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Studies on nucleoside 3', 5'-cyclic monophosphate 3'-phosphohydrolase from brain Wickson, Robert Douglas 1973

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' STUDIES ON NUCLEOSIDE 3',5'-CYCLIC MONOPHOSPHATE 31-PHOSPHOHYDROLASE FROM BRAIN by Robert Douglas Wickson B.Sc.(Hons.), University of Calgary, 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Pharmacology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1973 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of P h a r m a c o l o g y  The University of British Columbia Vancouver 8, Canada (i) ABSTRACT A nucleoside 3',5'-cyclic monophosphate 3'-phosphohydrolase was partially purified from bovine cerebral cortex. The enzyme isolated hydrolyzed both cyclic AMP (K m = 30 uM) and cyclic GMP (K m = 3 uM). In the I | absence of metal ions, no activity was observed. Divalent cations (Mg = I | | | | | Mn > Co > Ni ) stimulated activity with the activator constants being I | very low (e.g., 4.5 uM for Mg ). Cyclic GMP inhibited cyclic AMP hydrolysis and vice versa. Cyclic AMP inhibition of cyclic GMP hydrolysis was competitive*with a of 25 uM (cf., = 30 uM) indicating the presence of a single enzyme. 3-Isobutyl-l-methylxanthine inhibited cyclic AMP hydrolysis competitively with a of 1.5 uM. Gel f i l t r a t i o n of the partially purified enzyme showed a decrease in the apparent molecular weight in the presence of I | | | | | Mg and a further decrease in the presence of Mg plus Ca The phosphodiesterase present in the crude homogenate was inhibited by EGTA and activated by Ca . During DEAE-cellulose chromatography, a heat-stable protein factor was separated from the enzyme. In the absence of this factor, no stimulation by Ca was observed. Phosphodiesterase was shown to require both this protein activator and Ca in order to be stimulated. A model for the activation was proposed based upon the data obtained from kinetic experiments. Increasing concentrations of Ca decreased the apparent activator constant for protein activator while increasing protein I [ activator concentrations decreased the apparent activator constant for Ca I | The observed activation by Ca and protein activator suggested a means by which downward sloping Lineweaver-Burk plots could be obtained. In the case of phosphodiesterase, downward sloping Lineweaver-Burk plots have been interpreted by other workers as negative cooperativity. ( i i ) In the crude homogenate, there appeared to be a low K m phosphodiesterase specific for cyclic AMP in addition to the high K m enzyme partially purified. Calculations suggested that the high K m enzyme was at least as important as the low K m enzyme in hydrolyzing cyclic AMP. In addition, these calculations suggested that phosphodiesterase activity was not in a 10 to 100-fold excess over adenyl cyclase (or guanyl cyclase) activity. Phosphodiesterase and adenyl cyclase activities are probably delicately balanced, with any change in either of their a c t i v i t i e s capable of effecting changes in cyclic AMP concentrations. ( i i i ) TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS 11 CHEMICAL ASSAYS 12 DATA CALCULATIONS 12 RESULTS I. Assay of Nucleoside 3',5'-Cyclic Monophosphate 12 31-Phosphohydrolase II. Purification of Nucleoside 3',5'-Cyclic Monophosphate 16 3'-Phosphohydrolase from Bovine Cerebral Cortex III. Purification Results 18 IV. Separation of a Protein Activator using DEAE-Cellulose 24 Chromatography V. Preparation of the Protein Activator 30 VI. Kinetic Nature of the Activation of Phosphodiesterase 31 by Ca and Protein Activator VII. Multiple Forms of Phosphodiesterase 46 VIII. Determination of the Michaelis Constants for 55 Phosphodiesterase from Various Fractions in the Purification Procedure IX. Some Properties of the Partially Purified Enzyme 65 DISCUSSION 74 BIBLIOGRAPHY 112 APPENDIX 117 (iv) LIST OF TABLES Comparison of the rates of hydrolysis of cyclic AMP and cyclic GMP for the high K and low K phosphodiesterases present in the crude homogenate. (v) LIST OF FIGURES No. Page 1 Effect of EGTA and Ca on phosphodiesterase activity at 21 various steps of purification. 2 DEAE-cellulose chromatography of an ammonium sulphate 26 fraction. 3 Effect of adding aliquots of peak II (tube #80) to aliquots 29 of peak I (tubes #40,44,48). 4 Effect of a large amount of protein activator (35 ug of 35 protein) on phosphodiesterase activity (3.8 ug of protein). I | 5 Effect of protein activator and Ca on the K and V 37 c -l J A » M m max for cyclic AMP. I | 6 Effect of protein activator and Ca on the K and V 39 r , . „ > m m max for cyclic GMP. 7 Effect of cyclic GMP on affinity of phosphodiesterase for 42 protein activator. I | 8 Effect of protein activator and Ca on the hydrolysis of 44 cyclic GMP. I | 9 Effect of Ca on the apparent K for protein activator. 48 a I | 10 Effect of protein activator on the apparent K& for Ca . 50 I | | | 11 Effect of Mg and Ca on the apparent molecular weight 53 of phosphodiesterase. 12 Assay to determine K for cyclic AMP. 57 13 Assay to determine for cyclic GMP. 59 14 of acetone powder supernatant fraction for cyclic AMP. 62 15 Km of acetone powder supernatant fraction for cyclic GMP. 64 16 of 1.0% sodium deoxycholate solubilized enzyme. 67 17 Effect of divalent cations on phosphodiesterase activity. 70 18 Inhibition of cyclic AMP hydrolysis by 3-isobutyl-l- 73 methylxanthine (SC2964). 19 Inhibition of cyclic GMP hydrolysis by cyclic AMP. 76 (vi) No. Page 20 Determination of K' . 84 Ca 21 Determination of K' . 86 Ap 22 Determination of K. and K„ . 89 Ap Ca I | 23 Model C: steps involving Ca (Ca) and protein activator 91 (Ap) addition to the enzyme (E). 24 Lineweaver-Burk plot showing a possible method of 96 obtaining a downward curvature in the presence of a single non-cooperative enzyme. A - l Model A. 120 A-2 Model B. 125 A-3 Model C. 128 (vii) ABBREVIATIONS phosphodiesterase nucleoside 3',5'-cyclic monophosphate 3'-phosphohydrolase ACTH adrenocorticotropic hormone TSH thyroid-stimulating hormone cyclic AMP adenosine 3',5'-cyclic monophosphate 5'-AMP adenosine 5'-monophosphate ATP adenosine 5'-triphosphate cyclic GMP guanosine 3',5'-cyclic monophosphate GTP guanosine 5'-triphosphate cyclic NMP nucleoside 3',5'-cyclic monophosphate 5'-NMP nucleoside 5 *-monophosphate Ro 20-1724 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone SQ20009 l-ethyl-4-(isopropylidene-hydrazino)-lH-pyrazalo[3,-4-b]pyridine-5-carboxylic acid ethyl ester hydrochloride SC2964 3-isobutyl-l-methylxanthine EGTA ethyleneglycol-bis-(8-aminoethyl ether)N,N'-tetraacetate EDTA ethylenediaminetetraacetate Tris tris(hydroxymethyl)aminomethane S substrate E enzyme A activator Ap protein activator Ca Ca ES enzyme-substrate complex EAp enzyme-protein activator complex (viii) enzyme-Ca complex I | protein activator-Ca complex I | enzyme-protein activator-Ca complex I | enzyme-protein activator-Ca -substrate complex maximal velocity Michaelis constant inhibitor constant activator constant dissociation constant of S from ES (= [E][S]/[ES]) dissociation constant of Ca from ApCa (= [Ap][Ca]/[ApCa]) dissociation constant of ApCa from EApCa (« [E][ApCa]/[EApCa]) dissociation constant of Ap from EAp (= [E][Ap]/[EAp]) dissociation constant of Ca from ECa (= [E][Ca]/[ECa]) dissociation constant of Ap from EApCa (= [ECa][Ap]/[EApCa]) dissociation constant of Ca from EApCa (= [EAp][Ca]/[EApCa]) micro-nano-(ix) ACKNOWLEDGEMENTS It is a pleasure to thank Dr. George I. Drummond for his advice and aid in performing this work. The kinetic analysis was aided greatly by several useful and helpful discussions with Dr. Basil Roufogalis and Dr. David Godin. The author wishes to express his appreciation to Mr. Glenn Collins for a l l the services he has provided over the last year, and in particular, for photographing the diagrams. Most of a l l , the continuing support and encouragement provided by my wife Virginia is greatly appreciated. In addition, I thank her for the preparations of the diagrams for photography. The financial support of the National Research Council of Canada in the form of a 1967 Science Scholarship is gratefully acknowledged. - 1 -INTRODUCTION A heat stable factor, identified as adenosine 3',5'-cyclic monophosphate (cyclic AMP), was discovered in l i v e r homogenates by Sutherland and Rail (1.2) . This nucleotide was found to mediate the increase in phosphorylase activity observed during cellular stimulation by the hormones epinephrine and glucagon (1). These workers also found that cyclic AMP was hydrolyzed to 5'-AMP by a phosphodiesterase and was formed from ATP by tissue particles (2.3) . Subsequent studies showed that intracellular concentrations of cyclic AMP increased during epinephrine-induced activation of glycogenolysis in heart and skeletal muscle (4,5). A rise in cyclic AMP levels was also observed during lipolysis in fat cells following stimulation by a number of hormones, such as the catecholamines, ACTH, glucagon, TSH and prolactin (6,7). These observations led to the proposal of the "second messenger" hypothesis, which was developed to explain the relationship between cyclic AMP and hormone action (8). The hormone, or f i r s t messenger, is envisaged to interact with the membrane at an extracellular site giving rise to an activation of adenyl cyclase, the enzyme which catalyzes the conversion of ATP to cyclic AMP and inorganic pyrophosphate (9,10). The increased activity of adenyl cyclase gives rise to an increase in the intracellular concentration of cyclic AMP, the second messenger, which then activates the c e l l to carry out i t s specific function(s). A somewhat similar system i s believed to operate in the formation of guanosine 3',5'-cyclic monophosphate (cyclic GMP) from GTP, the reaction being catalyzed by guanyl cyclase (11). Intracellular levels of cyclic GMP have been shown to increase in response to acetylcholine in heart (12), oxytocin in the uterus (13) and oxotremorine in brain (14). The role which this cyclic nucleotide plays in the c e l l remains to be thoroughly - 2 -investigated; however, Goldberg ej: a l . (13) have postulated that cyclic AMP and cyclic GMP may act as opposing, but not equal, forces in the control of cellular metabolism. Following hormonal input to a c e l l , increased cyclic nucleotide levels are believed to activate specific protein kinases (15-17). Protein kinase is an enzyme which is capable of phosphorylating other proteins by means of phosphate transfer from ATP. The binding of cyclic AMP to the protein kinase regulatory subunit releases the catalytic subunit (18-21), which is then free to icatalyze phosphorylation reactions. Protein kinases have been shown to phosphorylate a number of different protein substrates in a variety of tissues (15,16,22-24). An example is phosphorylase b_ kinase in rabbit skeletal muscle (23). When phosphorylase b_ kinase is phosphorylated by protein kinase, i t is converted to an activated form, which then converts phosphorylase b_ to &, resulting in a stimulation of glycogen breakdown (25). As mentioned earlier, Sutherland and Rail (2) discovered an enzyme activity in tissue extracts which was capable of hydrolyzing cyclic AMP to 5'-AMP. This enzyme, later identified as a nucleoside 3',5'-cyclic monophosphate 3'-phosphohydrolase (phosphodiesterase) was found in many tissues (2,26,27). The enzyme required magnesium ions for activity and was inhibited by methylxanthines. Further characterization of phosphodiesterase from rabbit tissues was carried out by Drummond and Perrott-Yee (26), who found that the highest levels of enzyme activity occurred in brain, followed by kidney, heart, spleen, l i v e r and skeletal muscle. Within the nervous system, cerebral cortex had the highest activity followed by cerebellum, pons and medulla. In each tissue studied, the phosphodiesterase activity was reported to be 10 to 100-fold greater than that of adenyl cyclase (28,29). - 3 -I n t h e s e s t u d i e s , h o w e v e r , s a t u r a t i n g c o n c e n t r a t i o n s o f c y c l i c AMP w e r e u s e d . The h y d r o l y s i s r a t e o f c y c l i c AMP i n v i v o i s p r o b a b l y much l o w e r s i n c e t h e c o n c e n t r a t i o n o f t h i s compound i n t h e c e l l i s i n t h e m i c r o m o l a r r a n g e . B r e c k e n r i d g e and J o h n s t o n (30) d e t e r m i n e d t h e p h o s p h o d i e s t e r a s e a c t i v i t y i n d i f f e r e n t r e g i o n s o f r a b b i t b r a i n . They s u g g e s t e d t h a t s t r u c t u r e s w i t h l o w l e v e l s o f p h o s p h o d i e s t e r a s e may be p r e d o m i n a n t l y m y e l i n a t e d c e l l p r o c e s s e s ( o p t i c n e r v e , w h i t e m a t t e r o f c e r e b e l l u m and d o r s a l co lumns o f s p i n a l c o r d ) o r may be d e n s e c e l l u l a r a r e a s ( r e t i n a and v e n t r a l co lumns o f s p i n a l c o r d ) . The c a u d a t e n u c l e u s and t h e h y p o t h a l a m u s w e r e a r e a s w h i c h c o n t a i n e d h i g h p h o s p h o d i e s t e r a s e a c t i v i t y . No g e n e r a l c o r r e l a t i o n e x i s t e d be tween p h o s p h o d i e s t e r a s e and a d e n y l c y c l a s e a c t i v i t i e s i n t h e b r a i n a r e a s e x a m i n e d . More r e c e n t l y , F l o r e n d o e t a l . (31) have u t i l i z e d a h i s t o c h e m i c a l t e c h n i q u e f o r t h e i n t r a c e l l u l a r l o c a l i z a t i o n o f p h o s p h o d i e s t e r a s e . T h i s method r e l i e s on t h e p r e c i p i t a t i o n o f l e a d p h o s p h a t e f o l l o w i n g t h e h y d r o l y s i s o f 5 ' - A M P t o i n o r g a n i c p h o s p h a t e and a d e n o s i n e . L e a d p h o s p h a t e i s t h e n v i s u a l i z e d by e l e c t r o n m i c r o s c o p y . When s e c t i o n s f r o m r a t c e r e b r a l c o r t e x we re i n c u b a t e d i n a medium c o n t a i n i n g c y c l i c AMP, t h e l e a d p h o s p h a t e fo rmed was l o c a l i z e d a l m o s t e x c l u s i v e l y i n p o s t s y n a p t i c n e r v e e n d i n g s , w i t h most o f t h e r e a c t i o n p r o d u c t o c c u r r i n g i n t h e i m m e d i a t e v i c i n i t y o f t h e a r e a o f t h i c k e n i n g o f t h e p o s t - s y n a p t i c membrane. The r e m a i n d e r o f t h e l e a d p h o s p h a t e was u s u a l l y o b s e r v e d i n c l o s e a s s o c i a t i o n w i t h smooth e n d o p l a s m i c r e t i c u l u m and p o s s i b l y w i t h m i c r o t u b u l e s . A x o n a l s t r u c t u r e s had l i t t l e o r no r e a c t i o n p r o d u c t a s s o c i a t e d w i t h them. Drummond and P e r r o t t - Y e e (26) h a d o r i g i n a l l y r e p o r t e d t h a t p h o s p h o d i e s t e r a s e f r o m r a b b i t b r a i n was e x c l u s i v e l y a s o l u b l e enzyme. U s i n g - 4 -a subcellular fractionation technique, Cheung and Salganicoff (32) found the enzyme from rat brain to be partly soluble and partly particulate. The mitochondrial fraction contained 40% of the total activity while the microsomal and 100,000 x g supernatant fractions accounted for 20% and 30% respectively. Subfractionation of the mitochondrial fraction using a discontinuous sucrose gradient revealed that the bulk of the activity was present in the subfractions rich in nerve endings. De Robertis et al.(33) and Gaballah and Popoff (34) reported similar results. Studies on adenyl cyclase in the 1960's stimulated a search for inhibitors of phosphodiesterase, since, in tissue homogenates, the degradation of cyclic AMP interfered with the assay for adenyl cyclase activity. Even in relatively purified adenyl cyclase preparations, some phosphodiesterase contamination i s always present. Theophylline and caffeine have been widely used to inhibit phosphodiesterase during adenyl cyclase assays (9-11,33). Caffeine (K ± = 3 mM) and theophylline (K ± = 0.11 mM) were found to be competitive inhibitors of rat and rabbit brain enzymes respectively (35,36). 3-Isobutyl-l-methylxanthine, a potent inhibitor of lipoly s i s in fat cells (37), is known to inhibit the brain enzyme (38). Honda and Imamura (36) reported that a series of phenothiazine derivatives (promazine, chlorpromazine, perphenazine, fluphenazine and prochlorperazine) and chlorprothixene inhibited cyclic AMP hydrolysis. Perphenazine displayed non-competitive behavior with a of 20 uM. Reserpine and diethylaminoethyl-1-reserpine also inhibited the rabbit brain enzyme. The latter exhibited competitive inhibition with a of 0.55 uM. Tri c y c l i c antidepressants (imipramine, desmethylimipramine and amitriptyline), meprobamate and chlorthiazide were not inhibitory when tested at concentrations of 50 uM. - 5 -Using mouse brain homogenates, imipramine, desmethylimipramine and nortriptyline at 1 to 5 mM concentrations were inhibitors of phosphodiesterase (39). The rat brain enzyme i s also inhibited non-competitively by papaverine, a smooth muscle relaxant, the being 42 uM (40). Other compounds, such as those designated Ro 20-1724 (41) and SQ20009 (42) are potent inhibitors of the dog and rat brain enzymes respectively. Citrate, inorganic pyrophosphate, nucleoside triphosphates and EDTA are inhibitors of the bovine brain enzyme (35,43,44). Inhibition due to these agents has been attributed to their a b i l i t y to sequester metal ions, in particular Mg (35,45). Miki and Yoshida (46) reported EGTA to also be an inhibitor of rat brain phosphodiesterase, presumably due to i t s a b i l i t y to chelate Ca O'Dea et a l . (47) and Cheung (48) have reported inhibition of cyclic AMP hydrolysis when protein kinase was present in the assay. In the presence of cyclic AMP-dependent protein kinase, a decreased rate of cyclic AMP hydrolysis occurred. This inhibition has been attributed to cyclic AMP binding to protein kinase and thus making i t unavailable for hydrolysis. This inhibition of cyclic AMP hydrolysis by protein kinase may be an important regulatory mechanism in vivo. Roberts and Simonsen (39) and Harris et a l . (49) have shown that cyclic GMP was an inhibitor of cyclic AMP hydrolysis by brain phosphodiesterase. Contrary to these reports, several authors (50-52), using various rat tissues, reported a stimulation of cyclic AMP hydrolysis by cyclic GMP. Using concentrations of 1 uM cyclic AMP as substrate and 2 uM cyclic GMP as activator, Beavo et a l . (50) reported a two-fold stimulation of cyclic AMP hydrolysis by a 20,000 x g particulate fraction from rat brain while the 20,000 x g supernatant fraction showed no stimulation. The rat li v e r enzyme - 6 -was activated by cyclic GMP concentrations i n the range of 0.1 to 20 uM and was inhibited by cyclic GMP concentrations in excess of 20 uM for both the particulate and supernatant enzymes. Russell et a l . (52), using phosphodiesterase prepared from rat l i v e r , also reported cyclic GMP activation for the soluble enzyme, but no activation for the enzyme present in the particulate fraction. An activation by cyclic GMP would indicate an allosteric site for the binding of this nucleotide. Metal ions (2,26,27) have long been recognized as activators of the brain enzyme. Early studies showed that divalent cations were required for I | phosphodiesterase activity. With regard to the rabbit brain enzyme, Mg I | was the most active Ion tested and could be partially replaced by Mn and I | Co (26). In contrast to these reports, Cheung (44), using an enzyme from bovine brain, has reported considerable phosphodiesterase activity in the I | absence of added metal ions. The addition of Mg resulted in a mild I | | | stimulation of 50% at optimal concentrations. Mn and Co showed maximal I | activity with Mg having lesser activity. Butcher and Sutherland (27) showed that imidazole had a stimulatory effect on the rate of cyclic AMP hydrolysis. Using mouse brain homogenates, Roberts and Simonsen (39) have studied the effect of a variety of imidazole derivatives on phosphodiesterase activity. Imidazole-4-acetic acid, imidazole-4-carboxylic acid, histidine and l-methylimidazole-4-acetic acid were the most potent stimulators of those compounds studied. Early work by Drummond and Perrott-Yee (26) showed that large and unexplained losses of phosphodiesterase activity occurred during efforts to purify the enzyme. Using bovine brain, Cheung (53,54) reported that a semi-purified enzyme (but not the crude enzyme) was activated by snake venom - 7 -or trypsin. He also discovered a heat-stable non-dialyzable factor in crude brain extracts which activated the enzyme and which could be separated from the enzyme by DEAE-cellulose chromatography. The addition of the factor back to purified phosphodiesterase preparations stimulated activity 5.5 fold (55), but produced no response when added to phosphodiesterase present in a crude homogenate. This indicated that the activating factor was present in excess of phosphodiesterase in the homogenate. Cheung (55) has attributed the large losses of enzyme activity during purification to the separation of this activating factor from the enzyme. The factor was destroyed by trypsin, but not by deoxyribonuclease or ribonuclease indicating that i t was a protein. Since then, the protein activator from bovine brain (56) and heart (57) has been purified to homogeneity. The isolated protein activating factor is stable to boiling at pH 1.7 and has a molecular weight between 25,000 and 40,000 a.u. It apparently does not bind cyclic AMP, but has been reported to decrease the K of phosphodiesterase for cyclic AMP and increase the m maximal velocity of the reaction. Kakiuchi and Yamazaki (58), in 1970, reported the stimulation of ++ phosphodiesterase from dialyzed rat brain supernates by minute amounts of Ca I | This stimulation occurred only when Mg was present at concentrations of 1 mM or greater. The presence of EGTA (0.1 to 0.3 mM) reduced enzyme activity by 65%, and the addition of trace amounts of Ca in excess of EGTA restored activity. Later they isolated a protein which enhanced stimulation due to Ca (59). When the protein activating factor was present, enzyme was I | stimulated by lower concentrations of Ca than it was in the absence of this factor (60). The maximal stimulation was also increased. Much recent evidence suggests that phosphodiesterase exists in multiple forms. Kakiuchi et al . (61) separated two forms of phosphodiesterase using - 8 -Sepharose-6B gel filtration. Both forms required Mg for activity, but the higher molecular weight enzyme was not stimulated by Ca while the lower molecular weight enzyme was. The existence of more than one molecular form of phosphodiesterase has been reported in a variety of tissues (50,52, 61,72). In bovine heart, Hardman and Sutherland (62) first showed a second enzyme which was more specific for uridine 3',5'-cyclic monophosphate (cyclic UMP) and more sensitive to inhibition by theophylline and activation by imidazole. Employing starch-gel electrophoresis, Monn and Christiansen (71) demonstrated four bands of activity for cyclic AMP hydrolysis using rat and rabbit brain supernates, while Campbell and Oliver (70), using polyacrylamide gel electrophoresis, were able to show only a single peak of enzyme activity for cyclic AMP degradation. However, these latter workers did demonstrate multiple forms of phosphodiesterase in other tissues. Using agarose gel fi ltration, Thompson and Appleman (68,69) separated three peaks of phosphodiesterase activity from sonicated rat brain supernates. One peak was eluted in the void volume of the column, while the remaining two peaks were eluted at 1.6 (peak II) and 1.8 (peak III) void volumes. Peak II hydrolyzed both cyclic AMP and cyclic GMP, while peak III hydrolyzed only cyclic AMP. Michaelis constants of 104 uM and 12.9 uM were reported for cyclic AMP and cyclic GMP respectively for the peak II enzyme (a cyclic AMP-cyclic GMP phosphodiesterase) while a of 2.0 uM for cyclic AMP was reported for the peak III enzyme (a cyclic AMP phosphodiesterase). Using peak II phosphodiesterase, the of each nucleotide as an inhibitor of hydrolysis of the other corresponded to its value indicating the presence of one enzyme capable of hydrolyzing both cyclic AMP and cyclic GMP. Because the K for cyclic GMP was lower than that for cyclic AMP, Thompson - 9 -and Appleman have suggested that the high Km enzyme (peak II) might be principally a cyclic GMP phosphodiesterase. Both of the enzymes reported by Thompson and Appleman (68,69), that is peak II and peak III, displayed downward sloping Lineweaver-Burk plots (73) (or upward sloping Eadie plots (74)). .This,type.of non-linearity is generally thought to be indicative of two independent catalytic sites or a negatively cooperative enzyme. Two independent catalytic sites could be either two separate enzymes or two sites on one enzyme. The concept of negative cooperativity was originally introduced to account for the binding of nicotinamide adenine dinucleotide (NAD) to glyceraldehyde 3-phosphate dehydrogenase (75). Here the binding of the first ligand is believed to inducea change in the conformation of the enzyme making the binding of a second ligand unfavorable. Russell est a l . (76) have attributed the downward curvature of the Lineweaver-Burk plots observed with phosphodiesterase to the existence of a negatively cooperative enzyme. This conclusion was based upon a computer analysis of their data which showed a better f i t on non-linear least squares analysis for the case of a negatively cooperative enzyme than for two enzymes. Theoretical aspects of downward sloping Lineweaver-Burk plots have been discussed by several authors (77-81). Almond and Niemann (76) have shown that systematic assay errors ( i .e . , undercorrection for the blank in an assay) can give rise to this phenomenon. Botts (78) and Harper (79,80) give theoretical considerations to various kinetic equations which can result in downward sloping Lineweaver-Burk plots. In the consideration of a multi-site enzyme model, Engel and Ferdinand (81) have attributed abrupt transition in Lineweaver-Burk plots to not only a negatively cooperative - 10 -component of enzyme activity, but also to a positive cooperativity at higher substrate levels. They indicated that following the negatively cooperative component, the addition of a third or subsequent ligand may be more favorable than i t previously was, that is , a positive cooperativity could occur. When this project was begun in 1970, phosphodiesterase had not been purified nor had there been many investigations as to its precise role in the control of cyclic AMP and cyclic GMP levels. Since that time, the literature on this enzyme has become voluminous. The work presented here began with an attempt to purify the enzyme from bovine cerebral cortex. A form of the enzyme was partially purified which possessed properties similar to those of the high enzyme (peak II) of rat brain described by Thompson and Appleman (68). Some properties of the enzyme (Vmax> Km > metal ion requirements and hydrolysis rates for cyclic AMP and cyclic GMP) were studied. A heat-stable protein was isolated which required Ca to activate the phosphodiesterase. The nature of the activation was studied and a kinetic model proposed. Studies on the activation of phosphodiesterase revealed a possible explanation for the occurrence of downward sloping Lineweaver-Burk plots (76). Using the Michaelis constants and the maximal velocities experimentally determined, calculations showed that phosphodiesterase was probably not present in a 10 to 100-fold excess over adenyl cyclase. Thompson and Appleman (68,69) have reported the presence of a low K cyclic AMP specific phosphodiesterase (peak III). This enzyme is believed to be the enzyme directly involved in the control of cyclic AMP concentrations in the ce l l . Calculations indicated that the enzyme partially purified here (a high enzyme capable of hydrolyzing both cyclic AMP and cyclic GMP) hydrolyzed - 11 -c y c l i c AMP as r a p i d l y as t h e l o w K m c y c l i c AMP p h o s p h o d i e s t e r a s e ( 6 8 , 6 9 ) . T h i s i n d i c a t e d t h a t t h e h i g h enzyme may c o n t r i b u t e v e r y s i g n i f i c a n t l y t o t h e h y d r o l y s i s o f c y c l i c AMP a t c o n c e n t r a t i o n s w h i c h may e x i s t ixt_ v i v o . The i m p o r t a n t r o l e w h i c h p h o s p h o d i e s t e r a s e u n d o u b t e d l y p l a y s i n t h e c o n t r o l o f c y c l i c AMP and c y c l i c GMP c o n c e n t r a t i o n s i s d i s c u s s e d . MATERIALS T r i t i a t e d c y c l i c AMP (24 C i / m m o l e ) and c y c l i c GMP ( 4 . 3 C i / m m o l e ) we re p u r c h a s e d f r o m New E n g l a n d N u c l e a r , B o s t o n . The e t h a n o l was e v a p o r a t e d u n d e r r e d u c e d p r e s s u r e b e f o r e u s e . S o l u t i o n s o f l a b e l l e d c y c l i c n u c l e o t i d e s we re d i l u t e d w i t h t h e i r r e s p e c t i v e u n l a b e l l e d compounds t o t h e d e s i r e d s p e c i f i c a c t i v i t y and we re t h e n d i l u t e d t o a p p r o p r i a t e c o n c e n t r a t i o n s . The aqueous s o l u t i o n s w e r e s t o r e d a t - 2 0 ° C . U n l a b e l l e d c y c l i c AMP and c y c l i c GMP, as w e l l as EGTA and T r i s , we re o b t a i n e d f r o m S igma C h e m i c a l C o . , S t . L o u i s . A s a m p l e o f 3 - i s o b u t y l - l -m e t h y l x a n t h i n e (SQ2964) was a g i f t f r o m G . D . S e a r l e and C o . , A r l i n g t o n H e i g h t s , I l l i n o i s . M e t a l s a l t s we re p u r c h a s e d f r o m K&K L a b o r a t o r i e s , P l a i n v i e w , New Y o r k . D E A E - c e l l u l o s e was p u r c h a s e d f r o m Eas tman O r g a n i c C h e m i c a l s , R o c h e s t e r , and p r e p a r e d as f o l l o w s : t w e n t y grams o f t h e c e l l u l o s e we re p l a c e d i n one l i t r e o f 1 .0 N NaOH and a l l o w e d t o s e t t l e . The s l u r r y was t h e n f i l t e r e d by l i g h t s u c t i o n t h r o u g h a c o a r s e s i n t e r e d g l a s s f u n n e l and t h o r o u g h l y washed w i t h w a t e r . The c e l l u l o s e was t h e n r e s u s p e n d e d i n 0.5 N H C l and t h e w a s h i n g p r o c e d u r e r e p e a t e d . The m a t e r i a l was a g a i n supended i n 1 .0 N NaOH and t h o r o u g h l y washed w i t h w a t e r . F i n e s w e r e removed b e f o r e u s e . B i o G e l A 1 . 5 m was p u r c h a s e d f r o m C a l b i o c h e m , L o s A n g e l e s ; f i n e s w e r e - 12 -removed b e f o r e u s e . The a g a r o s e g e l was p a c k e d i n a K 2 5 / 1 0 0 co lumn p u r c h a s e d f r o m P h a r m a c i a (Canada) L t d . , M o n t r e a l . CHEMICAL ASSAYS P r o t e i n c o n c e n t r a t i o n was r o u t i n e l y measured by t h e o p t i c a l method o f W a r b u r g and C h r i s t i a n ( 8 2 ) , u s i n g a Beckman DU s p e c t r o p h o t o m e t e r . F o r a l l s o l u t i o n s c o n t a i n i n g p a r t i c u l a t e m a t t e r and t h o s e f r a c t i o n s w i t h ^ 2 8 0 n m ^ 2 6 0 n m < 1 . 0 , p r o t e i n was d e t e r m i n e d by t h e method o f Lowry e t a l . ( 8 3 ) , u s i n g b o v i n e se rum a l b u m i n as s t a n d a r d . C a l c i u m was d e t e r m i n e d u s i n g a V a r i a n A s s o c i a t e s T e c h t r o n A t o m i c A b s o r p t i o n S p e c t r o p h o t o m e t e r s e t a t 4 2 2 . 7 n m . T h e s e d e t e r m i n a t i o n s we re k i n d l y p e r f o r m e d by M r . E . E . Q u i s t , F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s . DATA CALCULATIONS A l l L i n e w e a v e r - B u r k p l o t s (73) w e r e drawn as t h e b e s t l i n e by e y e , w h i l e t h e l i n e s f o r a l l E a d i e p l o t s (74) and E a d i e t y p e p l o t s we re done by l i n e a r r e g r e s s i o n . Mos t o f t h e d a t a w e r e p r o c e s s e d t h r o u g h t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a ' s d u p l e x IBM 3 6 0 / 6 7 c o m p u t e r ; t h e r e m a i n d e r we re c a l c u l a t e d u s i n g a Compucorp 140 S t a t i s t i c i a n d e s k c a l c u l a t o r . RESULTS I. A s s a y o f N u c l e o s i d e 3 ' , 5 ' - C y c l i c Monophospha te 3 ' - P h o s p h o h y d r o l a s e P r i o r t o t h e commencement o f t h e p r e s e n t w o r k , t h e a s s a y s most commonly emp loyed f o r n u c l e o s i d e 3 ' , 5 ' - c y c l i c monophosphate 3 ' - p h o s p h o h y d r o l a s e i n v o l v e d t h e p h o t o m e t r i c d e t e r m i n a t i o n o f i n o r g a n i c p h o s p h a t e ( 2 7 , 3 0 , 3 5 ) . I n s u c h a s s a y s y s t e m s , s n a k e venom, w h i c h c o n t a i n s h i g h c o n c e n t r a t i o n s o f 5 ' - n u c l e o t i d a s e , i s added t o e a c h a s s a y t u b e and t h u s when 5 ' - A M P i s p r o d u c e d by p h o s p h o d i e s t e r a s e , t h e n u c l e o t i d a s e f u r t h e r d e g r a d e s i t t o a d e n o s i n e - 13 -and inorganic phosphate. Such assay methods have a major limitation in that physiological concentrations of substrate (in the uM range) cannot be employed because of the lack of sensitivity of the phosphate determination. A spectrophotometric assay in which the enzyme reaction was coupled with 5'-adenylate deaminase had previously been used in this laboratory (26). This assay method which is based on the decrease in absorbance at 265 nm during the deamination of 5'-AMP to inosine 5'-monophosphate (5'-IMP) similarly lacks sensitivity and precludes the use of substrate concentrations in the uM range. During the past three years, more sensitive radiochemical assay methods (63,69,84,85) have appeared as radioactively labelled cyclic nucleotides became commercially available. These assay methods provide the sensitivity necessary to measure reaction velocities employing substrate in the physiological or micromolar range. A radioactive assay method based on paper chromatographic separation of the reaction products from the substrate was developed for the present work. The assay for phosphodiesterase was carried out at 30°C. Each incubation tube contained 25 mM Tris-HCl, pH 7.5 or 50 mM imidazole, pH 7.0; 5 mM MgSO^ (except i n the case of metal ion studies); cyclic AMP or cyclic GMP (approximately 5 x 10^ dpm/tube); and enzyme. Water or aqueous solutions of compounds under study were added to a fi n a l volume of 0.2 ml. The reaction was routinely started by the addition of substrate except in the case of assays to determine values where enzyme was added to i n i t i a t e the reation. Control tubes contained a l l components with either boiled enzyme solution or an equivalent volume of water. After incubation, which was usually 10 min when cyclic GMP was used as substrate and 30 min when using cyclic AMP as substrate, the reaction was stopped by the addition of 25 ul of glacial - 14 -acetic acid. A portion of the sample, usually 200 u l , was applied in a 2 cm streak to Whatman 3MM paper (23 cm wide x 50 cm long) along with carrier compounds (0.05 umoles of cyclic NMP, 5'-NMP and nucleoside), and chromatographed (descending) 1 6 - 1 8 hours using a solvent system composed of 1.0 M ammonium acetate: 95% ethanol (3:7 v/v). After development, the chromatograms were visualized under ultraviolet light. Spots were cut out and radioactivity determined in the 5'-NMP, cyclic NMP and nucleoside areas. Each spot (approximately 4 cm wide x 6 cm long) was cut out, cut in half, and each 4 cm x 3 cm portion placed in a liquid s c i n t i l l a t i o n v i a l with the 4 cm edge being horizontal. The papers were placed uniformly in the vials in the form of a loose curl ( C X > ) . To each v i a l was added 18 ml of a cocktail consisting of 4 g PPO (2,5-diphenyloxazole) and 50 mg POPOP (1,4-bis-[2-(5-phenyloxazolyl)]-benzene) per l i t r e of toluene. Radioactivity was determined using a Nuclear Chicago Isocap/300 liquid s c i n t i l l a t i o n spectrometer. The papers were then removed from the vi a l s , and the liquid s c i n t i l l a t i o n f l u i d checked for radioactive contamination. Few (less than 0.5%) of the counts were present in the liquid s c i n t i l l a t i o n f l u i d , indicating that the radioactivity was not eluted from the paper. In the more purified enzyme preparations, contaminating 5'-nucleotidase activity had been removed and in these cases, radioactivity i n excess of the blank was present only in the 5'-NMP spot. Calculation of reaction v e l o c i t y — The disintegrations per minute in the three spots (5*-NMP, cyclic NMP and nucleoside) represented greater than 95% of the radioactivity applied to the chromatogram. The total number of disintegrations per minute in the three spots was constant, independent of the distribution of the radioactivity between the spots. Reaction velocities - 15 -were calculated from the counts per minute using two methods. The f i r s t involved a conversion of the counts per minute to disintegrations per minute using a standard curve for tritium counted on paper. Knowing the specific radioactivity of the added cyclic nucleotide, the amount of product in nanomoles could be calculated. This was then converted to nanomoles/mg protein per min. The second method employed the percentage conversion of the cyclic nucleotide to product. The percentage breakdown of the substrate was converted to nanomoles of substrate hydrolyzed and therefore to nanomoles of product formed. The reaction velocity (nanomoles/mg protein per min) could be calculated. Both methods gave identical results. Validity of the assay— As long as substrate was saturating, the formation of product (5'-NMP plus nucleoside) was a linear function of time for up to 5 hours at 30°C. This was the case for concentrations of protein in the assay between 4 and 100 ug per tube. When quantities of protein between 0.4 and 4 ug per tube were used, the reaction velocity was linear with time for one hour. At these lower concentrations of protein, denaturation of the enzyme seemed to occur. When enzyme concentration was varied at saturating levels of substrate (using a phosphodiesterase eluted from a BioGel A1.5m column), the reaction velocity was linear up to 120 ug of protein. At lower levels of substrate, deviation from linearity with time and protein concentration was observed due to depletion of substrate. Determination of K^— The enzyme assay was used to determine the kinetic parameters C^mSLX a n < i K ) of the enzyme at various stages during purification. In these assays, a constant level of enzyme was used over the complete substrate range. The specific radioactivity of the cyclic nucleotide was varied as required in order to maintain a sample to blank counts per minute - 16 -ratio of at least two. At low substrate, this method produces a significant breakdown of the substrate (up to 35% in our assays). Since this high percentage breakdown of substrate occurs, the assay Is not linear with time. Under these circumstances, the data were analyzed using the method of Lee and Wilson (86). Essentially, the average substrate rather than the i n i t i a l , is plotted against the average (measured) velocity. The average substrate is calculated as the arithmetic average of the i n i t i a l substrate plus the substrate present when the reaction i s stopped. II. Purification of Nucleoside 3',5'-Cyclic Monophosphate 3'-Phosphohydrolase from Bovine Cerebral Cortex When this work was begun, extensive purification of the enzyme had not been achieved. Many attempts (26,35,46) had been made, but the yields and purification were never substantial. Because brain possesses the highest phosphodiesterase activities (26), we chose to purify the enzyme from this source. Bovine brains were obtained from a local slaughterhouse, packed in ice and transported to the laboratory, where blood vessels and the lower structures were removed from the cerebra. The cerebra were used either fresh or frozen at -80°C with no apparent differences i n the resulting preparations. Preparation of acetone powder— Fresh or frozen cerebra were homogenized in 10 volumes of acetone (prechilled to -15°C) using a Sorvall Omni-Mixer at maximum velocity. Homogenization was carried out for three 45 sec periods with 30 sec cooling periods between. The homogenate was centrifuged at 16,000 x g for 30 min at -15°C. The pellets were rehomogenized (using the above method) in two volumes of prechilled acetone and recentrifuged at - 17 --15°C. The pellets were placed in a dessicator and dried under vacuum, first using a water aspirator and finally a rotary o i l pump. The acetone powder prepared in this way (with an approximate yield of 0.2 g per g wet weight of brain) could be stored at -20°C for up to one year with no apparent loss in activity. Preparation of initiallextract— A l l further procedures were carried out at 2°C. As required, a sample of the acetone powder was homogenized in 20 volumes of 5 mM Tris-HCl, pH 7.5, using a Sorvall Omni-Mixer at maximum velocity. Homogenization was carried out for three 45 sec periods with 30 sec cooling periods between. The homogenate was centrifuged at 37,000 x g for 30 min. The pellets were rehomogenized (using a Potter-Elvehjem homogenizer) in 10 volumes of 5 mM Tris-HCl, pH 7.5 and recentrifuged. The supernatant fluids from both centrifugations were combined and used in further purification. In early work, the remaining pellet material was routinely discarded. Precipitation with ammonium sulphate— Phosphodiesterase was precipitated by bringing the solution to 60% saturation with stirring using solid ammonium sulphate, maintaining the pH near 7.0 with 2.0 N KOH. The solution was stirred for 30 min, and then allowed to stand for one hour. The suspension was centrifuged at 37,000 x g for 30 min. The supernate (which contained no measurable phosphodiesterase activity) was discarded; the pellet was suspended in a minimum volume of 5 mM Tris-HCl, pH 7.5, and dialyzed overnight against 4 changes of 5 mM Tris-HCl, pH 7.5 (4 litres per change). The dialyzed preparation was divided into aliquots (usually about 20 ml) and stored at -20°C. DEAE-cellulose chromatography— A sample of the dialyzed ammonium - 18 -sulphate fraction (usually about 700 mg protein) was applied to a DEAE-cellulose column (2 cm in diameter x 30 cm long) previously equilibrated with 5 mM Tris-HCl, pH 7.5. The column was washed with 5 mM Tris-HCl, pH 7.5 unt i l the washout peak had subsided to 10% of i t s peak protein content. The column was then eluted with a linear salt gradient. Equal volumes of 5 mM Tris-HCl, pH 7.5 and 500 mM potassium phosphate, pH 7.0 (one l i t r e each) were used to produce the gradient. The flow rate was generally 1.0-1.5 ml/min and fractions were collected for 10 min periods using an LKB fraction collector. Enzyme activity reached a maximum when the salt concentration was 100 mM potassium phosphate. The contents of the tubes containing peak enzyme acti v i t i e s were concentrated using an Amicon PM-10 u l t r a f i l t e r with a pressure of 30 psi of The concentrate was dialyzed thoroughly against 5 mM Tris-HCl, pH 7.5 and divided into aliquots (4 ml or less) for storage at -20°C. Gel f i l t r a t i o n — A BioGel A1.5m column (exclusion limit = 1.5 x 10^ a.u.) was equilibrated with 5 mM Tris-HCl - 5 mM MgSO^ , pH 7.5. A sample of the enzyme concentrated from the DEAE-cellulose chromatography was thawed, enough MgSO^ was added to yield a 5 mM concentration, and the solution applied to the column, which was 2.5 cm in diameter and 88 cm long. The sample was followed by application of 4 ml of 10% sucrose in 5 mM Tris-HCl - 5 mM MgSO^ , pH 7.5. The enzyme was eluted (ascending) using 5 mM Tris-HCl - 5 mM MgSO^ , pH 7.5. The contents of the tubes containing peak enzyme activity were pooled and concentrated using an Amicori PM-10 u l t r a f i l t e r at a pressure of 30 psi of N£ before storage at -20°C. III. Purification Results Purification and Y i e l d — Previous workers (26,35,46,55) had reported - 19 -large losses in enzyme activity during attempted purification. Cheung (55) attributed these losses to the removal of a protein activating factor. In our work, as w i l l be shown later, a heat-stable factor was also separated during DEAE-cellulose chromatography. In crude supernates derived from cerebral cortex, other workers (46,58) showed a marked inhibition of phosphodiesterase activity by EGTA, presumably due to chelation of Ca present in the supernate. In the present work, enzyme which had been purified through the DEAE-cellulose column showed no inhibition by EGTA nor activation by Ca lik e l y due to the removal of the protein activator to be described later. However, a l l fractions prior to DEAE-cellulose chromatography were inhibited by EGTA. This inhibition was studied using phosphodiesterase from different steps in the purification procedure and is shown in Fig. 1. The results showed a marked inhibition of phosphodiesterase activity by EGTA (solid bars). However, this inhibition could be overcome by the addition of Ca in excess of EGTA in the assay mix (striped bars). At each successive step in purification through the ammonium sulphate precipitation, the percentage inhibition by EGTA decreased. Since the purified enzyme by i t s e l f was not inhibited by EGTA nor stimulated by Ca , assays to determine purification and yields were performed in the presence of 500 uM EGTA. By performing the assays in this manner, the basal unstimulated levels of phosphodiesterase activity were compared. Apparent losses of activity due to changes in sensitivity of the enzyme to Ca were minimized in this manner. Both;-the crude homogenate and the enzyme partially purified from BioGel A1.5m gel f i l t r a t i o n hydrolyzed both cyclic AMP and cyclic GMP. Purification of the enzyme was followed using either 1 mM cyclic AMP or cyclic GMP as - 20 -Fig. 1: Effect of EGTA and Ca on phosphodiesterase activity at various steps of purification. The experiments were performed at pH 7.0, using 4.9 uM cyclic GMP as substrate. Phosphodiesterase was assayed in the [ | absence of EGTA and Ca (open bars), in the presence of 0.5 mM EGTA and I | absence of Ca (solid bars), and in the presence of 0.5 mM EGTA and 5 mM CaCl2 (striped bars). The fractions assayed were: A - crude homogenate of the acetone powder, B - 37,000 x g supernate, C - 37,000 x g pellet, D - supernate from the ammonium sulphate precipitation step (dialyzed), and E - pellet from the ammonium sulphate precipitation step (resuspended and dialyzed). v ( n m o l e s / m g p r o t e i n / m i n ) DO Ol ro o i m - 22 -substrate in the presence of 500 uM EGTA. In both cases, a 15-fold purification was achieved with a yield of 30%. Following the i n i t i a l 37,000 x g centrifugation, the activity was divided almost equally between the supernate and the pellet. Since the pellet material was routinely discarded, this centrifugation resulted in a 50% loss in total activity, as only 50% of the activity present in the original homogenate of the acetone powder was in the supernate. Of the 50% of the activity in the supernate, 60% of this (30% of the activity present in the original homogenate) was recovered after the BioGel A1.5m gel f i l t r a t i o n step. Since 60% of the activity present in the 37,000 x g supernate was recovered, the enzyme which was partially purified seemed to be a major component of the supernatant phosphodiesterase. Acetone powder preparation versus other methods of preparation— The i n i t i a l step in our purification procedure i s a homogenization of the cerebra in acetone. The acetone powder was chosen because a) i t produced no loss in activity during the acetone treatment, b) i t removed a great deal of the l i p i d in the i n i t i a l extraction, and c) i t provided a simple technique for preparing a very stable readily available source of the enzyme. The acetone powder could be stored at -20°C for up to one year with no apparent loss in activity. Following the acetone powder preparation, a 1:20 homogenate (using 5 mM Tris-HCl, pH 7.5) was prepared. For comparative purposes, whole cerebra were also homogenized in 10 mM Tris-HCl, pH 7.5 or 0.32 M sucrose. The three methods of homogenization appeared to give identical enzyme preparations. The total activity per gram of brain tissue (measured in the presence of 500 uM EGTA) and the relative distribution between the supernate and pellet following 37,000 x g centrifugation (approximately 50% of the - 23 -activity in each fraction) was similar, apparently independent of the method of i n i t i a l homogenization. A l l three preparations gave the same qualitative enzyme activity profiles when chromatographed on BioGel A1.5m in the presence of 5 mM Tris-HCl - 5 mM MgSO^ , pH 7.5, and showed the same relative specificity for cyclic AMP and cyclic GMP. As discussed later, the K m values for cyclic AMP and cyclic GMP in the 37,000 x g supernate agree very closely with those of Thompson and Appleman, who used a method of direct homogenization of brain in aqueous buffer (68,69). Phosphodiesterase prepared from the 37,000 x g p e l l e t — In later work, phosphodiesterase present in the 37,000 x g pellet derived from the acetone powder homogenate was studied. The pellet (as previously mentioned) contained approximately 50% of the enzyme activity i n i t i a l l y present in the homogenate and thus represented a major loss of activity during purification. In an attempt to isolubilize, the enzyme, pellets were suspended in 3 volumes of 1.0% sodium deoxycholate in 5 mM Tris-HCl, pH 7.5 and allowed to stand for 30 min. The suspension was centrifuged at 100,000 x g for one hour and the supernate removed using a Pasteur pipette. Following 100,000 x g centrifugation, 80% of the activity present in the 37,000 x g pellet fraction now appeared in the supernate. Only 20% of the activity present in the 37,000 x g pellet material was not solubilized by treatment with 1.0% sodium deoxycholate. The sodium deoxycholate solubilized enzyme was applied to a BioGel A1.5m column. The enzyme activity profile, as well as the relative rates of hydrolysis for cyclic AMP and cyclic GMP, were identical to those obtained using the 37,000 x g supernatant enzyme. This similarity in properties of the pellet and supernatant enzymes suggests that the pellet enzyme may be the soluble enzyme which has somehow been trapped or adsorbed - 24 -onto the particulate matter either during the acetone powder preparation or during subsequent homogenization. Other purification techniques attempted— Several other techniques were tried in an attempt to provide greater purification of the enzyme. The enzyme was purified to the end of the ammonium sulphate precipitation step, and this enzyme fraction used in the following attempts. Ethanol precipitation (up to 62% ethanol), pH precipitation (from pH 4.0 to 6.5 in steps of 0.5 pH units) and heat treatment (up to 65°C for up to 30 min) proved ineffective in achieving any purification and often resulted in large losses of activity. Considerable work was done attempting to use hydroxylapatite in columns and batchwise. The results were inconsistent, sometimes a 10-fold purification was achieved with a 70% yield while at other times, a 0.7-fold purification with a 10% yield was obtained. Ion exchange resins, (CM-cellulose and cellulose phosphate) were tried, but proved to be ineffective. IV. Separation of a Protein Activator using DEAE-Cellulose Chromatography Prior to the ammonium sulphate precipitation, the enzyme could be stimulated 6-fold by Ca (from the EGTA inhibited levels). The ammonium sulphate fraction'was applied to the DEAE-cellulose column. Following concentration of the phosphodiesterase from the tubes containing peak -in-activity (peak I ) , a maximal Ca -stimulation of only 1.3 fold was observed. Experiments were performed in order to determine the reason for the loss of sensitivity to Ca during chromatography. A DEAE-cellulose column was run and the results are shown in Fig. 2. This figure shows a typical phosphodiesterase activity profile. Since the ammonium sulphate fraction - 25 -Fig. 2: DEAE-cellulose chromatography of an ammonium sulphate fraction. Phosphodiesterase activity was measured at pH 7.5 in the absence of CaC^ (0) and in the presence of 5 mM CaC^CA). Substrate was 3 mM cyclic GMP, but 1 uM or 30 uM cyclic AMP gave identical results. The absorbance at 280 nm is shown by the open squares (•). 20 40 60 80 100 Tube Number - 27 -was activated by Ca , and phosphodiesterase concentrated from the peak activity was not, an assay of the enzyme in the fractions eluted from the column was performed. Each fraction from the column was assayed in the absence (open circles) and presence (open triangles) of 5 mM CaC^- In the absence of Ca , the enzyme activity exhibited a profile characteristic of a single enzyme. With Ca present in the assay mix, a second peak (II) of phosphodiesterase activity appeared. The possibility existed that two I | enzymes were present and that peak II was an enzyme species requiring Ca for activity. It occurred to us that the position of peak II in the elution profile was similar to that observed by Cheung for the heat-stable protein activator which he had separated from the phosphodiesterase (56). He had shown that the addition of this protein activator to the semipurified phosphodiesterase resulted in a marked stimulation of cyclic AMP hydrolysis. An experiment was performed in which aliquots of peak II (tube #80) were combined with aliquots of peak I (tubes #40,44,48) and assayed in the absence or presence of Ca . Prior to the assay, the aliquots from both peaks were dialyzed to remove potassium phosphate. The results of this experiment are shown in Fig. 3. The addition of Ca alone in the assay for peak I activity had no effect on cyclic GMP hydrolysis (compare open and angle striped bars). When an aliquot of peak II was added to that of peak I in the absence of I | Ca , a small increase in enzyme activity was observed (compare open and solid bars). This activity (solid bars) was equivalent to the sum of the acti v i t i e s of the aliquots from peak I and peak II measured in the absence I | | | of Ca . When Ca was added in the assay mix along with phosphodiesterase from peak I and peak II, a 6-fold increase in the total activity was observed (compare solid and horizontal striped bars). This stimulation was - 28 -Fig. 3: Effect of adding aliquots of peak II (tube #80) to aliquots of peak I (tubes #40,44,48). The assay was performed at pH 7.0 in the presence of 100 uM EGTA using 3 uM cyclic GMP as substrate. An aliquot of peak I was assayed alone (open bars), in the presence of 5 mM CaC^ (angle striped bars), in the presence of 3 ug of peak II protein (solid bars) and in the presence of 3 ug of peak II protein plus 5 mM CaC^ (horizontal striped bars). - 29 -- 30 -more than the additive effect predicted from summing peak I (unstimulated) I | and Ca -stimulated peak II activities. The results suggested that the I | stimulation of the combined aliquots in the presence of Ca (horizontal striped bars) could be due to an activating factor present in peak II which was stimulating phosphodiesterase (of peak I) which had tailed into the fractions in which the activator was eluted. Cheung (56) had reported that the protein activator was stable to boiling for 5 min. An aliquot of peak II was boiled to see i f i t contained a similar factor. By boiling an aliquot of peak II, a l l phosphodiesterase activity (when measured with or without added Ca in the assay mix) was destroyed. However, when the boiled peak II extract was added to peak I activity, a 6-fold stimulation of cyclic AMP or cyclic GMP hydrolysis in ++ ++ the presence of Ca was observed. In the absence of Ca , no stimulation was seen. This indicated that peak I was a phosphodiesterase which could I | be activated by Ca i f a heat-stable factor in peak II was also present in the assay. Kakiuchi and Yamazaki (60) had reported that a heat-stable protein increased the effectiveness of Ca in producing a stimulation of I | phosphodiesterase activity, but that nonetheless Ca alone was capable of producing stimulation. The results presented here are in sharp contrast to those of Cheung (55,56), Teo et a l . (57) and Kakiuchi and Yamazaki (58,60), since these data demonstrate the necessity of having both Ca and protein activator present for stimulation of phosphodiesterase activity. V. Preparation of the Protein Activator As shown above, an apparent second peak of phosphodiesterase activity - 31 -(peak II) was observed when Ca was added to the assay mix. This apparent peak of activity was due to phosphodiesterase present in the t a i l i n g edge of peak I being stimulated by a heat-stable protein activator. Phosphodiesterase activity in peak II was destroyed by boiling, but the heat-stable activator remained intact. The peak II stimulatory factor was purified as follows: extracts were pooled, concentrated using an Amicon PM-10 u l t r a f i l t e r at a pressure of 30 psi of JH^ and then boiled for 5 min. The boiled extract was divided into one ml aliquots and stored at -20°C. This concentrate which was devoid of phosphodiesterase activity was used as the activator source for a l l later experiments. VI. Kinetic Nature of the Activation of Phosphodiesterase by Ca and Protein Activator Data calculations for activation studies— During studies of the protein activator and Ca -stimulation of phosphodiesterase, the percentage breakdown of substrate varied over a wide range (4-40%). This, of course, depended upon the amounts of activators present in a given assay tube. As uM ranges of substrate (usually 4.9 uM for cyclic GMP or 30 uM for cyclic AMP) were employed in this study, and these concentrations were not saturating, the reaction velocity was not linear. A more reliable estimate of the reaction velocity at any one substrate level was necessary. As the reaction velocities were measured, substrate level in individual tubes varied (see the data processing for assays to determine K^ values or reference 86). To obtain a more meaningful comparison of data accumulated over a wide range of hydrolysis, the measured velocity was used to calculate the velocity which would have been observed i f the i n i t i a l substrate concentration could have - 32 -been maintained throughout the time course of the assay ( i n i t i a l velocity). Essentially, the i n i t i a l velocity is calculated using the measured velocity, the average substrate, and the of the enzyme for substrate. A simple modification of the Michaelis-Menten equation gives a reasonable estimate of the i n i t i a l velocity: v [S], K + [S] v . = a v 8 X ±_ _ _JB avg. ( 1 ) avg m i where i refers to i n i t i a l values and avg to the measured velocity and average substrate values. The i n i t i a l velocities calculated as above are the velocities plotted in the following graphs. Activation by a large quantity of protein activator— In the studies described thus far using DEAE-cellulose column chromatography, an activation of peak I phosphodiesterase was observed only in the presence of both protein activator and Ca . The requirement for both Ca and protein activator for stimulation had not been previously reported. In the studies of Cheung (55,56), Teo et a l . (57) and Kakiuchi and Yamazaki (60), large quantities (15 ug or greater) of the protein activator had been used. Further studies were designed to examine the relationship between protein activator and Ca more closely. In a l l of these studies on the activation I | by Ca and protein activator, similar results were obtained using either 4.9 uM cyclic GMP or 30 uM cyclic AMP as substrate. Peak I phosphodiesterase was used as an enzyme source; the protein activator was prepared as described in section V of Results. A l l assays were performed using 50 mM imidazole, pH 7.0 as buffer. - 33 -Figure 4 shows the effect of Ca and a large amount of protein activator (35 ug) on phosphodiesterase activity. In the presence or absence I | of EGTA, Ca by i t s e l f had no effect on activity (angle striped bars) and Ca plus protein activator produced a 6-fold stimulation (horizontal striped bars). In the absence of EGTA, the presence of protein activator alone produced near maximal activation of cyclic GMP hydrolysis (solid bar, l e f t panel). This stimulation was totally abolished by the addition of EGTA (compare solid bars in l e f t and right panels). This indicated that, either EGTA had an inhibitory effect on the protein activator, or more li k e l y , there was sufficient Ca contamination in the assay mix to produce a response. The latter explanation was substantiated by our findings that, I | as measured by atomic absorption spectrophotometry, Ca was present as a I | contaminant at a level of 5 uM. This indicated that Ca contamination of the assay reagents could account for the results of Cheung (55,56) and Teo et a l . (57). I | Effect of Ca and protein activator on K and V — Cheung (56) and _ m max ° Teo elt a l . (57) had reported an increase in the aff i n i t y of phosphodiesterase for cyclic AMP in the presence of protein activator. Figures 5 and 6 show the effect of protein activator and Ca on the af f i n i t y and maximal velocity of the enzyme employing cyclic AMP and cyclic GMP as substrate, respectively. Both figures indicate an increase in the maximal velocity, with no change in the dissociation constant for substrate. The K remained constant as m evidenced by the common 1/[cyclic AMP] intercept of the Lineweaver-Burk plot shown in Fig. 5 and the parallel lines (common slopes) of the Eadie plot shown in Fig. 6. Thus, contrary to the reports by Cheung (56) and Teo et a l . (57), the af f i n i t y of the enzyme for substrate did not change in the presence - 34 -Fig. 4: Effect of a large amount of protein activator (35 ug protein) on phosphodiesterase activity (3.8 ug protein). This experiment was performed using 4.9 uM cyclic GMP as substrate in the absence (left panel) and presence (right panel) or 250 uM EGTA. Phosphodiesterase was assayed alone (open bars), in the presence of 5 mM CaCl2 (angle striped bars), in the presence of protein activator (solid bars) and with 5 mM CaC^ plus protein activator (horizontal striped bars). v (nmoles/mg protein/min) o c O 00 o ro o - 36 -Fig. 5: Effect of protein activator and Ca on the K and V for ° m max cyclic AMP. Phosphodiesterase (3.8 ug) was assayed in the absence (0) and presence (A) of protein activator (1.4 ug protein) plus CaCl2 (5 mM), - 38 -Fig. 6: Effect of protein activator and Ca on the K and V for b v m max cyclic GMP. Phosphodiesterase (3.8 ug protein) was assayed in the absence (A) and presence (0) of protein activator (1.4 ug protein) plus CaC^ (5 mM), - 40 -of the protein activator. Effect of substrate on affinity of the enzyme for protein activator— Using fixed cyclic AMP concentrations, Cheung (56) and Teo et a l . (57) varied the amount of protein activator in their assay. At each successively higher cyclic AMP concentration, the apparent affinity of phosphodiesterase for protein activator increased. The affinity of phosphodiesterase for the protein activator ( in the presence of 5 mM CaC^) is shown in Figure 7, using cyclic GMP as substrate. The rationale for the type of plot used (v versus (v-v0)/[protein activator]) is given in the Appendix and wil l be described more fully in the Discussion. Essentially, this type of plot should be used to determine the activator constant i f an enzyme has some basal activity in the absence of activator. This is the case for phosphodiesterase. Parallel lines were observed at the different fixed substrate levels used; indicating that increased substrate produced no change in the affinity of the enzyme for protein activator. I | j | Effects of Ca and protein activator— Since both Ca and protein activator appeared to be required to stimulate phosphodiesterase, studies were initiated to examine the possible relationship between the two activators in the stimulation of cyclic NMP hydrolysis. Assays were performed at a constant substrate level (4.9 uM for cyclic GMP and 30 uM for cyclic AMP). In one experiment the effect of varying protein activator was examined I | at several fixed Ca concentrations. In another experiment, several fixed -+-+ amounts of protein activator were used and Ca concentration was varied. Figure 8 shows the effect of varying protein activator on activation at different fixed Ca concentrations (panel A) and the effect of varying Ca at different fixed protein activator concentrations (panel B). Both - 41 -Fig. 7: Effect of cyclic GMP on affinity of phosphodiesterase for protein activator. Phosphodiesterase (3.8 ug protein) was assayed in the presence of 1.13 ug of protein activator and 5 mM CaC^. Cyclic GMP concentrations (in uM) are indicated on each curve. ( v - v 0 ) / Protein Activator (jjg) - 43 -Fig.18: Effect of protein activator and Ca on the hydrolysis of cyclic GMP. Phosphodiesterase (3.8 ug protein) was assayed in the presence of 100 uM EGTA for the control tubes (0) only. In panel A, protein activator was present at varying concentrations and assays contained CaC^ (in mM) at concentrations indicated by the numbers on each curve. In panel B, CaC^ was varied in the presence of fixed amounts of protein activator (in ug protein) indicated by the numbers on each curve. Substrate was 4.9 uM cyclic GMP. Protein Activator (jig) Ca + +(mM) - 45 -experiments show a dose dependency of each activator on the velocity of the reaction. Addition of increasing concentrations of Ca in the assay resulted in an apparent increase in the affinity of phosphodiesterase for the protein activator as well as an increased maximal velocity of cyclic GMP (and cyclic AMP) hydrolysis. Similarly, as the amount of protein activator I | was increased, the apparent K& for Ca decreased and the maximal stimulation of cyclic NMP hydrolysis increased. I | The effect of Ca and protein activator on the activation of phosphodiesterase differed from models described for simple metal ion activators (87). Dixon and Webb describe the case in which an enzyme is totally inactive in the absence of metal ion. This is clearly not the case here where a measurable basal level of activity exists in the absence of activators. To aid in the kinetic analysis, model rate equations were derived using the method of Cha (88). These derivations are described in detail in the Appendix. Three models have been derived and are shown in Fig. A- l to A-3 in the-Appendix. A l l three models predict linear Eadie plots. These plots should show parallel lines, whose slopes are independent of the concentration of either activator and have variable intercepts dependent upon the amount of each activator present (see Fig.6). However, plots to determine the activator constants for protein activator and Ca are not so simple. A plot of 1/v versus 1/[A] (analogous to a Lineweaver-Burk plot) or v versus v/[A] (analogous to an Eadie plot) is predicted not to give a straight line and did not produce one when the experimental data were plotted. However, in the case of a l l three models, a rearrangement of the rate equation can be made to enable the determination of the activator constants. This new equation predicts a linear plot when v versus (v-v0)/[A] is plotted - 46 -where v 0 i s the velocity in the absence of activator(s) at a fixed substrate concentration. The data, previously plotted in Fig. 8, were plotted according to this form and the results are shown in Fig. 9 and 10. Figure 9 shows that the apparent K for protein activator (the negative value of the slope) j, | decreases in the presence of increasing fixed concentrations of Ca • The I | apparent K for Ca decreases as the amount of protein activator added a increases, as shown in Fig. 10. Both graphs, as well as having variable slopes, are observed to have variable intercepts. I | The data indicate that the Kfl for Ca decreases as the amount of protein activator increases and the apparent K & for protein activator decreases as the amount of Ca rises. The maximal velocity (the intercepts of Fig. 9 I | and 10) also increase as the level of Ca and protein activator increase. The meaning of the changes in the apparent activator constants for Ca and protein activator w i l l be discussed more ful l y in the Discussion in context with the models derived in the Appendix. VII. Multiple Forms of Phosphodiesterase Using gel f i l t r a t i o n , Kakiuchi et a l . (61) and Thompson and Appleman (68,69) have separated multiple molecular forms of the brain cerebral cortex I | | | phosphodiesterase. A Ca -dependent and a Ca -independent form of the enzyme were separated using Sepharose-6B gel f i l t r a t i o n (61). The work of Thompson and Appleman (68,69) showed two peaks of activity, a peak with activities which hydrolyzed both cyclic AMP and cyclic GMP (peak II) and one specific for cyclic AMP (peak III). In our work agarose gels were routinely eluted with 5 mM MgSO^  present. During one elution, MgSO^ , was omitted from the column and the sample. This experiment showed a different enzyme activity profile than that observed with MgSO^ present and a series of experiments were performed to elucidate the nature of this difference. - 47 -Fig. 9: Effect of Ca on the apparent K for protein activator. Phosphodiesterase (3 .8 ug protein) was assayed in the presence of fixed concentrations (in mM) of CaC]^, which are indicated on each curve. Protein activator was varied. Substrate was 4 .9 uM cyclic GMP. 120 ( V - V Q ) / P r o t e i n Activator (jig) - 49 -Fig. 10: Effect of protein activator on the apparent K for Ca SL Phosphodiesterase (3.8 ug protein) was assayed in the presence of fixed amounts (in ug) of protein activator, which are indicated on each curve. CaCl„ was varied. Substrate was 4.9 uM cyclic GMP. v ( nmoles/mg protein/min) - os -- 51 -To clarify the nature of this different behavior of the enzyme in the absence or presence of Mg , phosphodiesterase was chromatographed on a BioGel A1.5m column under various ionic conditions. A l l experiments were performed using a single enzyme preparation (a DEAE-cellulose column eluate) which was concentrated, divided into 1.6 ml aliquots and stored at -20°C. The experiments were performed using a single column 2.5 cm in diameter x 88 cm long, which was not repacked between runs. The column was thoroughly equilibrated with the buffer to be used before each experiment. In cases where an experiment was run in the absence of Mg or Ca , a chelating agent (EDTA or EGTA) was passed through the gel, followed by a large volume of buffer without the chelating agent. The results of three separate experiments using various ionic conditions during elution of phosphodiesterase are depicted in Fig. 11. In the absence of metal ions (open circles), the phosphodiesterase appeared in the void volume of the column (i .e . , 154 ml). In the presence of 5 or 10 mM MgSO^  (or MgC^), a decrease in the apparent molecular weight occurred (open triangles). The apparent molecular weight was further decreased in the presence of 5 mM MgSO^  - 5 mM CaC^ (open ++ squares). The decrease in the apparent molecular weight due to added Ca appeared to be an effect due specifically to Ca and not due to a nonspecific ionic strength increase, since 10 mM MgSO^  did not give the same result. The column was not calibrated, and thus the apparent molecular weights could not be determined. The results showing an apparent decrease in molecular I | weight by Mg are in agreement with those recently reported by Schroder and Rickenberg (89). They found a decrease in the molecular weight of phosphodiesterase from bovine liver when gel filtration was performed in the I | presence of 10 mM Mg , as compared to the molecular weight in the presence of 0.1 mM Mg"1"1". - 52 -Fig. 11: Effect of Mg and Ca on the apparent molecular weight of phosphodiesterase. Concentrates (16 mg protein) from DEAE-cellulose columns were chromatographed on BioGel A1.5m. The column (2.5 cm x 88 cm) was eluted with 5 mM Tris-HCl, pH 7.5 in the absence of ions (O) , in the presence of 5 or 10 mM MgSO^  or MgCl2 (A), and in the presence of 5 mM MgSO^  plus 5 mM CaCl2 (•). The protein profile is given by the closed circles (•). The assay was performed at pH 7.5 using 4.9 uM cyclic GMP as substrate. - 54 -These above experiments had been performed with columns which had been carefully equilibrated. By varying the condition in the column (leaving trace amounts of Ca , or leaving ions in the mixture in which the sample was added), it was possible to obtain combinations of one, two or a l l three enzyme activity peaks. For example, a column was equilibrated with 5 mM Tris-HCl - 5 mM MgSO^ , pH 7.5. Previously, the column had been eluted with 5 mM Tris-HCl - 5 mM MgSO^  - 5 mM CaCl 2 > pH 7.5 and had not been washed with EGTA. As a result some contamination by Ca was present in the gel. Upon elution of phosphodiesterase from the column, two peaks of activity (i.e. at 194 ml and 225 ml) were observed. These two peaks corresponded to the I [ activity peaks observed in the presence of Mg alone (194 ml) and in the I | | | presence of Mg and Ca (225 ml). The occurrence of any or a l l three of these peaks in a single column eluate emphasizes the caution which must be exercised in eluting this enzyme during gel filtration. The multiple peaks of enzyme activity observed in gel filtration experiments were due to the presence of interconvertible forms of a single enzyme species. The species observed were dependent upon the ionic conditions (presence or absence of Mg and/or Ca ) in the column during elution. I | The three peaks were not stimulated by Ca alone. However, when both I | Ca and protein activator were present in the assay, a stimulation of 4-5 fold was observed in a l l cases. Identical results were obtained using either 3 uM cyclic GMP or 30 uM cyclic AMP as substrate. To ensure that the enzyme purified by the procedure described in section II of the Results was the same as the enzyme present in the crude homogenate, cruder enzyme preparations were chromatographed on the BioGel A1.5m column. Using a 37,000 x g supernatant fraction and enzyme which had been solubilized - 55 -from the 37,000 x g pellet material by 1.0% sodium deoxycholate, columns were eluted in the presence of 5 mM MgSO^  and 100 uM EGTA (to remove any contaminating Ca ). Gel filtration showed peaks of activity for both of these fractions at 194 ml, identical with those obtained using the DEAE-cellulose column concentrates. This indicated that the enzyme purified through the DEAE-cellulose column step was probably the same as that present in the original supernate. The coincident activity peak of the sodium deoxycholate solubilized enzyme with that of the supernatant enzyme further supported the idea that these enzymes might be one and the same. VIII. Determination of the Michaelis Constants for Phosphodiesterase from Various Fractions in the Purification Procedure The Michaelis constant, or K^, reported initially for brain cyclic AMP phosphodiesterase was in the order of 0.1 to 0.3 mM (36). Since tissue cyclic AMP levels are in the range of 1 uM or less, the high value for the enzyme seemed a paradox. Thompson and Appleman (68) reported two Michaelis constants for cyclic AMP (104 uM and 2 uM) and one for cyclic GMP (12.9 uM). This low K value for cyclic AMP (2 uM) was much closer to the m physiological concentration of this cyclic nucleotide. Using cyclic AMP as substrate, the Lineweaver-Burk plots obtained by these authors (68,69) exhibited downward curvature which they attributed to negative cooperativity (76). of the partially purified enzyme— Michaelis constants for cyclic AMP and cyclic GMP were determined for the phosphodiesterase purified 15-fold from BioGel A1.5m chromatography. A single K was observed for both cyclic m AMP (30 uM) and cyclic GMP (3 uM) as shown in Fig. 12 and 13 respectively. - 56 -Fig. 12: Assay to determine for cyclic AMP. The partially purified enzyme (8.7 ug protein) was assayed at pH 7.5. The inset shows the original v versus cyclic AMP plot while the main graph is an Eadie plot. - 58 -Fig. 13: Assay to determine K for cyclic GMP. The partially purified enzyme (8.7 ug protein) was assayed at pH 7.5. The inset shows the original v versus cyclic GMP plot while the main graph is an Eadie plot. _l I I I I 20 40 60 80 100 / ' c y c l i c GMP (>JM) - 60 -The maximal velocity for the cyclic nucleotides was about 300 nmoles/mg protein per min. H i l l plots (90), of log v/(V -v) versus log [S], yielded max slopes of 0.99 and 1.02 respectively for cyclic AMP and cyclic GMP. Since a H i l l slope of 1.0 is indicative of a non-cooperative enzyme, the values observed would indicate that phosphodiesterase is an enzyme which might obey simple Michaelis-Menten kinetics. These Michaelis constants were lower than those observed by Thompson and Appleman (68) for their high K m cyclic AMP -cyclic GMP phosphodiesterase (peak II), which displayed a K m of 104 uM for cyclic AMP and one of 12.9 uM for cyclic GMP. An investigation of K values from less purified enzyme fractions was undertaken to examine the reason for this discrepancy. K m of acetone powder supernatant f r a c t i o n — A determination using cyclic AMP as substrate was performed on the 37,000 x g supernatant fraction. The results of this study are shown in Fig. 14. A downward sloping Lineweaver-Burk plot i s seen indicating the presence of two or more independent catalytic sites or a negatively cooperative enzyme (75,76). The most purified enzyme had shown no signs of cooperativity ( H i l l slope = 0.99), and Thompson and Appleman (68) demonstrated the presence of two cyclic AMP phosphodiesterase activities which were separated by agarose gel f i l t r a t i o n . For these reasons, the data were analyzed assuming a two enzyme system rather than a single enzyme exhibiting negative cooperativity. The K and V of each enzyme were determined using the iterative method of m max J & Spears et a l . (91). The results indicated the presence of a high enzyme (K = 110 uM) and a low K enzyme (K = 2 uM). Their respective maximal m m J m r velocities were 60 and 3 nmoles/mg protein per min. Figure 15 shows the same acetone powder supernate assayed with cyclic - 61 -Fig. 14: Km of acetone powder supernatant fraction for cyclic AMP. Phosphodiesterase (9.2 ug protein) was assayed at pH 7.5. The upper left inset shows the original v versus cyclic AMP data. The main plot shows the Lineweaver-Burk plot of the data. The lower right inset is an expansion of the main plot in the substrate range from 8 uM to 0.5::mM. - 62 -- 63 -Fig. 15: of acetone powder supernatant fraction for cyclic GMP. Phosphodiesterase (9.2 ug protein) was assayed at pH 7.5 in the absence (0) and presence (A) of 0.25 mM EGTA. The inset shows the original v versus cyclic GMP data while the main plot is a Lineweaver-Burk plot of the data. - 65 -GMP as substrate. Here, only a single K and V were observed. In this m max experiment, the results indicated a decrease in the maximal velocity when EGTA was present in the assay mix (compare open circles with open triangles). In the presence or absence of EGTA, the K was determined to be 14 uM (common intercept on the 1/[cyclic GMP] axis). The results here indicated the presence of two enzyme activities for cyclic AMP hydrolysis and one for cyclic GMP. The Michaelis constants observed in the crude supernate for cyclic AMP and cyclic GMP are in good agreement with those published by Thompson and Appleman (68). From their data and the data here, a high enzyme capable of hydrolyzing both cyclic AMP and cyclic GMP (a cyclic AMP - cyclic GMP phosphodiesterase) and a low enzyme capable of hydrolyzing only cyclic AMP (a cyclic AMP phosphodiesterase) seem to exist in brain tissue. Similar results were obtained when the i n i t i a l homogenate was examined. of the sodium deoxycholate solubilized enzyme— The enzyme, which had been solubilized from the 37,000 x g pellet material by 1.0% sodium deoxycholate was also analyzed for i t s a b i l i t y to hydrolyze both cyclic AMP and cyclic GMP. The results are shown in Fig. 16. Only one of 30 uM was observed for cyclic AMP (open triangles) and one of 4.7 uM for cyclic GMP (open c i r c l e s ) . These Michaelis constants are in excellent agreement with those obtained from the enzyme purified 15-fold from the supernate (Fig. 12 and 13). This similarity in Michaelis constants indicates that the s.upernat-antiattd pellet enzymes may be the same phosphodiesterase. IX. Some Properties of the Partially Purified Enzyme At the time when this work was initiated, few papers had been published - 66 -Fig. 16: of 1.0% sodium deoxycholate solubilized enzyme. The assay was performed at pH 7.5 using 50 ug of protein. Substrate was cyclic AMP (A) and cyclic GMP (0). - 67 -( iniu / sa iou id ) A - 68 -regarding the general kinetic properties of the enzyme. The phosphodiesterase purified through; the BioGel A1.5m column step was examined to determine some properties of this enzyme and compare them with results reported elsewhere in the literature (26,27,35,44,46,50,68,69). Studies were undertaken concerning metal ion requirements, inhibition by 3-isobutyl-l-methylxanthine (SQ2964), the effect of cyclic AMP on cyclic GMP hydrolysis and the effect of cyclic GMP on cyclic AMP hydrolysis. Metal ion studies— Early workers (26,27) had shown that Mg was required for enzyme activity. Cheung (44) had reported contradictory results indicating considerable basal activity in the absence of added ions. Figure 17 shows the effect of some divalent cations on the rate of I | | | hydrolysis of cyclic AMP. Mg and Mn were found to have the greatest I | | | activity followed by Co and Ni . The activation constants determined by plotting 1/v versus 1/[metal ion] (87), were 4.5 uM for Mg , 5.7 uM for Mn , I | | | | | 9.0 uM for Co .arid 23 uM for Ni . Zn produced an interesting effect. In the presence of this ion, the activity rapidly rose reaching a maximum by 10 uM and then decline sharply. Neither the concentration of substrate used (4 uM, 24 uM or 1 mM cyclic AMP) nor the salt (MgSO^ versus MgC^) had any I | effect on the activation constant for Mg These results are not consistent with those of Cheung (44). Our data indicated that no activity was present in the absence of metal ions and is in agreement with the early published work (2,26,27). Because of the low K for the divalent cations, the addition of micromolar concentrations (or contamination by these ions) would produce a significant percentage of the activity attainable at saturating levels of these ions at any given substrate concentration. - 69 -Fig. 17: Effect of divalent cations on phosphodiesterase activity. The assay was performed at pH 7.5 using 12.3 ug of the partially purified enzyme. Substrate was 24 uM cyclic AMP. The metal ions used were: MgSO (O), MnCl2 ( A ) , CoCl2 (•), NiCl 2 (•) and ZnS04 ( A ) . Cation (mM) - 71 -Monovalent (Li , Na and K ), other divalent (Be , Cu and Ca ) and 3+ 3+ 3+ trivalent cations (La , Sc and Y ) a l l had no effect on phosphodiesterase activity. Inhibition by 3-isobutyl-l-methylxanthine— The ability of methylxanthines to inhibit phosphodiesterase from many tissues is well established (2,27,35, 36,37). Consequently, the inhibition by 3-isobutyl-l-methylxanthine (SC2964), a potent inhibitor of phosphodiesterase prepared from rat fat cells (37), is shown in Fig. 18. This compound is a competitive inhibitor (see inset) with an inhibition constant; of 1.5 uM as determined by a Dixon plot (92). These results are consistent with previous reports which have shown competitive inhibition by caffeine (35) and theophylline (27,36). Cyclic AMP inhibition of cyclic GMP hydrolysis and vice versa— Recent reports (50-52) have indicated that cyclic GMP is capable of activating cyclic AMP hydrolysis by an enzyme present in the 20,000 x g pellet fraction of rat r brain. Other workers (39,49) found that cyclic GMP inhibited cyclic AMP hydrolysis in brain extracts. In view of this, the effect of each cyclic nucleotideteon the hydrolysis of the other was studied. In the case of cyclic GMP inhibition of cyclic AMP hydrolysis, unlabelled cyclic GMP was used as an inhibitor and the rate of hydrolysis of tritiated cyclic AMP was measured. Only inhibition and no activation was seen at a l l concentrations of substrate (0.15 - 100 uM cyclic AMP) and inhibitor (0.14 -50 uM cyclic GMP) studied. The data could not be quantitated to obtain an inhibition constant for cyclic GMP. In order to obtain a reasonable inhibition of cyclic AMP hydrolysis (10-50%), only a small amount of cyclic GMP relative to the cyclic AMP concentration was required. However, at this concentration of cyclic GMP, the cyclic GMP was hydrolyzed at a significant rate relative - 72 -Fig. 18: Inhibition of cyclic AMP hydrolysis by 3-isobutyl-l-methylxanthine (SC2964). Phosphodiesterase (2.9 ug protein) was assayed at 7.5. In the inset, Lineweaver-Burk plots are shown with the concentration of SC2964 (in uM) indicated on each line. In the main plot, a Dixon plot is shown with the concentration of cyclic AMP (in mM) indicated on each line. - ZL -- 74 -t o t h e r a t e o f c y c l i c AMP h y d r o l y s i s . T h i s made i t i m p o s s i b l e t o m a i n t a i n a c o n s t a n t l e v e l o f i n h i b i t o r i n t he a s s a y , w i t h o u t i n h i b i t i n g t h e h y d r o l y s i s o f c y c l i c AMP a l m o s t t o t a l l y . T h i s p r o b l e m d i d n o t e x i s t d u r i n g measurements o f c y c l i c AMP i n h i b i t i o n o f c y c l i c GMP h y d r o l y s i s , s i m p l y b e c a u s e a t c o n c e n t r a t i o n s o f c y c l i c AMP r e q u i r e d t o i n h i b i t c y c l i c GMP h y d r o l y s i s , t h e p e r c e n t a g e b reakdown o f c y c l i c AMP ( t h e i n h i b i t o r ) was r e l a t i v e l y s m a l l . I n t h i s c a s e , a c o m p e t i t i v e i n h i b i t i o n was s e e n as shown i n t h e i n s e t o f F i g . 1 9 . A s d e t e r m i n e d f r o m t h e D i x o n p l o t , t h e f o r c y c l i c AMP was 25 uM, w h i c h i s i n good ag reement w i t h t h e K o f 30 uM o b s e r v e d f o r t h e p u r i f i e d enzyme ( F i g . 1 2 ) . T h i s s u g g e s t s t h e p r e s e n c e o f a s i n g l e enzyme c a p a b l e o f h y d r o l y z i n g b o t h c y c l i c AMP and c y c l i c GMP. T h i s i s i n ag reement w i t h t h e d a t a o f Thompson and App leman ( 6 8 ) , who showed i n h i b i t i o n o f c y c l i c AMP h y d r o l y s i s by c y c l i c GMP and v i c e v e r s a u s i n g a h i g h c y c l i c AMP - c y c l i c GMP p h o s p h o d i e s t e r a s e (peak I I ) . DISCUSSION N u c l e o s i d e 3 ' , 5 , ' - c y c l i c m o n o p h o s p h a t e - 3 ' - p h o s p h o h y d r o l a s e was p u r i f i e d 1 5 - f o l d f r o m b o v i n e c e r e b r a l c o r t e x . The p a r t i a l l y p u r i f i e d enzyme h y d r o l y z e d b o t h c y c l i c AMP and c y c l i c GMP. A h e a t - s t a b l e p r o t e i n f a c t o r was s e p a r a t e d f r o m t h e enzyme d u r i n g DEAE c e l l u l o s e c h r o m a t o g r a p h y . The enzyme was a c t i v a t e d I | when b o t h Ca and p r o t e i n a c t i v a t o r w e r e p r e s e n t i n t h e a s s a y m i x . Measu remen ts o f k i n e t i c p r o p e r t i e s o f t h e enzyme p e r m i t an e x a m i n a t i o n o f p o s s i b l e m o d e l s f o r t h e b i n d i n g o f C a and p r o t e i n a c t i v a t o r t o t h e enzyme. T h e s e same k i n e t i c p a r a m e t e r s c a n be u t i l i z e d i n p r e d i c t i n g t h e i m p o r t a n t r o l e t h a t p h o s p h o d i e s t e r a s e may p l a y i n t h e c o n t r o l o f c y c l i c AMP and c y c l i c GMP c o n c e n t r a t i o n s i n t h e c e l l . - 75 -Fig. 19: Inhibition of cyclic GMP hydrolysis by cyclic AMP. Phosphodiesterase (0.83 ug protein) was assayed at pH 7.5. In the inset, Lineweaver-Burk plots are shown with the concentration of cyclic AMP (in ' indicated on each line. In the main plot, a Dixon plot is shown with the concentration of cyclic GMP (in uM) indicated on each line. cycl ic AMP (>iM) - 77 -3-Isobutyl-1-methylxanthine inhibition— The results of methylxanthine inhibition are in agreement with early published work. Caffeine (35) and theophylline (27,36) displayed competitive inhibition towards cyclic AMP hydrolysis. This work shows 3-isobutyl-l-methylxanthine (SC2964) to have a similar competitive behavior (Fig. 18), however the potency of this inhibitor is much greater than that of the other methylxanthines studied. The for SC2964 was 1.5 uM, far lower than those for caffeine (K^ = 3 mM) and theophylline (K^ = 0.11 mM). The extremely potent inhibitory properties of this compound were first reported by Beavo jjt a l . (37), who showed that SC2964 promoted the greatest lipolytic response of any of the analogues studied. This compound is not generally used as an inhibitor of phosphodiesterase since i t is not commercially available, but does possess an interesting potential, especially for use in the assays of adenyl cyclase activity. In order to achieve a 90% inhibition of cyclic AMP hydrolysis, a concentration of 13.5 uM of SC2964 would be required as compared to a concentration of 1 mM for theophylline. Thus, much lower levels of this inhibitor would be required to virtually block cyclic AMP degradation due to phosphodiesterase. Metal ion activation— Cheung (44) reported considerable phosphodiesterase activity in the absence of added divalent cation and only a small stimulation at optimal metal ion concentrations. The present data (Fig. 17) show no activity in the absence of metal ions. This is in agreement with early published work (2,26,27), which showed a requirement for Mg for activity. I | The activator constants reported here (4.5 uM for Mg ) are very low. At I | 5 uM Mg , the enzyme would be more than half saturated with this cation. I | Since a small contamination by Mg (say 5-10 uM) would produce over half-saturation of the enzyme with this ion, i t is a likely possibility that - 78 -Cheung (44) had sufficient Mg contamination in his reagents to produce the results which he has published. I | | | | | The order of effectiveness of the metal ions was Mg a Mn > Co > I | | | Ni while Zn produced a somewhat atypical response. The metal ion behavior seemed specific for divalent ions, as none of the monovalent or trivalent cations tested had any effect. This finding alone negates some possible explanations for the mode of activation of cyclic AMP hydrolysis by divalent cations. The ionic radius of the ion seems to be important for I | those divalent cations which have activity. Excluding Mg , as i t is not a I | | | | | member of the transition series, the order of activity (Mn > Co > Ni ) corresponds to their appearance in the f i r s t transition series of the periodic table and to a decreasing order of ionic r a d i i (93). On the other hand, L i and Sc have virtually the same ionic r a d i i as Mg , but have no effect. That is to say, an ionic radius similar to an active ion does not guarantee that this ion w i l l be capable of activating phosphodiesterase. Another commonly noted property of ions, the charge to radius ratio, is 3+ I i identical for La and Mg , but these ions display divergent kinetic I [ | | | | behavior. Smaller (Be ) and larger (Ca ) divalent cations than Mg are ineffective. These data indicate that a nonspecific effect due to size or charge of the ion is not involved in the activation. However, these ions have different sizes of hydrated shells as well as different coordination numbers which may contribute to their potency as activators. Another point [ | to be considered when dealing with Mg as opposed to the transition metal I | ions is that Mg has no electrons in d-orbitals while the transition metals do. The orientation of the electron shells, therefore, is somewhat altered in these cases and this may help to determine exactly where Mg f i t s into - 79 -the order of effectiveness of the divalent cations for stimulating different enzymes. Inhibition by EGTA— The crude enzyme preparations showed an inhibition I | of phosphodiesterase activity by EGTA and a stimulation by excess Ca (Fig. 1). This i s in agreement with the work of Miki and Yoshida (46) and Kakiuchi and Yamazaki (58). During the purification of the enzyme, the percentage inhibition of phosphodiesterase activity by EGTA decreased. The inhibition by EGTA is believed to be due to the chelation of Ca in the crude fractions and the decline of the inhibition with the purity of the enzyme is probably a result of dilution of Ca in the enzyme preparation as purification proceeds. During purification, large losses in enzyme activity have been attributed to removal of protein activator (55), however, equally important I | i s the dilution of Ca . An apparent loss of 70% in enzyme activity (as evidenced by the 70% inhibition of the homogenate activity by EGTA) could be expected during purification even i f the protein activator was not removed. By using the EGTA inhibited levels of basal enzyme activity, i t is possible to obtain a more valid estimate of actual enzyme loss during purification. I | Protein activator and Ca stimulation of phosphodiesterase— During DEAE-cellulose chromatography, a protein factor was separated from the enzyme (Fig. 2). Following removal of this factor, stimulation of the I | enzyme by Ca was not observed (Fig. 3). Activation of phosphodiesterase was observed in the presence of both Ca and protein activator, but not in the presence of either one of these alone. These results differ from a l l previously published work. Cheung (53,55) originally reported stimulation by a protein factor. Kakiuchi and Yamazaki (58,60) reported Ca activation, - 80 -which was enhanced by a protein factor, but neither of these groups reported an absolute requirement for the two activators for stimulation of phosphodiesterase activity. Kakiuchi and Yamazaki (58) showed that uM I | concentrations of Ca were required for stimulation. A small contamination I | by Ca in the experiments of Cheung and a contamination by protein activator in the experiments of Kakiuchi and Yamazaki would produce the results observed by these workers. In this work, atomic absorption spectrophotometric analysis of the 50 mM imidazole - 5 mM MgSO^ buffer revealed a contamination I | of 5 uM Ca , enough to produce a maximal activation of cyclic NMP hydrolysis when protein activator is added in relatively large amounts (Fig. 4). In order to describe a mechanism by which Ca and protein activator might interact to produce a stimulation, a number of po s s i b i l i t i e s were examined as described in the Appendix. For the following discussion the reader should refer to the models given in Fig. A-l to A-3 in the Appendix. Model A (Fig. A-l) is the simplest scheme derivable and consists of only two enzyme species, that is enzyme (E) and enzyme-protein activator-Ca (EApCa), in the activation sequence. In this model, the protein activator f i r s t binds to Ca and this complex then adds to the enzyme. Model B (Fig. A-2) involves the formation of three enzyme species for the activation. Here, in addition to the two species described in Model A, enzyme-protein activator (EAp) can also form. A similar model (not shown) can be derived in which enzyme-Ca (ECa) can form, instead of EAp. Model C (Fig. A-3) requires the presence of four species (E, EAp, ECa and EApCa) for the activation by protein activator and Ca . This model is the same as Model B, except that now both EAp and ECa are capable of being formed. This activation of cyclic NMP hydrolysis requires the presence of both - 81 -activators. In analyzing the activation sequence, let us f i r s t consider the general case of having two activators, and k^. If the concentration of A^ is held constant and that of k^ varied, one can plot the reaction velocities obtained from the assay as v versus (v-v,,)/!^] where v i s the measured velocity, v 0 is the velocity in the absence of activator(s) and k^ i s the activator which is varied. This plot has a slope which i s the negative value of the apparent activator constant for k^. By repeating the experiment at new fixed concentrations of A^, one gets a family of lines, one line for each fixed concentration of A^ used. The reverse case, that is when the concentration of k^ is held constant and that of A^ varied, gives a second set of data which can be plotted similarly (v versus (v-v 0)/[A^]). This graph w i l l have a slope equal to the negative value of the apparent dissociation constant for A^. This general case for two activators can be applied to the data here concerning protein activator and Ca . A l l three models (A,B,C) previously described predict significantly different plots of this type (v versus (v-v 0)/[activator]). Model A predicts that graphs of v versus (v-v 0)/[Ca] and v versus (v-v„)/[Ap] should show a common intercept with variable slopes. The intercepts w i l l be independent of the amount of either of the activators present, but the slopes w i l l depend on the concentration of the activators. Specifically, the slopes of v versus (v-v„)/[Ca] w i l l depend upon the amount of protein activator present in the assay mix and the slope of v versus (v-v„)/[Ap] w i l l depend upon the concentration of Ca present. Model B predicts that both graphs should have variable slopes, but that the graph of v versus (v-Vo)/[Ca] should show a common intercept, independent of the amount of protein activator present. The graph of v versus (v-v Q)/[Ap] - 82 -should have both variable intercepts and slopes. Only Model C predicts variable intercepts and slopes for both of the graphs of v versus (v-v 0)/[A]. Referring to Fig. 9 and 10, i t is obvious that the data are consistent with Model C. Figures 9 and 10 both show variable intercepts and variable slopes. From the data, Model A and Model B can be excluded as mechanisms for the activation. Model C can explain the data, however, this model probably is not the only kinetic model capable of doing so. In the further consideration of Model C, the possibility exists that I | Ca (or protein activator) may have equal affinity for the enzyme and the I | enzyme-protein activator (or enzyme-Ca ) complex. This means that Kp = K' . If this were the case, parallel lines with variable intercepts are predicted on a v versus (v-v0)/[Ap] plot. This can be verified by substituting K = K' into equation A - l l of the Appendix. Parallel lines were not observed (Fig. 9). Similarly, ^ as evidenced by the variable slopes of Fig. 10. The analysis of the intercepts (equations A-13 and A-14) and slopes (equation A-15) of Fig. 9 and 10 predict that replots of these data should give values for the dissociation constants of protein activator and Ca for the enzyme. A replot of the intercepts of Fig. 9 is shown in Fig. 20. The slope of the line in Fig. 20 (see equation A-13), representing the negative value of K^a> is -3 uM. The replot of the intercepts of Fig. 10 is shown in Fig. 21. The slope of this graph (see equation A-14) gives K' Ap to be 0.2 ug. To obtain and K ^ , a different type of plot is required (see equation A-15). This requires a replot of the slope data of Fig. 9 or 10 and is a considerably more complex function. In order to replot the slope data, the determination of one of the primed constants, K.' or - 83 -Fig. 20: Determination of K ^ . This graph is a replot of the intercepts (a.) of Fig. 9 (see equation A-13 of the Appendix for the rationale). - 84 -- 85 -Fig. 21: Determination of K' . This graph is a replot of the intercepts (a2) of Fig. 10. The negative value of the slope (see equation A-14 of the Appendix) is K' . 120 80 160 240 ' 320 (a 2 -v 0 ) /Protein Activator (jig) - 87 -from Fig. 20 or 21 is required. The replot of the slopes of Fig. 10 is shown in Fig. 22. This plot of equation A-15 gives an intercept (1/K^) and a slope (1/*^) indicating that and are 4 ug and 80 uM respectively. A l l of the binding constants determined, of course, depend upon the purity of the protein activator and the enzyme preparation, as any I | extraneous binding of Ca or protein activator to any material other than the enzyme may alter the absolute values of the constants. Model C, as shown in the Appendix (Fig. A-3), is reproduced in Fig. 23. Only the portion of the model that deals with protein activator and Ca binding to the enzyme is shown. Not a l l of the possible pathways by which the enzyme could bind Ca and protein activator are indicated in the diagram. The dissociation constants for the individual steps are given in the figure and are as follows: K A p = 4 ug K C a = 80 uM KA P " °' 2 US KCa = 3 m The relationship between the constants (K > , K C a > K^) is indicative I | of a heterotropic cooperativity between protein activator and Ca for their I | binding to the enzyme. The binding of Ca i s enhanced in the presence of protein activator (i.e., K' = 3 uM and is smaller than K = 80 uM). Similarly, the binding of protein activator is enhanced by the presence of I | | | Ca (i.e., K! = 0.2 ug and is smaller than K, =4 ug). If Ca and Ap ° Ap protein activator bind together prior to addition to the enzyme, as opposed to a sequential binding of the two activators to the enzyme, the overall dissociation constant (K x K ) should be about 16 uM x ug (80 uM x A p + L # SL A p \J Si 0.2 ug). This overall dissociation constant could not be separated into - 88 -Fig. 22: Determination of KA and K ^ . This graph is a replot of the slopes (b2) of Fig. 10. The - l / b 2 intercept is l / K C a while the slope is 1/K (see equation A-15 of the Appendix). - 90 -Fig. 23: Model C: the steps involving protein activator (Ap) and Ca (Ca) addition to the enzyme (E). This diagram shows the steps in activation along with their dissociation constants as determined from Fig. 20 to 22. - 91 -_ K A p + Ca A _ Ap + Ca ^ ^ ApCa EAp K A p = 4>»9 * C a = 3 - M g E - EApCa K C a = 80jiM K A p = 0.2>jg ECa - 92 -i t s constituent parts by these kinetic methods. The determination of a l l of the constants (KAp> ^kp' KCa' KCa^ i n t h e context of Model C does not, however, assure that a l l of these binding steps occur. The measurements made were of the kinetic properties of the enzyme and are a reflection of the state of the enzyme, however, actual measurements of the binding steps were not made. The constants which have been determined predict only that i f these steps do occur, then the dissociation constants w i l l be those given. The determination of the constants by kinetic analysis does not define which steps occur. ++ The binding of Ca to protein activator in bovine heart has been 45 ++ measured (J.H. Wang, personal communication). Using Ca and a purified protein activator from bovine heart, Wang has determined a dissociation I j constant of 2.3 uM for the binding of Ca to the protein activator. Two I [ additional sites for Ca binding with dissociation constants of 11 uM were ++ also observed. Thus, the binding of Ca to protein activator i s known to occur. However, these data do not show that the complex (ApCa) can then bind to the enzyme in order to produce activation. Model C, which is a model consistent with the data, requires that E, EAp, ECa and EApCa a l l form, but again cannot identify which steps are important in the activation. The question s t i l l remains as to how and in what sequence, E, Ap and Ca combine to produce a stimulation of phosphodiesterase, and the problem of determining the exact sequence or sequences of events of physiological importance remain to be solved. Divalent cations and apparent molecular weight of phosphodiesterase— The enzyme elution profiles from the BioGel A1.5m gel f i l t r a t i o n indicate another aspect of how Ca may be involved with the enzyme (Fig. 11). The - 93 -gel f i l t r a t i o n data demonstrate the existence of i n t e r c o n v e r t i b l e forms of the p a r t i a l l y p u r i f i e d phosphodiesterase depending upon the divalent ions I | present. In the absence of Mg , a large molecular aggregate with no I | c a t a l y t i c a c t i v i t y e x i s t s . With Mg present, the apparent molecular weight declines dramatically and the enzyme exhibits a c t i v i t y . Further deaggregation I | | | occurred i n the presence of Mg plus Ca and no protein a c t i v a t o r , but the a c t i v i t y was not increased. I | The deaggregation of the enzyme by Ca during agarose gel f i l t r a t i o n suggests that t h i s may be a mechanism by which the binding of the p r o t e i n a c t i v a t o r to the enzyme i s f a c i l i t a t e d . These data may suggest that when I | Ca binds to the enzyme, a deaggregation occurs and then protein a c t i v a t o r I | binds to the enzyme-Ca complex. I | Protein a c t i v a t o r and Ca could be a means of c o n t r o l l i n g phosphodiesterase a c t i v i t y i n vivo. I f the assumption i s made that the p r o t e i n a c t i v a t o r i s i n the c e l l i n an unbound form and i t s concentration i s constant, the a c t i v i t y of phosphodiesterase would l i k e l y be governed by f l u c t u a t i o n s i n the I | i n t r a c e l l u l a r concentration of Ca . These fl u c t u a t i o n s could occur e i t h e r I | | | by a small i n f l u x of Ca or by a release of bound i n t r a c e l l u l a r Ca E i t h e r of these means of r a i s i n g Ca l e v e l s within the c e l l would thus provide a very e f f e c t i v e means of stimulating phosphodiesterase a c t i v i t y i n conjunction with the protein a c t i v a t o r . Possible explanation for downward sloping Lineweaver-Burk p l o t s — Working with an enzyme preparation i n which there are endogenous a c t i v a t o r ( s ) (or i n h i b i t o r s ) , such as i n the case of phosphodiesterase, presents s p e c i a l problems i n performing assays to determine the of the enzyme. The I | a c t i v a t i o n of phosphodiesterase by Ca and protein a c t i v a t o r provides a - 94 -possible explanation for the observations of downward sloping Lineweaver-Burk plots (50,68,69,76). Many authors (50,69) have performed assays to determine values in which a relatively constant percentage degradation of the cyclic nucleotide is maintained at a l l substrate concentrations. This is accomplished by increasing the enzyme concentration in the assay as the I | substrate increases. If an endogenous activator (such as Ca or protein activator) is present and contaminating the enzyme, this method of assaying to determine values could yield misleading results when the data were plotted. Suppose an assay to determine was performed on phosphodiesterase by varying the amount of enzyme in the assay. This enzyme is probably contaminated with Ca and protein activator in nonsaturating quantities, especially i f the enzyme preparation has not been purified through a DEAE-I [ cellulose column (see Fig. 1 showing the presence of contaminating Ca in the crude enzyme preparations). For convenience, reaction velocities used in this hypothetical assay are calculated using equation A-9a of the Appendix, as this equation gives a reasonable representation of the high phosphodiesterase purified from brain tissue. This equation (A-9a) is used in this example, but the findings hold true for most cases where activators are present at subsaturating levels (for a further discussion of activators, see Reiner (94)). The assumption used was that as the enzyme level increased, the contaminating activator concentrations increased accordingly. Enzyme reaction velocities are expressed as specific enzyme activities and plotted in Fig. 24. The inset shows the various Lineweaver-Burk plots predicted using different fixed enzyme concentrations. Each line represents a complete assay at a single fixed enzyme concentration (expressed as a multiple of E 0 ) . - 95 -Fig. 24: Lineweaver-Burk plot showing a possible method of obtaining a downward curvature in the presence of a single non-cooperative enzyme. The points are calculated from equation A-9a of the Appendix. The parameters used were as follows: = 29, v 2 = 104, Kg = 4, KA = 5.3, K' = 0.70, K = 21 and K' = 2.7. An enzyme concentration (designated Ap ua da I | E a) with a protein activator contamination of 0.1 and a Ca contamination of 1.0 was used as the lowest enzyme concentration. The inset shox^ s the Lineweaver-Burk plots, each line representing a single enzyme concentration (indicated as the multiple of E 0 on the line). The points taken in the main plot involve a single assay. The enzyme concentration was: E 0 (o), 2EC (A), 3Ee (•), 4E0 (•), and 5E0 ( A ) . - 97 -As can be seen, each line has a variable 1/v intercept (1/^ ) showing that the apparent increases as the amount of enzyme (and contaminating activator) increases. Each line, however, extrapolates back to a common 1/[S] intercept (common K value). This K value is 4.0 which is the value m m used to calculate the reaction velocities (see the parameter l i s t in the legend to Fig. 24). The main plot shows the effect of varying the amount of enzyme (and contaminating activator) during an assay to determine K^. When the substrate level was varied between 0.5 (1/[S] = 2.0) and 3.0 (1/[S] = 0.33), the enzyme concentration was designated E 0 (open circles). At this level of substrate (3.0), the enzyme concentration (and contaminating activator) was doubled (enzyme concentration is now 2E0). This enzyme concentration was used in the assay for substrate levels of 6.0 (1 /[S] = 0.17) and 9.0 (1/[S] = 0.11), as shown by the open triangles. At a substrate concentration of 10 or 20, the enzyme concentration was 3E0 (open squares). Finally the enzyme concentration was 4E„ (filled circle) and 5E0 (filled triangle) when the substrate was 30 (1 /[S] = 0.33) and 100 respectively. A perfect straight line can be drawn through the points up to [S] = 3.0. The 1/[S] extrapolation of this line gives a K of 4.0 (1/[S] = 0.25), which is the true K . m m However, an approximate second line can be drawn through the remainder of the points, giving a second false K of somewhere near 12 (1/[S] = 0.08). m This false value for the Km is a direct result of varying enzyme concentration during the assay. Since a K of 4.0 was the only value used to calculate the reaction velocities, no parameter corresponding to the Km of 12 was present in the calculations and, therefore, does not exist. The point of curvature and the steepness of the second line can be varied by changing - 98 -the parameters used in the calculations or by choosing different substrate levels at which changes in the enzyme concentrations are made. This example indicates that caution should be exercised in performing assays to determine K values which vary any parameter other than substrate, m especially in cases where endogenous activator (or inhibitor) might be present. In any case, where i t is a necessity to vary enzyme concentration during the assay, a large number of overlapping substrate points should be assayed at each new enzyme concentration used. This would give sufficient points of overlap to indicate the presence of two distinct lines (as shown in the inset of Fig. 24) with a common 1/[S] intercept rather than a curvature and two apparent Michaelis constants as shown in the main plot where no overlap of substrate concentrations is shown as an example. In the assays of phosphodiesterase activity in which enzyme concentration is varied during the assay, contamination of the enzyme by protein activator I | and Ca may provide an explanation for the occurrence of downward sloping Lineweaver-Burk plots. Negative cooperativity has been proposed as an explanation of this type of behavior for phosphodiesterase (76) and this explanation may indeed be correct; however, these authors vary enzyme concentration during their assays to determine K^. The data plotted in Fig. 24 indicates an alternate explanation for downward curvature of Lineweaver-Burk plots. Similarity of the 37,000 x g pellet and supernatant enzymes— During the i n i t i a l purification of the enzyme, the activity was found to be evenly distributed between the 37,000 x g pellet and supernate. A good portion (80%) of the 37,000 x g pellet enzyme could be solubilized by 1.0% sodium deoxycholate and this enzyme exhibited Michaelis constants for cyclic AMP and cyclic GMP which were identical to those of the partially purified - 99 -soluble enzyme. The enzyme activity profiles on BioGel A1.5m chromatography were also identical for the soluble and sodium deoxycholate solubilized preparations. This suggests that these are the same enzyme and that the soluble material may be trapped or adsorbed onto particulate matter by some means during the i n i t i a l purification steps. Alternately, the soluble enzyme could be the pellet enzyme which has been solubilized by the i n i t i a l purification steps. The distribution of the enzyme (partly soluble and partly particulate) i s in agreement with the work published elsewhere (32-34). values of the enzyme— Using cyclic AMP as substrate and a supernate derived from the acetone powder as an enzyme source, a downward sloping Lineweaver-Burk plot was observed (Fig;. 14). This i s attributed to the presence of two enzymes capable of hydrolyzing cyclic AMP (68,69). Using cyclic GMP as substrate, only one was observed. These observations of two Michaelis constants for cyclic AMP and one for cyclic GMP are in agreement with the results of Thompson and Appleman (68,69). They separated two enzyme activities during agarose gel f i l t r a t i o n ; one peak (II) hydrolyzed both cyclic AMP and cyclic GMP and the other (peak III) hydrolyzed cyclic AMP only. In this work, the enzyme purified 15-fold from bovine cerebral cortex exhibited a single K m for cyclic AMP (30 uM) and cyclic GMP (4 uM). It would seem, therefore, that this enzyme is the high cyclic AMP - cyclic GMP phosphodiesterase described by Thompson and Appleman (69). One enzyme may be responsible for the hydrolysis of both cyclic nucleotides. This i s supported by the observations that 1) cyclic AMP and cyclic GMP hydrolyzing activities could not be separated during column chromatography and 2) the cyclic nucleotides mutually inhibited each other's hydrolysis. - 100 -In the original crude homogenate, there existed a specific high affinity (low K )^ cyclic AMP phosphodiesterase and a second enzyme capable of hydrolyzing both cyclic AMP and cyclic GMP. During purification of the supernate, the high affinity cyclic AMP enzyme was removed from the second enzyme. This enzyme was not found in the final enzyme preparation from the BioGel A1.5m column. The reason that this low Km enzyme activity was not detected (and therefore lost) during the purification is believed to be that the cyclic AMP levels employed for routine assay during purification were not low enough to reveal its presence. In effect, a low cyclic AMP level (in the order of 1 uM or less) should have been used during the purification (instead of .the 30 uM or greater levels of cyclic AMP used) in order to follow the activity of this low K enzyme. Since this was not done, i t is not known at which stage this enzyme was separated from the main phosphodiesterase peak. It can only be reported that this enzyme activity did not appear when a thorough study was made of the high enzyme after the final purification step. A thorough examination of this low enzyme would be an important step in understanding more fully the hydrolysis of cyclic AMP. Thompson and Appleman (68) have reported Michaelis constants of 104 uM for cyclic AMP and 12.9 uM for cyclic GMP for the high Km cyclic AMP - cyclic GMP enzyme. A K of 2.0 uM for cyclic AMP was reported for the low cyclic AMP specific enzyme. These values are virtually identical with the Km values presented in this thesis for the phosphodiesterase activities present in the crude supernate (see section VIII of Results). However, upon measurement of the Michaelis constants of the partially purified enzyme, i t is apparent that the values have decreased for the high enzyme. They are now 30 uM - 101 -for cyclic AMP and 4 uM for cyclic GMP. This decrease in the Michaelis constants can li k e l y be attributed to a binding of the cyclic nucleotides to protein kinases or other binding sites present in the crude preparations, and these non-catalytic binding sites are removed during purification. As mentioned earlier, O'Dea et a l . (47) and Cheung (48) have reported that the protein kinase bound cyclic AMP was unavailable for hydrolysis by phosphodiesterase. A binding to such a site would lower the effective (free) concentration of the cyclic nucleotide. This would give rise to an apparent increase in the measured for phosphodiesterase. Comparison of phosphodiesterase and adenyl cyclase a c t i v i t i e s — Several workers (28,29) have reported that phosphodiesterase was present in a 10 to 100-fold excess over adenyl cyclase in many tissues, including brain. Beavo et a l . (84) have measured the rates of hydrolysis of cyclic AMP and cyclic GMP at 1 mM and 1 uM concentrations. Approximately equal rates of hydrolysis at 1 mM substrate and higher rates of cyclic GMP hydrolysis over cyclic AMP hydrolysis at 1 uM substrate were observed. This i s in agreement with the data here, except that the velocities reported by Beavo et a l . (84) are some 3-fold higher than the ones observed in this study. This is probably due to their dealing with an enzyme which was partially activated by Ca , since their measurements were made in the absence of EGTA. The reaction velocities measured here were in the presence of 500 uM EGTA. The often quoted 10 to 100-fold excess of phosphodiesterase over adenyl cyclase is a direct result of phosphodiesterase measurements being made at saturating levels of substrate (which are totally unphysiological) whereas the basal adenyl cyclase activities have been routinely measured at cellular levels of ATP and Mg . The kinetic properties of phosphodiesterase quite - 102 -likely are important in determining the ability of this enzyme to control cyclic AMP and cyclic GMP concentration in the cel l . In support of this, the kinetic parameters determined experimentally are utilized in order to demonstrate that this might occur. The Michaelis constants used are those observed in the crude homogenate as these may more accurately represent the approximate state in the ce l l . They would automatically account for effects of binding of the cyclic nucleotides to non-catalytic sites, such as protein kinase. If this extraneous binding could be quantitated, i t would, of course, be best to use the Michaelis constants determined for the purified enzyme and include the other binding constants for cyclic AMP to non-catalytic sites as separate entities in the calculations. One should also know the levels of free Ca and protein activator in the cel l . Since this data is not available, a state of the enzyme is assumed in which I | no Ca -protein activator stimulation is present (an obvious oversimplification) The cel l is assumed to have two phosphodiesterases, one specific for cyclic AMP and the other a cyclic AMP - cyclic GMP enzyme. Since no data other than and ^ m a x of the low cyclic AMP enzyme (peak III of Thompson and Appleman) is available in regard to inhibition or activation by cyclic GMP or any other endogenous factors, the enzyme was assumed to be a simple Michaelis-Menten enzyme. Therefore, a simple formula was used for the velocity calculations of this low cyclic AMP specific phosphodiesterase. The parameters which refer to this enzyme are designated by the subscript .1. In the case of the cyclic AMP - cyclic GMP phosphodiesterase, (peak II of Thompson and Appleman) cyclic GMP was assumed to inhibit cyclic AMP hydrolysis competitively and vice versa (see section IX of Results). For this enzyme, the subscript h refers to the enzyme parameters for cyclic AMP, and again a Michaelis-Menten form of velocity equation is used. Subscript G refers to parameters - 103 -of this enzyme for cyclic GMP. The rates of hydrolysis were calculated using the following formulae for cyclic AMP hydrolysis: V [cyclic AMP]/K V. [cyclic AMP] . . i + _5 <2) 1 + [cyclic AMP] + [cyclic GMP] K + [cyclic AMP] m K K "h mG and for cyclic GMP hydrolysis: V [cyclic GMP]/K mG mG v = (3) 1 + [cyclic AMP] + [cyclic GMP] K K '•G and are tabulated in Table I. For the calculation of the cyclic AMP hydrolysis rate, a basal level for cyclic GMP of 0.02 uM (95) was used when taking into account the inhibition by this cyclic nucleotide. Likewise, a basal level of 1 uM (96) was used as the concentration for cyclic AMP in the reverse situation. These concentrations were chosen as representative of cerebral cortex levels of the cyclic nucleotides under unstimulated conditions. The table gives the rates of hydrolysis calculated at various cyclic nucleotide levels. At 1 uM cyclic AMP thei. rate of hydrolysis of cyclic AMP is 26.5 nmoles/g wet weight per min. This is the same as observed experimentally and about 1/3 that observed by Beavo et a l . (84). Using these physiological cyclic AMP concentrations, the hydrolysis rate agrees extremely well with the rate of synthesis of cyclic AMP by adenyl cyclase (22 nmoles/g wet weight per min) published by Hardman and Sutherland (11). The rate of synthesis - 104 -TABLE I Comparison of the rates of hydrolysis of cyclic AMP and cyclic GMP for the high and low Km phosphodiesterases present in the crude homogenate. Experiments to determine Michaelis constants and maximal velocities were performed in the presence of 0.5 mM EGTA and at pH 7.5. The data shown in the table was calculated using the experimentally determined parameters and equations 2 (for cyclic AMP hydrolysis) and 3 (for cyclic GMP hydrolysis) of the text. The parmeters used were: V =1.58 umoles/g wet weight per "h min, V = 0.37 umoles/g wet weight per min, Vm = 1.62 umoles/g wet weight 1 G per min, K = 110 uM, K = 2 uM and K =14 uM. In the table, a l l "h m l mG velocities are expressed in nmoles/g wet weight per min and substrate concentrations in uM. - 105 -cyclic AMP hydrolysis velocity velocity cyclic GMP [S] due to % of due to % of VA hydrolysis high K ° m VA low K m VA enzyme enzyme .01 .14 44 .18 56 .33 1.1 .018 .26 44 .33 56 .59 2.1 .05 .72 44 .90 56 1.6 5.7 .1 1.4 45 1.8 55 3.2 11 .2 2.9 46 3.4 54 6.2 23 .5 7.1 49 7.4 51 14 55 .75 11 51 10 49 21 82 1.0 14 54 12 46 26 110 2.0 28 61 18 39 47 200 3.0 42 65 22 35 64 280 5.0 69 72 26 28 95 420 10.0 130 81 31 19 160 670 20.0 240 88 34 12 280 950 50.0 490 93 36 7 530 1260 100. 750 96 36 4 790 1420 500. 1300 97 37 3 1330 1570 - 106 -would be exactly counterbalanced by the rate of hydrolysis i f the cyclic AMP level was 0.75 uM. Phosphodiesterase and adenyl cyclase activities were measured experimentally at pH 7.5 and 7.4 respectively. The conditions under which the assays were performed may be totally different from those present in the ce l l , but the calculations do indicate that the actual enzyme parameters of the phosphodiesterase and the low cyclic AMP levels combine to produce a situation of approximately equivalent rates of hydrolysis and synthesis of cyclic AMP. It is intuitively obvious that the rate of synthesis of cyclic AMP by adenyl cyclase must equal the rate of hydrolysis of cyclic AMP by phosphodiesterase at any steady state level of cyclic AMP. These calculations indicate that this indeed is the situation. When the rate of cyclic GMP hydrolysis is considered, a rate at 0.02 uM cyclic GMP of 2.2 nmoles/g wet weight per min is calculated, approximately 1/12 that of cyclic AMP hydrolysis (at 1 uM cyclic AMP). The guanyl cyclase activity of brain as reported by Hardman and Sutherland (11) is 1.1-2.7 nmoles/ g wet weight per min (mean =1.7 nmoles/g wet weight per min) when measured In the presence of MnC^. Thus, here too the rate of hydrolysis is in the range that might be expected from the existing guanyl cyclase levels in brain. The kinetic constants of phosphodiesterase predict that the equilibrium concentration of cyclic AMP should be 0.75 uM and that for cyclic GMP should be about 0.02 uM, based upon the rate of synthesis of these nucleotides being equal to the rate of hydrolysis. The levels of cyclic AMP and cyclic GMP calculated in this manner are close to those present in brain (95,96). The calculations presented above indicate that adenyl cyclase (or guanyl cyclase) and phosphodiesterase activities are delicately balanced. Any shift in the activities of either or both of these enzymes would alter the concentration of cyclic AMP (or cyclic GMP) in the ce l l . - 107 -Relative rates of hydrolysis of cyclic AMP due to the high Km and low enzymes— Another important point arising from these calculations is the relative importance of the high K and low K enzymes for the hydrolysis of m m cyclic AMP. At 1 uM cyclic AMP, approximately 54% of the hydrolysis is due to the high enzyme. This percentage increases rapidly as cyclic AMP levels rise reaching 81% by 10 uM. This observation tends to negate the previous assignment (68) of the high enzyme to a relatively unimportant role in the control of cyclic AMP in the ce l l . In effect, this would indicate that both enzymes are about equally important at basal cyclic AMP levels, but during a stimulation of adenyl cyclase and the subsequent rise in cyclic AMP levels, the high enzyme becomes more and more important for the hydrolysis of this cyclic nucleotide, in proportion to the increase in its concentration. Granted, this may not properly describe the kinetics of the low enzyme in the cel l ; however, the activation of the high K enzyme by Ca and protein activator ixi vitro would seem to be a reasonable situation in vivo, and such an activation (in the absence of any change in the low enzyme) would accentuate the relative importance of the high K enzyme. On the other hand, the possibility exists that the cyclic GMP activation of cyclic AMP hydrolysis (50-52) reported may be a specific effect on the low Km cyclic AMP phosphodiesterase, although this possibility had not been examined. However, i f such were the case, i t could well prove to be an important aspect of the properties of the high affinity enzyme. Another possible explanation of cyclic GMP activation involves contamination of phosphodiesterase preparations by protein kinase. If protein kinase were present in the assay, the addition of cyclic GMP would displace some cyclic AMP from the protein kinase. This would increase the effective (free) concentration of cyclic AMP free to be hydrolyzed by phosphodiesterase. - 108 -Because of the rise in cyclic AMP concentrations, the rate of hydrolysis would increase due to the enzyme being more saturated with substrate (cyclic AMP). Thus, an apparent stimulation of cyclic AMP hydrolysis by cyclic GMP could be due to the presence of protein kinase in the assay. If an activation of adenyl cyclase occurred and the increased synthesis of cyclic AMP were maintained, a new steady state level of cyclic AMP would be reached at which this new adenyl cyclase activity would be counterbalanced with the new increased phosphodiesterase activity. The phosphodiesterase activity would be increased (barring activation) simply by the rise in cyclic AMP levels producing an enzyme more saturated with substrate. One should not be left with the impression that the high enzyme is more important in this case simply because the K is high. This is not the case - the important aspect m is the relationship that exists between the V and the K . Both of these max m must be high in relation to the low Km enzyme parameters. This can be further explained by a simple example. Suppose only a low K enzyme were present with a K of 2 uM and that the substrate level was 2 uM. If the substrate m were to rise 10 (or 100)-fold, the enzyme would only be able to double its hydrolysis rate regardless of the extent of the rise in substrate concentrations. On the other hand, an enzyme with a of 100 uM would respond to a 10-fold increase in cyclic AMP by essentially a 10-fold rise in activity. The system that appears to be present (a low K - low V cyclic AMP phosphodiesterase m max r r and a high - high v m a x cyclic AMP - cyclic GMP enzyme) would present a very efficient system in combination with adenyl cyclase for controlling cyclic AMP levels in the ce l l . It provides a possible explanation for the basal levels of cyclic AMP being about 0.75 to 1.0 uM since hydrolysis equals synthesis at this concentration of cyclic AMP. It is also a very effective system for returning cyclic AMP levels to normal quickly and effectively once the adenyl cyclase activity has returned to basal levels, especially with the - 109 -added impetus of a possible stimulation of the phosphodiesterase by Ca and protein activator. The control of cyclic GMP levels has been far less studied, but i t could be envisioned in a similar manner to that described for cyclic AMP. In this case, a single Michaelis-Menten enzyme was used to describe the phosphodiesterase. With a rise in cyclic GMP levels in heart, George ^t a l . (12) observed a fa l l in cyclic AMP levels. The cyclic GMP activation of cyclic AMP hydrolysis (possibly by the low Km cyclic AMP specific enzyme) would provide a convenient explanation for the coincident fa l l in cyclic AMP levels. A third point also arises from the data shown in Table I. Some authors (97-99) have reported inhibition of a low phosphodiesterase by various agents. In their studies, a low level of cyclic AMP (often as low as 1.0 x —8 10 M) was used and the resulting velocity assumed to represent the hydrolysis due to the low K^enzyme in crude supernates. Table I points out an obvious flaw in reasoning that using a low cyclic AMP level results in measurements —8 of the low enzyme. At 1.0 x 10 M cyclic AMP, the rate of hydrolysis due to the low enzyme is 56% of the total hydrolysis rate and essentially this should represent the ratio between the hydrolysis rates of the two phosphodiesterases regardless of how low the substrate level used is . This may not hold in the region where substrate _< enzyme levels since Michaelis-Menten pseudo first-order kinetics do not hold and second order kinetics appear, in which case, the rate should depend upon the number of catalytic sites of each kind present. Essentially the reaction becomes controlled by the rate of diffusion of the substrate onto the enzyme as well as the actual catalysis step. The calculations, therefore, indicate that i t is not valid to measure the low K enzyme by simply using a low substrate level, - 110 -unless the relative kinetic parameters have been determined and a significant contribution by the high enzyme to the hydrolysis rate can be ruled out. The data presented here provide a basis for describing a model for the integration of phosphodiesterase and adenyl cyclase into a system which would provide a means of control of cyclic AMP levels. The factors involved are as follows: I | 1) Mg is required for phosphodiesterase activity, but i t s low K Si would suggest that this ion is not involved in the control of the enzyme in the c e l l . 2) two phosphodiesterases, a high - high ^ m a x enzyme capable of hydrolyzing both cyclic AMP and cyclic GMP and a low K m - low ^ m a x enzyme specific for cyclic AMP, are believed to be present. The I | high K m enzyme can be activated by Ca and protein activator. L i t t l e i s known regarding the low K enzyme, but i t may provide the explanation for cyclic GMP activation of cyclic AMP hydrolysis, although no experimental evidence exists for this. 3) The high enzyme appears capable of hydrolyzing cyclic AMP at least as rapidly as the low K m enzyme at basal cyclic AMP levels. At elevated cyclic AMP concentrations, the high K m enzyme would appear to hydrolyze a major percentage of this cyclic nucleotide, in order to return i t s concentration to normal. I | 4) Ca causes a deaggregation of the high K phosphodiesterase and this interconversion of molecular forms may be a key to the mechanism of the Ca -protein activator stimulation. 5) Phosphodiesterase activity i s not in a 10 to 100-fold excess of adenyl cyclase activity in the c e l l , and these enzymes probably control cyclic AMP levels by the delicate balance which exists between their a c t i v i t i e s . - I l l -A system of adenyl cyclase (or guanyl cyclase) and phosphodiesterase such as proposed would seem capable of effecting changes in cyclic AMP (or cyclic GMP) concentrations in an orderly and controlled manner. This is an over simplification of the situation regarding cyclic AMP metabolism in vivo. However, the data presented indicate that phosphodiesterase and : adenyl cyclase are equally important in the control of cyclic AMP concentrations in the ce l l . - 112 -BIBLIOGRAPHY 1. Rail, T.W., Sutherland, E.W. and Berthet, J. , J. Biol. Chem., 224, 463 (1957). 2. 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Pharmacol., 273, 62 (1972). 99. Solomon, S.S., J. Lab. Clin. Med., 79, 598 (1972). - 117 -APPENDIX The Appendix deals with the derivation of the rate equations for three [ | models describing the activation of phosphodiesterase by Ca and protein activator. These models were derived in order to provide a possible explanation of the data presented in Fig. 5 to 10. In this Appendix, the following abbreviations have been used: S substrate (cyclic AMP or cyclic GMP) P product (5'-AMP or 5'-GMP) E enzyme (phosphodiesterase) ES enzyme-substrate complex Ap protein activator Ca Ca"1^ EAp enzyme-protein activator complex I | ECa enzyme-Ca complex I | ApCa protein activator-Ca complex I | EApCa enzyme-protein activator-Ca complex EApCaS enzyme-protein activator-Ca -substrate complex Kg dissociation constant of S from ES (= [E][S]/[ES]) ^Ap+Ca dissociation constant of Ca from ApCa (= [Ap][Ca]/[ApCa]) ^ApCa dissociation constant of ApCa from EApCa (= [E][ApCa]/[EApCa]) K dissociation constant of Ap from EAp (= [E][Ap]/[EAp]) Ap K dissociation constant of Ca from ECa (= [E][Ca]/[ECa]) L»a K' dissociation constant of Ap from EApCa (= [ECa][Ap]/[EApCa]) Ap K ^ dissociation constant of Ca from EApCa (= [EAp][Ca]/[EApCa]) The method of Cha (88) was used to derive the rate equations. Inherent in the derivation is the assumption of a rapid equilibrium between the - 118 -various enzyme species or a combined steady state and rapid equilibrium system. In addition, the data shown in Fig. 5 to 7 allow certain other simplifying assumptions to be incorporated into the derivations. 1) The K of the enzyme for cyclic AMP and cyclic GMP does not change I j in the presence of protein activator and/or Ca . Only the maximal velocity increases in the presence of both protein activator and Ca** (Fig. 5 and 6). 2) The apparent activation constant for protein activator does not change as the substrate concentration is varied (Fig. 7). Model A represents the simplest case, with only two enzyme species involved in the activation as shown in Fig. A - l . As an illustration, this model is described in some detail. Using the method of Cha, total enzyme concentration is represented as the sum of a l l of the individual species: [ E ] t o t a l = [ E l + [ E S ] + £ E A P C a l + [EApCag] The rate equation essentially states that the velocity is a function of the enzyme in the form of ES and EApCaS, since these are the only forms which yield product. Therefore, the rate equation is : v = k^ES] + k2[EApCaS] where k^ is the rate constant in the absence of protein activator and/or I | Ca and is the rate constant in the presence of protein activator plus I | Ca . The fraction of each enzyme species present is determined by the - 119 -Fig. A - l : Model A. Each double headed line (T—*) indicates a rapid equilibrium step with the dissociation constant indicated. Each heavy single headed arrow (-»•) indicates an irreversible rate limiting catalysis step with the rate constant indicated alongside. - 120 Ap „ K Ap+Ca _ ^ + Ca ^ * ApCj KApC< ^ EApCa A "<s K A p C i EApCaS J<2 - 121 -concentration of i t s individual components and the dissociation constants between them. A fraction (f) can be defined showing the amount of a particular enzyme species present, for example: f(E) - [ E ] / [ E ] t o t a l f ( E S ) = J M I s i / [ E W KS f ( E A p C a ) , M C A E C a , / [ E w . I M , / [ E w ApCa ApCa Ap+Ca f ( E A p C a S ) = [ EHAp][Ca][S] total ApCa Ap+Ca S Since these are a l l of the enzyme species, the sum of the numerators of the four preceeding equations must be the total enzyme present. The rate equation, as described above, then is a function of the fraction of each catalytically active species present and is given by: v = A i i S i + k 2 [Apj[Ca] [S] \ [ B ] t o t a l \ KS KApCaKAp+CaKs/ f + 1$± + CAp][Ca] + [Ap][Ca][S]  KS KApCaKAp+Ca KApCaKAp+CaKS It can be noted that in actual fact the denominator i s the sum of a l l of the enzyme species and,- therefore, the [ E ] f c o t a l ' T n i s last equation can be - 122 -rearranged to the form: [S] Vl KAPCa KAp+Ca + VAp ] [Ca] v = — — x c K (A-l) K S + M KA P C a KA P + C a + [A*>] t C a ] where refers to the velocity maximum in the absence of either or both of the activators and refers to the maximal velocity in the presence of both activators. This notation applies to the remaining mddels as well. Equation A - l can be rearranged to the form which can be used for Eadie plots in order to determine the K for substrate: = ! i ^ i l V ^ . i l K (A.2) For the activator plots, a velocity (v0) is defined as the velocity in the absence of one or both of the activators at a given substrate concentration. Therefore: v 1[s] Vo = Kg + [S] Equation A - l can then be rearranged into the forms: V«v 0 K, K v «. _2 v^v. x ApCa Ap+Ca ( A _ 3 ) V. [Ap] [Ca] - 123 -and V - V p K 'ApCa Ap+Ca [Ap] (A-4) v = x [Ca] Equations A-3 and A-4 predict linear plots of v versus (v-v 0)/[A] i f the second activator i s held constant. Since these equations predict a common intercept of V^Vo/V^ for v versus (v-v„)/[Ap] and v versus (v-v 0)/[Ca] and this is not observed experimentally (Fig. 9 and 10), a simple model like Model A is not sufficient to explain the data. The second model proposed is shown in Fig. A-2 and designated Model B. Here three species (E, EAp:anSi! EApCa) are proposed. This model essentially states that Ap can bind to the enzyme alone, either through direct attachment or by a loss of Ca from EApCa. Here an additional piece of experimental data is u t i l i z e d . The addition of protein activator alone did not decrease the enzyme activity (Fig. 4). This means that, i f EAp forms, then this form must be catalytically active and react to form product at a maximal rate of V^. If this were not the case, and EAp was not catalytically active, an inhibition of activity should have been, but experimentally was not, observed. The velocity equation for this model is given by Equation A-5. v X V l ( K C a K A P + K C a [ A P ] ) + V 2 [ A P ] C C a ] (A-5) Kg + [S] K K A„ + K' [Ap] + [Ap][Ca] As with equation A - l , this equation can be rearranged to give the Eadie form - 124 -Fig. A-2: Model B. Each double headed line — i n d i c a t e s a rapid equilibrium step with the dissociation constant indicated. Each heavy single headed arrow (-»•) indicates an irreversible rate limiting catalysis step with the rate constant indicated alongside. E+P EAp v- — EApCa KCa . ^ K Ap+Ca Ap + Ca - ApCa * E A p C a S \ EApCa + P - 126 -of the equation: V l K C a ( K A p + tApl) +V 2 [A P][Ca3 v = c - x K (A-6) K* a (K A p + [Ap]) + [Ap][Ca] [S] Eadie type forms can also be obtained for the plotting of activator concentrations. v . K ; a + V 2 v 0 [ C a ] / V 1 K A P K Ca ( A v = - x c (A-7) K^a + [Ca] [Ap] + [Ca] V 9 V ° „ w K A n + tAp]) v = _2 v ^ x Ca Ap ( A _ 8 ) V x [Ca] [Ap] Equations A-6 and A-8 predict plots similar to those of equations A - l and A-3 respectively, while equation A-7 predicts a plot with variable slopes and intercepts dependent upon the concentration of Ca used in the assay. The plot predicted by equation A-8 is not obtained (Fig. 10). For Model B, the identical equations are obtained in the case where ECa, but not EAp, forms. The arguments are identical, with the substitution of Ca for Ap and Ap for Ca in the equations (A-5 to A-8). Model B did not totally explain the data and Model C was next proposed (Fig. A-3). Here, a l l four enzyme species (E, EAp, ECa and EApCa) can form. The rate equations can be derived and depending upon the pathway chosen are: - 127 -Fig. A-3: Model C. Each double headed line —indica tes a rapid equilibrium step with the dissociation constant indicated. Each heavy single headed arrow (->-) indicates an irreversible rate limiting catalysis step with the rate constant indicated alongside. Ap + Ca x - * ApCa E + P ECa + - 129 -[S] ^ V l KCa ( KAp KCa + K C a [ A p ] + K A p [ C a 3 ) + V 2 K C a [ A p ] [ C a ]  KS + t S ] X KCa ( KApKCa + K C a [ A p ] + K A P [ C a ] ) + K C a [ A p ] [ C * ] (A-9a) [S] V l K A P ( K Ap K Ca + K C a [ A p ] + K A p [ C a ] ) + V 2 K A p [ A p ] [ C a ] v = = x KS + [ S ] K ; p ( K A p K C a + K C a [ A p ] + K A p [ C a ] ) + VApl[Cal (A-9b) [S] ^ VlKApCaKAP +Ca ( KApKCa + K C a [ A p ] + K A P [ C a ] ) + V 2 K A p K C a [ A p ] [ C a ]  KS + [ S ] X KAPCaKAP +Ca ( KApKCa + K C a [ A p ] + K A p [ C a ] ) + K A p K C a [ A p ] f C a ] (A-9c) A l l of these equations are identical. Equation A-9a takes the pathway E ->• EAp -*• EApCa; A-9b takes the pathway E •* ECa •* EApCa; and A-9c takes the pathway E -*• EApCa in order to reach EApCa from E. These equations can be rearranged as before. For an Eadie plot, equation A-9a rearranges to: V l K Ca ( K A P K Ca + + VCa]) + V2WAp] ^  _ _y_ x ^ KCa ( KApKCa + K C a [ A p ] + K A p [ C a ] ) + K C a [ A p ] [ C a ] [ S ] X " CA-10) To obtain the Eadie type equation for protein activator, equation A-9a rearranges to: v = v 0 Kl + V.VotCal/V. Ca 2 *• 1 v-v0 KCa + [ C a ] [ A p ] x ^Ap K Ca ( K Ca + [ C a ] )  K C a ( K C a + [ C a ] ) (A-ll) - 130 -and rearrangement of equation A-9b to yield an Eadie type form for the Ca data yields: ^ _ v.K; p +V 2v , [Ap]/V 1 _ v _ V o ^ * C a K Ap ( K Ap + tApl) K A p + [Ap] [Ca] X K A p (K A p + [Ap]) (A-12) Plots of v versus v/[S] (equation A-10), v versus (v-vQ)/[Ap] (equation A-ll) and v versus (v-v0)/[Ca] (equation A-12) are shown in Fig. 5, 6, 9 and 10. This set of equations (A-9a to A-10) is internally self-consistent in that in the absence of one or both activators, they reduce to: v 1[s] v = K s + [S] and v = V - — x K 1 [S] s These represent the simple Michaelis-Menten formulations in the absence of one or both activators. Equations A - l l and A-12 contain combinations of constants in the components of the equations representing the intercepts and slopes. In order to attain a graphical analysis of the equations to obtain values of the individual dissociation constants, these intercepts and slopes can be plotted. From the intercepts (a )^ of the plot for equation A - l l (Fig. 9), - 131 -a new equation can be obtained by rearrangement: V ? v 0 a . - V o a. = - - — - — x K' (A-13) 1 \ [Ca] C a and a new equation can be obtained from the intercepts (a.^) of equation A-12 (Fig. 10): V 9 v 0 a_-v0 a_ = -s— - -2 x K» (A-14) 2 V 1 [Ap] AP The plots for equations A-13 and A-14 are shown in Fig. 20 and 21 of the Discussion. The use of the slopes to obtain and requires a somewhat more complex rearrangement. For these purposes, an equation in the form of A-12 is derived from equation A-9a. The slope (b2) of this new equation is then given by: b KCa KCa ( KA P + W> 2 K A P K Ca + W ^ 1 which can be rearranged to the form: 1 . + [ A P I X A + J_V Ca KAp \ b2 K Ca/ (A-15) b2 KC  "  - 132 -This equation requires the use of a constant (K' ) obtained from a previous replot of equation A-13 (Fig. 20). The plot of the slopes of Fig. 10 according to equation A-15 is shown in Fig. 22. The model (Model C) explains the observed data, however, as with a l l kinetic analysis, models which have not been proposed may exist and explain the data. 

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