz, J. L. and Walter, R. (1971). Proc. Nat. Acad. Sci. (USA), 68, 1963. 28. Bar, H. P., Hechter. O., Schwatz I. L. and Walter R. (1970). Proc. Nat. Acad. Sci. (USA), bT_, 7. 29. Bar, H. P. and Hechter, O. (1969). Proc. Nat. Acad. Sci. (USA), _63, 350. 30. Birnbaumer, L. and Rodbell, M. (1969). J. Biol. Chem., 244, 3477. 31. Rodbell, M., Birnbaumer, L. and Pohl, S. L. (1970). J. Biol. Chem., 245, 718. 32. Hechter, O., Yoshinaga, K., Halkerston, I. D. K., Cohn, C. and Dodd, P. (1966). in O. Walaas (Editor), Molecular Basis  of Some Aspects of Mental Activity, Vol. 1, p. 291, Academic Press, New York. 89 Birnbaumer, L., Pohl, S., Michiel, H., Kraus, J. and Rodbell, M. (1970). in P. Greengard and E. Costa (Editors), Advances  in Biochemical Psychopharmacology, Vol. 3, p. 186, Raven Press, New York. Rodbell, M., Birnbaumer, L. and Pohl, S. L. (1971). in M. Rodbell and P. Condliffe (Editors), Fogarty International Centre Proceedings, No. 4, United States Government Printing Office, Washington. De Robertis, E., Rodriguez de Lores Arnaiz, G., Alberici, M., Butcher, R. W. and Sutherland, E. W. (1967). J. Biol. Chem., 242, 3487. Drummond, G. I. and Perrott-Yee, S. (1961). J. Biol. Chem., 236, 1126. Appleman, M. M. and Kemp, R. G. (1966). Biochem., Biophys. Res. Commun., 24, 564. Triner, L., Vulliemaz, Y., Schwartz, I. and Nahas, G. G. (1970). Biochem. Biophys. Res. Commun., 40, 64. ~ Honda, F. and Imamura, H. (1968). Biochem. Biophys. Acta, 151, 267. Brooker, G., Thomas, L. J. Jr. and Appleman, M. M. (1968). Biochem., _7» 41 77. Perlman, R. and Pastan, I. (1968). Biochem. Biophys. Res. Commun., 30, 656. Wicks, W. O. (1969). J. Biol. Chem., 224, 3941. Pastan, I. and Perlman, R. L. (1969). J. Biol. Chem., 244, 2226. Greengard, P. (1969). Biochem. J., 115, 19. Cori, G. T. and Cori, C. F. (1945). J. Biol. Chem., 158, 321. Cori, G. T. (1945). J. Biol. Chem., 158, 333. Sutherland, E. W. and Wosilait, W. D. (1955). Nature, 175, 169. Walsh, D. A., Perkins, J. P. and Krebs, E. G. (1968). J. Biol. Chem., 243, 3763. 90 49. Soderling, T. R. and Hickenbottom, J. P. (1970). Fed. P r o c , 29, 601. 50. Corbin, J. D. , Reimann, E. M. , Walsh, D. A. and Krebs, E. G. (1970). J. Biol. Chem., 245, 4849. 51. Huttunen, J. K., Steinberg, D. and Mayer, S. E. (1970). Proc. Nat. Acad. Sci. (USA), _67, 290. 52. Krebs, E. G. and Walsh, D. A. (1969). FEBSSymp., J_9, 121. 53. Kuo, J. F. and Greengard, P. (1969). Proc. Nat. Acad. Sci. (USA), 64, 1349. 54. Kuo, J. F. and Greengard, P. (1969). J. Biol. Chem., 244, 3417. 55. Tao, M., Salas, M. L. and Lipmann, E. (1970). Proc. Nat. Acad. Sci. (USA), bl_, 408. 56. Gill, G. N. and Garren, L. D. (1970). Biochem. Biophys. Res. Commun., 36, 328. 57. Whittaker, V. P. (1969). in A. Lajtha (Editor), Handbook of  Neurochemistry. Vol. 2, p. 327, Plenum Press, New York. 58. Eccles, J. C. (1964). The Physiology of Synapses, Springer Verlag, New York. 59. Katz, B. (1962). Proc. Roy. Soc. B, 155, 455. 60. Whittaker, V. P. (1965). In J. A. V. Butler and H. E. Huxley (Editors), Progress in Biophysical and Molecular Biology, Vol. 15, p. 39, Pergamon Press, New York. 61. Johnson, G. A., Boukma, S. J., Lahti, R. A. and Mathews, J. (1972). Fed. P r o c , 31, 513 Abs. 62. Goldberg, N. D., Lust, W. D., O'Dea, R. F., Wei, S. and O'Toole, A. G. (1970). in P. Greengard and E. Costa (Editors), Advances in Biochemical Psychopharmacology, Vol. 3, p. 67, Raven Press, New York. 63. Sattin, A. (1971). J. Neurochem., j_8, 1087. 64. • Williams, R. H., Little, S. A. and Ensinck, J. W. (1969). Amer. J. Med. Sci., 258, 190. 91 65. Weiss, B. and Costa, E. (1968). Biochem. Pharm., _17, 2107. 66. Chou, W. S., Ho, A. K. S. and Loh, H. H. (1971). Nature, 233, 280. 67. Weiss, B. and Costa, E. (1968). J. Pharm. Exp. Therap., 161, 310. 68. Rail, T. W. and Kakiuchi, S. (1966). in O. Walaas (Editor), Molecular Basis of Some Aspects of Mental Activity, Vol. 1, p. 417, Academic Press, New York. 69. Isaac, P. and Grahame-Smith, D. G. (1972). Proc. Biochem. Soc, 14P. 70. Swislocki, N. I. (1972). Fed. P r o c , 31. 911 Abs. 71. Klainer, L. M., Chi, Y. M. , Friedberg, S. L. , Rail, T. W. and Sutherland, E. W. (1962). J. Biol. Chem., 277, 1239. 72. Drummond, G. I., Severson, D. L. and Duncan, L. (1971). J. Biol. Chem., 246, 4166. 73. McCune, R. W., Gill, T. H., Von Hungen, K. and Roberts, S. (1971). Life Sci., 10, Part II, 443. 74. Kebabian, J. W. and Greengard, P. (1971). Science, 174, 1346. 75. Kakiuchi, S. and Rail, T. W. (1965). Fed. P r o c , 24, 150. 76. Kakiuchi, S. and Rail, T. W. (1968). Mol. Pharm., 4, 367. 77. Kakiuchi, S. and Rail, T. W. (1968). Mol. Pharm., 4, 379. 78. Shimizu, H., Daly, J. W. and Creveling, C. R. (1969). J. Neurochem., 16, 1609. 79. Shimizu, H., Creveling, C. R. and Daly, J. W. (1970). J. Neurochem., 17, 441. 80. Forn, J. and Krishna, G. (1970). Fed. P r o c , _29, 480. 81. Shimizu, H., Tanaka, S., Suzuki, T. and Matsukada, Y. (1971). J. Neurochem., 18, 1151. 82. Fumagalli, R., Bernareggi, V., Berti, F. and Trabucchi, M. (1971). Life Sci., 10, Par t i , 1111. 92 83. Sattin, A. and Rail, T. W. (1967). Fed. P r o c , 26, 707. 84. Sattin, A. and Rail, T. W. (1970). Mol. Pharm., _6, 13. 85. Kakiuchi, S. Rail, T. W. and Mcllwain, H. (1969). J. Neurochem., 16, 485. 86. Shimizu, H., Creveling, C. R. and Daly J. W. (1970). Mol. Pharm. , 6, 184. 87. Seeds, N. W. and Gilman, A. G. (1971). Science, 174, 292. 88. McAfee, D. A., Schorderet, M. and Greengard, P. (1971). Science, 171, 1156. 89. Gilman, A. G. and Nirenberg, M. (1971). Nature, 234, 356. 90. Schmidt, M. J., Palmer, E. C., Dettbarn, W. D. and Robison, G. A. (1970). Dev. Psychobiol., 3, 53. 91. Rail, T. W. and Sattin, A. (1970). in P. Greengard and E. Costa (Editors), Advances in Biochemical Psychopharmacology,. Vol. 3, p. 113, Raven Press, New York. 92. Siggins, G. R., Hoffer, B. J. and Bloom, F. E. (1969). Science, 165, 1018. 93. Siggins, G. R., Hoffer, B. J. and Bloom, F. E. (1971). Brain Res., _25, 535. 94. Siggins, G. R., Oliver, A. P., Hoffer, B. J. and Bloom, F. E. (1971). Science, 171, 192. 95. Birks, R. and Macintosh, F. C. (1961). Can. J. Biochem. Physiol., 39, 787. 96. Krnjevic, K. and Miledi, R. (1958) J. Physiol., 141, 291. 97. Breckenridge, B. McL., Burn, J. H. and Matschinsky, F. M. (1967). Proc. Nat. Acad. Sci. (USA), _57, 1893. 98. Goldberg, A. L. and Singer, J. J. (1969 ). P r o c Nat. Acad. Sci. (USA), _64, 134. 99. Bray, J. J., Kon, C. M. and Breckenridge, B. (1971). Brain Res., 26, 385. 93 100. Klein, D. C. (1969). Fed. P r o c , 28, 734, 101. Berg, G. R. and Klein, D. C. (1970). Fed. P r o c , _29, 615. 102. Klein, D. C. , Berg, G. R., Weller, J. and Glinsmann, W. (1970). Science, 167, 1738. 103. Klein, D. C., Berg, G. R. and Weller, J. (1970). Science, 168, 979. 104. Goldberg, N. D. and O'Toole, A. G. (1969). J. Biol. Chem., 244, 4458. 105. Weller, M. and Rodnight, R. (1970). Nature, 225, 187. 106. Gilman, A. G. (1970). Proc. Nat. Acad. Sci. (USA), 67, 305. 107. Krishna, G., Weiss, B. and Brodie, B. B. (1968). J. Pharm. Exp. Therap., 163, 379. 108. Bucher, T. and Pfleiderer, G. (1955). in/sTp. Colowick and N. O. Kaplon (Editors), Methods in Enzymology, Vol. 1, p. 435, Academic Press, New York. 109. Miyamoto, E., Kuo, J. F. and Greengard, P. (1969). J. Biol. Chem., 244, 6395. 110. Appleman, M. M., Birnbaumer, L. and Torres, H. N. (1966). Arch. Biochem. Biophys. , 116, 39. 111. Ellman, G. L., Courtney, K. D., Andres, V. Jr. and Feather-stone, R. M. (1961). Biochem. Pharm., 7, 85. 112. Gray, E. G. and Whittaker, V. P. (1962). J. Anat. , 96, 79. 113. Cotman, C. W. and Matthews, D. A. (1972). Biochem. Biophys. Acta, 249, 380. 114. French, S. W., Todoroff, T., Norum, M. L. and Ihrig, T. J. (1972). Exp. Mol. Path., J_6, 16. 115. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). J. Biol. Chem., 193, 265. 116. Robison, G. A., Butcher, R. W., <£)ye, I., Morgan, H. E. and Sutherland, E. W. (1966). Mol. Pharm. 1, 168. 94 117. Robison, G. A., Exton, J. H. , Park, C. R. and Sutherland, E. W. (1967). Fed. P r o c , 26, 257. 118. Nam, D. H. and Mayer, S. E. (1968). Mol. Pharm., 4, 61. 119. Abdel-Latif, A. A. (1966). Biochim. Biophys. Acta, 121, 406. 120. Autilio, L. A., Appel, S. H., Pettes, P. and Gambetti, P. L. (1968). Biochem., ]_, 2615. 121. Perkins, J. P. and Moore, M. M. (1971). J. Biol. Chem., 246, 62. 122. Rodbell, M., Birnbaumer, L., Pohl, S. L. and Krans, M. J. (1971). J. Biol. Chem., 246, 1877. 123. Severson, D. L. (1972). Ph.D. Thesis, University of British Columbia. \ 

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