ARCHIVES
OF
BIOCkEYISTRY
AND
BIOPHYSICS
182,
124-133 (I9771
Rat Brain Adsnylate Further THOMAS Department
Studies
on Its Stimulation
J. LYNCH,
of Biochemistr$,
E. ANN
Cyclase by a Ca2+-Binding
TALLANT,
AND
WA1 YIU
St. Jude Children’s Research Hospital and University the Health Sciences, Memphis, Tennessee 38101 Received January
Protein* CHEUNG of Tennessee Center for
5, 1977
Bovine or rat brain adenylate cyclase (EC 4.6.1.1) solubilized by Lubrol-PX, a nonionic detergent, requires a Ca*+-binding protein activator for full activity (Cheung et al., 1975, Biochem. Biophys. Res. Commun. 66,1055-1062). We now show that particulate rat brain adenylate cyclase also required the activator for maximum activity. A brain particulate fraction was extracted with a hypertonic NaCl solution containing [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. This procedure removed preferentially the activator, making adenylate cyclase activator deficient and, consequently, dependent on an exogenous activator for maximum activity. The activator increased the V of adenylate cyclase without affecting’its apparent K, for ATP. In the presence of the activator, the enzyme was more stable against thermal inactivation, suggesting that the activator probably induced a conformational change to the enzyme. F- and 5’-guanylylimidodiphosphate [GMP-p(NH)p] greatly stimulated brain adenylate cyclase. Adenylate cyclase activity obtained in the presence of the activator and F- was comparable to the summed activities of the two agents assayed separately, indicating that their effects were additive. Similarly, the effects of the activator and GMP-p(NH)p were additive. These results suggest that the action of the activator is independent of the other two ligands. Since the activator is present in excess over adenylate cyclase, the cellular flux of CaZ+ is believed to be important in modulating the enzyme activity. The role of the Cal+/ activator is discussed with respect to cyclic AMP metabolism in brain.
Although CAMP’ is known to mediate the action of many polypeptide hormones and catecholamines, the mechanism by which its intracellular level is regulated on a moment-by-moment basis is not yet understood. The effect of CAMP is rapid and the increase of CAMP in target cells in response to a stimulus is usually shortlived, often appearing as a peak response (1). There are two enzymes or enzyme sys-
terns that control the tissue level of the cyclic nucleotide. Adenylate cyclase (EC 4.6.1.1), associated with the plasma membrane, catalyzes the formation of CAMP, and cyclic 3’,5’-nucleotide phosphodiesterase (EC 3.1.4.17), partly particulate and partly soluble, catalyzes its degradation. An endogenous Ca2+-binding protein activator first discovered for bovine brain phosphodiesterase (2) also stimulates brain adenylate cyclase (3, 4). In the presence of Ca2+, the activator assumes a more helical structure and combines with the apoenzyme to form an enzyme * activator complex (5). Lowering the Ca2+ concentration dissociates the two proteins and returns the enzyme activity to its basal level. Stimulation of adenylate cyclase by the activator is immediate and reversible and appears similar to that found with phosphodiesterase (6-8). The sequence of
t This work was supported by a grant from the U.S. Public Health Service (NS080591 and by a grant-in-aid from the American Cancer Society (ACS IN 990 1 Abbreviations used: CAMP, cyclic AMP; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; DEAE, diethylaminoethyl; DTT, dithiothreitol; GMP-p(NH)p, 5’guanylylimidodiphosphate; ECTEOLA-cellulose, epichlorohydrin triethanolamine cellulose. 124 Copyright
8 1977 by Academic
Press, Inc.
All rights of reproduction in any form reserved.
ISSN 0003-9861
ACTIVATOR.Ca2+-DEPENDENT reactions leading to activation picted as follows. (Activator)
+ Ca*+ inactive
(Activator*.
ADENYLATE
may be deCa*+) active
(Enzyme) + (Activator*. Ca*+) + active less active (Enzyme*. Activator*. Ca*+) active
HI
M
The asterisk depicts a different conformation. The stoichiometry of the components in these equations has not been established; the equations simply designate the interaction of these components. Stimulation of adenylate cyclase by the activator was demonstrated with a LubrolPX-solubilized preparation (3, 4). Since adenylate cyclase is associated with the plasma membrane, and the detergent-solubilized enzyme may have properties different from those of the membrane enzyme, it is important to demonstrate the same effect directly on the membrane enzyme in the absence of the detergent. In this communication we describe the preparation of a particulate adenylate cyclase without the use of a detergent and largely free of its endogenous activator, the stimulation of the particulate enzyme by the activator, and its effect on the stimulation of adenylate cyclase by F- or by 5’guanylylimidodiphosphate. A preliminary note has appeared (9). EXPERIMENTAL
PROCEDURES
Chemicals and reagents. [3H]ATP (specific activity, 18 Ci/mmol) and r3H]cAMP (specific activity, 27 Ci/mmol) were obtained from Schwarz/Mann. Amberlite IRP-58 (260-400 mesh), a phenolic polyamine, was a generous gift from Dr. Anthony Pitochelli of Rohm and Haas. The resin was washed sequentially with 0.25 N NaOH, H20, 0.5 N HCl, and then exhaustively with H,O. The resin was suspended in H,O to give a 33% slurry (1 vol of resin to 2 vol of H,O). Lubrol-PX, pyruvate kinase, phosphoenolpyruvate, and ECTEOLA-cellulose were obtained from Sigma. Dowex 5OW-X8 (ZOO-400 mesh) was obtained from either Sigma or Bio-Rad. It was washed with 0.5 N NaOH, H20, 0.5 N HCl, and then exhaustively with H,O. The resin was suspended in 1 mM HCl. 5’-Guanylylimidodiphosphate [GMPp(NH)p] was obtained from ICN and 3-isobutyl-lmethylxanthine was from Aldrich Chemical Company. All chemicals were of reagent grade. Preparation of activator-deficient particulate ade-
CYCLASE
125
nylate cyclase. Step A: Sprague-Dawley rata weighing 160-180 g were decapitated and the whole brains were removed. They were either used fresh or stored at -90°C. The tissue was homogenized in a glass homogenizer with a Teflon pestle in 10 v01 of 20 mM Tris-HCl, pH 7.5 (Buffer A). The homogenate was centrifuged at 1OOOgfor 15 min. The precipitate was homogenized in 10 vol of Buffer A and was collected by centrifugation at 1OOOgfor 15 min. This step was repeated twice. The preparation was designated as unwashed particles. Step B: The sediment from Step A was further washed in 10 vol of 20 mM Tris-HCl (pH 7.5) containing 1 M NaCl and 2 mM EGTA (Buffer B). The suspension was centrifuged at 15,000g for 10 min. The washing was repeated twice. Step C: To remove the NaCl and EGTA, the pellet was then washed twice with 20 mM Tris-HCl (pH 7.5) containing 3 mM DTT (Buffer C). After the second wash, the pellet was resuspended in Buffer C to a final protein concentration of 5-10 mg/ml. The yield of washed particles from 1 g of brain tissue was lo-20 mg of protein. The sample was divided into small fractions which were stored at -90°C. Adenylate cyclase in this preparation was activator delicient and required an exogenous activator for maximum activity. The extent of stimulation was two- to threefold. Unless otherwise stated, all experiments were performed using this preparation. Alternatively, the sediment from Step A was washed four times in 10 vol of 20 mM Tris-HCl (pH 7.5), 3 mM DTT, 1 mM EGTA, and 0.05% Lubrol-PX (Buffer D). The rest of the procedure was as described in the preceding paragraph. Buffer D was more effective in removing the activator; the stimulation of detergent-washed adenylate cyclase by an exogenous activator was usually more than fivefold. Assay of adenylate cyclase. Crude preparations of adenylate cyclase were contaminated with CAMP phosphodiesterase and ATPase. The assay system, therefore, contained 3-isobutyl-1-methylxanthine and caffeine to inhibit phosphodiesterase, carrier CAMP to protect the newly synthesized [3H]cAMP from degradation, and an ATP regeneration system to counteract the effect of ATPas’e. The reaction mixture in a final volume of 0.1 ml contained 40 mM Tris-HCl (pH 7.5), 2 mM 3-isobutyl-l-methylxanthine, 40 mM caffeine, 5 mM MgC12, 2 to 4 mM CAMP, 1 mM 13HlATP (500,000 cpm/tube), 80-140 pg of protein, 4 mM phosphoenolpyruvate, and 40 pg/ml of pyruvate kinase. The final concentration of D’lT in the incubation ranged from 0.6 to 1.5 mM. When present, the concentration of the exogenous activator was 5 pg of protein/tube. Other additions are indicated in the legends. The reaction was usually started by the addition of the enzyme. At the end of the incubation at 37”C, usually lasting 10 min, the reaction was terminated by adding 50 ~1 of 1 N HCl. The reaction mixture was neutralized with 300 ~1 of
126
LYNCH,
TALLANT,
0.233 M Tris. Unreacted ATP was precipitated by the sequential additions of 80 ~1 each of 0.25 M ZnSOl and 0.25 M Ba(OH), (10). The reaction mixture was quantitatively transferred to a Dowex BOW-X8 cationic exchange resin column (6.5 x 6 cm), which was washed with 6 ml of 1 mM HCl. The eluant was discarded. The column was eluted with 3.5 ml of H,O and the eluant was collected into a scintillation vial. Cyclic AMP recovery was monitored photometrically at 256 nm; the recovery was 60-70%. The eluant was evaporated to dryness at 80°C. The dried sample was taken up in 0.4 ml of H,O. Seven milliliters of Bray’s solution was added for counting. All data were corrected for their CAMP recoveries. The background count was less than 0.1% of the amount of radioisotope added initially. The specific activity of adenylate cyclase is defined as picomoles of CAMP formed per milligram of protein per minute at 37°C under the assay conditions. All determinations were done in duplicate and were validated several times with different enzyme preparations. Assay of cyclic nucleotide phosphodiesterase. Phosphodiesterase was assayed by a two-stage procedure (11) modified after Thompson and Appleman (12). The reaction mixture contained 40 mM TrisHCl (pH 8.0), 5 mr+i MgCIZ, 50 PM CaCl,, 1 mM 13H]cAMP, and an appropriate amount of enzyme in a total volume of 0.1 ml. The reaction was initiated by the addition of substrate. After 10 min at 3O”C, the reaction was terminated by placing the tubes in a boiling water bath for 30 s. The tubes were cooled to 30°C and 20 ~1 of snake venom (Crotalus atrox, 1.0 mg/ml), as a source of 5’-nucleotidase to convert 5’AMP to adenosine, was added to start the second incubation. After 10 min, 1 ml of an IRP-58 slurry was added to terminate the reaction and the tubes were centrifuged. The resin binds the unreacted CAMP and little or no adenosine. A fraction of the supernatant fluid, which contained 13H1adenosine, was counted in a liquid scintillation spectrometer. Preparation of activator. A Ca2+-binding protein activator was purified from bovine brain to the stage of DEAE-cellulose column chromatography (13). Assay of activator. The activator was measured by its ability to stimulate an activator-deficient phosphodiesterase purified from bovine brain (14). Briefly, a sample to be tested was heated at 95°C for 1 min to inactivate phosphodiesterase and then assayed for the activator in a reaction mixture containing 40 mM Tris-HCl (pH 8.0), 5 mM MgC12, 50 PM CaCl,, 7 pg of activator-deficient phosphodiesterase, the heated sample, and 1 mM 13H1cAMP. The remaining procedure and assay were identical to that of phosphodiesterase in the preceding section. Determination ofprotein. Protein was determined according to Lowry et al. (15) with bovine serum albumin as a standard or according to Warburg and Christian (16).
AND
CHEUNG RESULTS
Preparation of an Activator-Deficient ticulate Adenylate Cyclase
Par-
Although stimulation of brain adenylate cyclase by the Ca2+-binding protein, was previously demonstrated with a detergentsolubilized preparation, it was deemed important to show the same activator response on the membrane enzyme prepared without the use of a detergent. Adenylate cyclase is associated with the plasma enmembrane; a detergent-solubilized zyme may have properties quite different from those of the enzyme associated with the membrane. Moreover, detergents affect adenylate cyclase activity in a variable manner (17-221, which may mask or distort the real effect of the activator. Therefore, we first set out to prepare a particulate adenylate cyclase that is responsive to the activator. Two observations are relevant in this regard. First, solubilization of adenylate cyclase by a detergent facilitates the dissociation of the endogenous activator from the enzyme in an anionic exchange column. The activator, being an acidic protein, was retained by the column and was clearly separated from the enzyme, which emerged with the void volume of the column virtually free of the activator (3, 4). Separation of the activator from phosphodiesterase was achieved similarly (14). Second, EGTA suppressed adenylate cyclase and phosphodiesterase activity to a basal level by dissociating the enzymeactivator complex (6-8). A hypertonic salt solution also depressed phosphodiesterase to its basal level, presumably by dissociating the enzyme *activator complex (23). Preliminary experiments suggested that brain adenylate cyclase was more tightly associated with a membranous fraction than was the activator. We reasoned that washing the membranes with a hypertonic salt solution would remove preferentially the activator. A brain particulate fraction was washed with 20 mM Tris-HCl (pH 7.5) containing 1 M NaCl and 2 mM EGTA. After two to three washes, all removable activator appeared to have been extracted from the membranes. The removal of the activator made the washed particulate en-
ACTIVATOR.
Ca2+-DEPENDENT
zyme activator deficient and dependent on an exogenous activator for full activity (Table I). However, the extent of stimulation was relatively small compared to that obtained with the detergent-solubilized enzyme. The apparently smaller stimulation was presumably due to a less effective removal of the endogenous activator from the particles by the hypertonic solution, as there was some residual activator in the washed particles but not in the detergentsolubilized enzyme. Alternatively, brain particles were washed with a buffer solution containing 20 mM Tris-HCl (pH 7.51, 3.0 mM DTT, 1 mM EGTA, and 0.05% Lubrol:PX. This medium removed essentially all of the endogenous activator, as the final washed particles showed no detectable activator activity. Adenylate cyclase activity in this preparation was higher than that of the detergent-solubilized enzyme and the extent of stimulation was comparable. The specific activities of both the detergentsolubilized and the detergent-washed enzymes were lower than the specific activity of the NaCl-washed particles. These results demonstrated that the endogenous activator could be removed from membrane adenylate cyclase without the use of a detergent and that the activatordeficient membrane enzyme was responsive to an exogenous activator. Although the extent of stimulation was relatively small, this was not an inherent characteristic of the particulate enzyme, since it could be made to respond as avidly as the detergent-solubilized enzyme, provided that the endogenous activator was effectively removed, for example, by washing the enzyme with a low concentration of Lubrol-PX. Differential Effect of EGTA on Adenylate Cyclase Activity of Holoenzyme or Apoenzyme EGTA, which has a greater affinity for Ca2+ than for Mg2+ (24), inhibits markedly adenylate cyclase activity; the inhibition is fully reversed by a molar excess of Ca2+ (25). EGTA inhibits the enzyme presumably by chelating the Ca2+ required for the formation of the active enzyme. activator
ADENYLATE
127
CYCLASE TABLE
I
STIMULATION OF RAT BRAIN ADENYLATE CYCLASE BY A C$+-BINDING PROTEIN ACTIVATORS Preparation
Addition
Adenylate CYclase (P,mgoil/
Residual activator
min) NaCl-washed partitles Detergent-washed particles Detergent-solubilized enzyme
None Activator None Activator None Activator
579 1602 173 1073 76 485
(+I (-) (-)
a Adenylate cyclase activity was determined in the presence or absence of an exogenous activator purified from bovine brain (13). It has been established that the activator lacks species specificity and that the activator from bovine brain cross-reacts with adenylate cyclase from rat brain, and vice versa (14). The NaCl- or detergent-washed particles were prepared as described under Experimental Procedures. The detergent-solubilized enzyme was prepared from an ECTEOLA-cellulose column (3). The concentration of the exogenous activator was 5 pgltube. Ten micrograms of each enzyme preparation was heated for 1 min and then assayed for the activator activity with the phosphodiesterase system as described in the text. The NaCl-washed particulate enzyme after heat denaturation stimulated phosphodiesterase twofold; no activator activity was detected in the other two preparations.
complex (9). However, the possibility that EGTA directly inhibits the enzyme was not excluded. The availability of an apparently activator-free adenylate cyclase offers an experimental system to examine this. As shown in Table II, EGTA markedly suppressed the enzyme activity of the unwashed particles, slightly decreased that of the NaCl-washed particles, but did not alter that of the detergent-washed particles. Since the unwashed particles contained the activator (91, and the detergentwashed particles were activator free, these results demonstrated unequivocally that EGTA inhibited the activity of the holoenzyme and not that of the apoenzyme. The slight inhibition of the NaCl-washed particles was due to the presence of some residual activator (see Table I). It should be noted that the amount of residual activator varied from preparation to preparal
128
LYNCH, TABLE
TALLANT,
II
DIFFERENTIAL EFFECT OF EGTA ON ADENYLATE CYCLASE ACTIVITY OF WASHED AND UNWASHED PARTICLES ‘OF RAT BRAINY Preparation
Unwashed
Addition
particles
NaCl-washed
particles
Detergent-washed titles
par-
None EGTA None EGTA None EGTA
Activity (pmoll mglmin) 597 218 377 267 138 138
a Adenylate cyclase was assayed in the presence or absence of 200 PM EGTA as described in the text. Unwashed and washed particles were prepared as described in the text.
tion, as did the extent of EGTA inhibition on the cyclase activity. Sensitivity to ca2+
of Adenylate
Cyclase Activity
Bradham (25) first demonstrated that brain adenylate cyclase required Ca2+ in addition to Mg2+ for maximum activity. Figure 1A shows the titration of adenylate cyclase with EGTA. The enzyme activity diminished with increasing concentrations of EGTA and, at 40 PM EGTA, the activity was reduced to a basal level. Determination of calcium with atomic absorption spectrophatometry indicated that the majority of the Ca2+ was derived from the reagents. Because of the contamination of Ca2+ in the reaction mixture, a molar excess of EGTA was usually added when the true basal adenylate cyclase was to be determined. To test the sensitivity of adenylate cyclase to a change in Ca2+ concentration, enough EGTA (40 PM) was added to the incubation mixture to chelate the endogenous Ca2+.The abscissa in Fig. 1B refers to the concentration of Ca2+ added to the reaction mixture. The concentration of exogenous Ca2+ giving half-maximum stimulation was about 20 PM and that giving maximum stimulation was about 50 PM. A concentration higher than 200 pM caused inhibition. In this experiment, the activator was present in excess over adenylate cyclase, a condition believed to be prevalent in vivo (6).
AND
CHEUN,G
Effect of Activator on the Kinetic stunts of Adenylate Cyclase
Con-
The effect of the activator on the kinetic constants of adenylate cyclase was analyzed with the Lineweaver-Burk plot shown in Fig. 2. In the presence of the activator, the V was increased about threefold, from 615 to 1729 pmol/mg/min. The apparent Km for ATP, however, remained unchanged at 270 PM. Thus, the primary effect of the activator is to increase the velocity of the enzyme rather than its affmity for the substrate. Effect of Activator ity of Adenylate
on the Thermal
Stabil-
Cyclase Adenylate cyclase activity is unstable, especially at elevated temperatures. The thermal stabilities of the holoenzyme (in the presence of Ca2+ and an exogenous’ activator) and the apoenzyme tin the presence of EGTA) are compared in Fig. 3. The enzyme was preincubated at 47°C for various times before being assayed at 37°C. A
1,000 BOO 600. /1 O ,LG--Em
180 Co”
CONCENTRATION (uMl
Fro. 1. Effect of EGTA (A) and Ca2+ (B) on adenylate cyclase activity. The reaction mixture was supplemented with an exogenous protein activator (5 pg/tube). (A) ‘Ike concentration of EGTA was varied as indicated. (Bl EGTA (40 PM) was added to chelate the endogenous Caz+. The concentrations of Cal+ on the abscissa refer to Caz+ added to the reaction mixture.
ACTIVATOR.Ca2+-DEPENDENT
FIG. 2. Double-reciprocal plot of adenylate cyclase in the presence or absence of the activator. The reaction mixture contained 20 PM Ca2+ in the presence of the activator or 60 FM EGTA in the absence of the activator (5 pg/tube). Mg*+ was kept constant at 5 mM; ATP varied from 10 pM to 2 mM.
ADENYLATE
129
CYCLASE
After 2 min, the holoenzyme lost 20%.of its activity, whereas the apoenzyme lost 40%. Although the difference appeared small, it was reproducible; a similar differential was noted in five experiments using different preparations. Parallel controls were run with the enzyme preincubated with Ca2+but no activator and with both activator and EGTA. The cyclase activities were all less stable than the activity’of the holoenzyme (data not shown). This suggested that the holoenzyme was more thermally stable than the apoenzyme. Stability of an enzyme reflects its conformation; the activator. Ca2+ presumably induced a conformational change on the enzyme, making it more thermally stable. Alternatively, the differential thermal stability may result from the action of a Ca2+-dependent proteolytic enzyme on adenylate cyclase. This possibility appears unlikely as adenylate cyclase was more stable in Ca2+ than in EGTA, contrary to the expectation of a Ca2+-dependent proteolytic enzyme. Additive Effect of Activator Adenylate Cyclase
and F- on
One of the salient features of mammalian adenylate cyclase is its stimulation in vitro by F- (1). Table III shows the activity of adenylate cyclase in the presence of the activator, F-, or both. The activity of the enzyme obtained in the presence of the two agents was comparable to the sum of the FIG. 3. Effect of activator on the thermal stability of adenylate cyclase. The experiment was done in two stages. During the preincubation at 47”C, the reaction mixture (1.0 ml) contained 20 mM Tris-HCl (pH 7.5), 3 mg of protein, and, when present, 100 pg of the activator. In the tube containing the activator, 80 PM Cal+ was present (holoenzyme); in that containing no activator, 400 PM EGTA was present (apoenzyme). At the times indicated, 50 ~1 was transferred to a clean glass tube kept at 0°C. At the end of the thermal treatment, the chilled tubes were transferred to a water bath kept at 37°C and 50 ~1 of the substrate and other reaction components was added to initiate the enzyme reaction. Adenylate cyclase was assayed with 5 fig of activator/tube and 40 pM Ca2+ or with 200 PM EGTA. The data are representative of five experiments using different enzyme preparations.
TABLE ADDITIVE CYCLASE
Additions
None Activator FActivator
III
STIMULATION OF RAT BRAIN ADENYLATE BY THE PROTEIN ACTIVATOR AND BY F-”
+ F-
Adenylate cyclase activity (pmoll mglmin) 436 1602 2302 3435
Increase over basal activity (Pm-$Vwl 1166 1866 2999
c1Adenylate cyclase was assayed in the presence of its activator, F-, or both. Where indicated, NaF was 10 mhi. Exogenous Ca*+ (20 PM) was included when the activator was present; in its absence, EGTA was added to give a final concentration of 100 WM.
130
LYNCH,
TALLANT,
two activities obtained individually with each agent, indicating that the effect of the two agents was additive. This was not unexpected because the mode of action of the two agents appears different: The activator interacts with the enzyme to form an active enzyme. activator complex, whereas F- is believed to act directly on the catalytic subunit (26). In the experiment summarized in Table III, optimal concentrations of F- and the activator were used. In other experiments with a suboptimal concentration of the two ligands, additive effects were also observed (data not shown). Additive Effect of Activator and 5’-Guanylylimidodiphosphate on Adenylate Cyclase
Rodbell et al. (27) first reported that low concentrations of GTP (0.05 to 50 pM) augmented the effect of glucagon on hepatic adenylate cyclase activity. 5’-Guanylylimidodiphosphate, GMP-p(NH)p, a derivative of GTP, stimulated the enzyme even more effectively (28). Londos et al. (29) further showed that the derivative stimulates adenylate cyclase of all mammalian tissues examined. Rat brain adenylate cyclase is also markedly stimulated by low concentrations of the nucleotide; maximum stimulation was obtained at 10 pM GMP-p(NH)p, with 50% stimulation at about 3 FM (data not shown). GTP also stimulated the enzyme, but much less effectively. Table IV compares the effect of GMPp(NH)p with that of the activator. The stimulated activity of adenylate cyclase by GMP-p(NH)p was usually greater than that by the activator. The activity in the presence of optimal concentrations of the two agents was comparable to the summed activities of the two agents assayed individually, indicating that the effect of the two agents was additive. An additive effect was also seen with a suboptimal concentration of the two ligands (data not shown). Lack of Effect of Activator on Brain Particulate Phosphodiesterase
Rat or bovine brain phosphodiesterase is partly soluble and partly particulate (30,
AND
CHEUNG
31). Table V compares the effect of EGTA or the activator on the soluble and particulate brain phosphodiesterase. As shown previously (14), the activator markedly increased the activity of the soluble enzyme; TABLE
IV
ADDITIVE EFFECT OF PROTEIN ACTIVATOR AND GMPp(NH)p ON RAT BRAIN ADENYLATE CYCLAMEN Addition
None Activator GMP-p(NH)p Activator + p(NH)p
Adenylate cyclase activity (pmol/mg/ min)
Increase over basal activity (pmol/mg/ min)
357 730 1426 1919
373 1068 1562
GMP-
a Adenylate cyclase was assayed in the absence or presence of its activator, GMP-p(NH)p, or both. The concentration of the protein activator was 5 pg/ tube and that of GMP-p(NH)p was 100 PM. Exogenous Ca2+ (50 PM) was included when the activator was present. However, EGTA was not added to the control tube because preliminary experiments showed that this enzyme preparation contained very little residual activator, and the cyclase activity was essentially notkhibited by EGTA. TABLE
V
LACK OF EFFECT OF PROTEIN ACTIVATOR ON BRAIN PARTICULATE PHOSPHODIESTERASE~ Experiment
Additions
Soluble phosphodiesterase activity (nmol/fractionlmin)
Particulate phosphodiesterase activity (nmol/fractionlmin)
1
None EGTA Activator
17 ND 49
20 22 21
2
None EGTA Activator
73 77 298
140 129 142
a A particulate and a soluble phosphodiesterase from rat or bovine brain were assayed in the presence or absence of an exogenous protein activator or EGTA. The concentration of the activator was 5 pg/ tube and that of EGTA was 200 PM. The soluble enzyme was purified to the stage of the DEAEcellulose column chromatography and was activator deficient (14). The particulate phosphodiesterase is the same.NaCl-washed particles used as a source of activator-deficient aclenylate cyclase. Experiment 1 and Experiment 2 used rat and bovine brain, respectively. ND, not determined.
ACTIVATOR.Ca2+-DEPENDENT
ADENYLATE
CYCLASE
131
probably more Ca2+is available to adenylate cyclase in the plasma membrane than to phosphodiesterase in the cytoplasm. Since the activator confers stability to adenylate cyclase against thermal inactivation, it is believed that the apoenzyme, when it forms an enzyme *activator complex, assumes a different conformation. Based on the results here and elsewhere, we may conclude that the sequence of reactions depicting the stimulation of brain cytoplasmic phosphodiesterase by the protein activator, shown by Eqs. 111and [2], applies also to membrane adenylate cyclase. Stimulation of adenylate cyclase by Fand by GMP-p(NH)p is comparable in several respects. First, the two ligands actiDISCUSSION vate adenylate cyclase. in all mammalian Previous studies showed that a Ca2+- tissues (26, 29); second, stimulation is greater in broken cell preparations than in binding protein activator stimulated brain intact cells (26, 35); third, under certain adenylate cyclase which was solubilized by Lubrol-PX (3, 4). The present studies conditions, stimulation appears irreversible (26, 35). On the other hand, F- appears demonstrate that the activator also stimulates the enzyme associated with the mem- to act directly on the catalytic subunit of branes. A detergent-solubilized adenylate adenylate cyclase, whereas GMP-p(NH)p cyclase is apparently uncoupled from its interacts with a specific regulatory subhormone receptor (32); since the activator unit distinct from the hormone receptor stimulates both the particulate and the (36). The additive effect of the activator detergent-solubilized enzyme, it follows and F- or GMP-p(NH)p suggests that the that the activator interacts with the apo- action of the activator is independent of enzyme independent of the hormone re- that of the two ligands. ceptor. It does not necessarily follow, howIn comparing the stimulation of adenylever, that the effect of the activator is ate cyclase by the activator with that of divorced from that of the hormone. Brain cytoplasmic phosphodiesterase, several saadenylate cyclase is usually not very re- lient features may be noted. First, formasponsive to neurohormones (33). tion of the enzyme *activator complex is The mechanism of stimulation of ade- Ca2+ dependent, although the two enzyme nylate cyclase and that of phosphodiestersystems may display different sensitivities ase by the protein activator appear to be toward Ca2+. Second, the primary effect of qualitatively identical: In the presence of the activator was on the V of the reaction Ca2+, the activator interacts with the and not on the apparent K, of the subapoenzyme to form an active en- strate. Thus, both enzymes may be classizyme *activator complex, and removal of fied as “V type” (37). Third, although the Ca2+ dissociates the complex into its com- activator affects the thermal stability of ponents. With a saturating concentration the holoenzyme, the effects on the two sysof the activator, the concentrations of Ca2+ tems were opposite: Adenylate cyclase was needed to give half-maximum stimulation more stable whereas phosphodiesterase of adenylate cyclase and phosphodiesterwas less stable in the presence of the actiase were about 20 (Fig. 1B) and 5 ,UM (13), vator (5, 38, 39). respectively. The concentration of free The stimulation of adenylate cyclase Ca2+ in the cytoplasm is about lO-'j M. and cytoplasmic phosphodiesterase by the Following depolarization, there is an in- same Ca2+-binding protein raises the flux of Ca2+ (34) into the nerve cell and question of its biological utility. In analogy
in contrast, it did not alter the activity of the particulate enzyme. The particulate phosphodiesterase had been washed exhaustively with a hypertonic NaCl-EGTA solution to remove the endogenous activator and was the same preparation used for adenylate cyclase, which was stimulated by an exogenous activator. Moreover, EGTA did not inhibit the activity of particulate phosphodiesterase. These results show that the particulate phosphodiesterase is activator independent. The response of the soluble and not the particulate phosphodiesterase to the activator is of particular interest in considering the brain metabolism of CAMP relative to the cellular flux of Ca2+ (see Discussion).
132
LYNCH,
TALLANT,
to the scheme depicted for phosphodiesterase, the activity of adenylate cyclase may be regulated by the cellular flux of Ca2+ through the plasma membrane or the release of membrane-associated Ca2+ in response to stimuli, resulting in an increase in intracellular CAMP. Ca2+ subsequently arriving in the cytosol stimulates the cytoplasmic phosphodiesterase, which then returns the elevated intracellular CAMP level to its prestimulated level. The sequential stimulation of adenylate cyclase and cytoplasmic phosphodiesterase would allow a transient elevation of cellular CAMP in response to stimuli. On the other hand, the cytoplasmic phosphodiesterase also catalyzes the hydrolysis of cGMP; in fact, at a micromolar concentration of the substrates, the rate of cGMP hydrolysis is greater than that of CAMP. The cellular flux of Ca2+, therefore, could cause an increase of CAMP or a concomitant decrease of cGMP. Since the cytoplasmic but not the membrane phosphodiesterase is responsive to the Ca2+/activator, the two enzymes may play complimentary roles. The membrane enzyme in close proximity to adenylate cyclase may be important in preventing an excessive accumulation of CAMP in or adjacent to the plasma membrane, whereas the cytoplasmic enzyme may be particularly crucial when the level of CAMP is elevated following stimulation of the nerve cells. REFERENCES 1. ROBISON, G. A., BUTCHER, R. W., AND SUTHERLAND, E. W. (1971) Cyclic AMP, Academic Press, New York. 2. CHEUNG, W. Y. (1967) B&hem. Biophys. Res. Commun. 29, 478-482. 3. CHEUNG, W. Y., BRADHAM, L. S., LYNCH, T. J., LIN, Y. M., AND TALLANT, E. A. (1975) Biothem. Biophys. Res. Conmun. 66, 1055-1062. 4. BROBTROM, C. O., HUANG, Y. C., BRECKENRIDGE, B. McL., AND WOLFF, D. J. (1975)Proc. Nat. Acad. Sci. USA 72, 64-68. 5. LIU, Y. P., AND CHEUNG, W. Y. (1976) J. Biol. Chem. 251, 4193-4198. 6. LYNCH, T. J., TALLANT, E. A., AND CHEUNG, W. Y. (1976) Biochem. Biophys. Res. Commun. 68, 616-625. 7. LIN, Y. M., LIU, Y. P., AND CHEUNG, W. Y. (1975) FEBS Lett. 49, 356-360.
AND
CHEUNG
8. TESHIMA, Y., AND KAKIUCHI, S. (1974) Biochem. Biophys. Res. Commun. 56, 489-495. 9. LYNCH, T. J., TALLANT, E. A., AND CHEUNG, W. Y. (1976) Fed. Proc. 35, 1633 (abstr.). 10. KRISHNA, G., WEISS, B., AND BRODIE, G. J. (1968) Pharmacol. Exp. Ther. 163, 379-385. 11. LYNCH, T. J., TALLANT, E. A., ANDCHEUNG, W. Y. (1975) Biochem. Biophys. Res. Commun. 65, 1115-1122. 12. THOMPSON, W. J., AND APPLEMAN, M. J. (1971) Biochemistry 10, 311-316. 13. LIN, Y. M., LIU, Y. P., AND CHEUNG, W. Y. (1974) J. Biol. Chem. 249, 4943-4954. 14. CHEUNG, W. Y. (1971) J. Biol. Chem. 246, 28592869. 15. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 16. WARBURG, O., AND CHRISTIAN, W. (1941) Bio&em. Z. 310, 384-421. 17. NEER, E. J. (1973) J. BioZ. Chem. 248,3742-3744. 18. MIDDLEMIBS, D. N., AND FRANKLIN, T. J. (1975) FEBS Lett. 55, 225-228. 19. PERKINS, J. P., AND MOORE, M. M. (1971) J. Biol. Chem. 246,62-68. 20. DYE, I., ANDSUTHERLAND, E. W. (1966)Bioehim. Biophys. Acta 127, 347-354. 21. JOHNSON, R. A., AND SUTHERLAND, E. W. (1973) J. Biol. Chem. 248, 5114-5121. 22. BROSTROM, M. A., BROBTROM, C. A., BRECKENRIDGE, B. M., AND WOLFF, D. J. (1976) J. Biol. Chem. 251, 4744-4750. 23. LIN, Y. M. (1973) Doctoral dissertation, The University of Tennessee Center for the Health Sciences, Memphis, Tennessee. 24. HOLLOWAY, J. H., AND REILLEY, C. N. (1960) Anal. Chem. 32, 249-256. 25. BRADHAM, L. S. (1972) Biochim. Biophys. Acta 276, 434-443. 26. PERKINS, J. P. (1973) Advan. Cyclic Nucleotide Res. 3, l-64. 27. RODBELL, M., KRANS, H. M. J., POHL, S. L., AND BIRNBAUMER, L. (1971) J. Biol. Chem. 246, 1872-1876. 28. HARWOOD, J. P., Liiw, H., AND RODBELL, M. (1973) J. Biol. Chem. 248, 6239-6245. 29. LONDOS, C., SOLOMON, Y., LIN, M. C., HARWOOD, J. P., SCHRAMM, M., WOLFF, J., AND RODBELL, M. (1974) Proc. Nat. Acad. Sci. USA, 71, 3087-3090. 30. CHEUNG, W. Y., AND SALGANICOFF, L. (1967) Nature (London) 214, 90-91. 31. DE ROBERTIS, E., ARNAIZ, G. R. D. L., ALBERICI, A., BUTCHER, R. W., AND SUTHERLAND, E. W. (1967) J. Biol. Chem. 242, 3487-3493. 32. PUCHWEIN, G.‘, PFEUFFER, T., AND HELMREICH, E. J. M. (1974) J. Biol. Chem. 249, 3232-3240. 33. KLAINER, L. M., CHI, Y. M., FRIEDBERG, S. L.,
ACTIVATOR*Ca2+-DEPENDENT ROLL, T. W., AND SUTHERLAND, E. W. (1962) J. Biol. Chem. 237, 1239-1243. 34. BAKER, P. F., HODGKIN, A. L., ANDRIDGWAY, E. B. (1971) J. Physiol. 218, 709-755. 35. LEFKOWITZ, R. J., AND CARON, M. G. (1975) J. Biol. Chem. 205, 4418-4422. 36. PFEUFFER, T., AND HELMREICH, E. J. M. (1975) J. Biol. Chem. 250, 867-876.
ADENYLATE
CYCLASE
133
37. MONOD, J., WYMAN, J., AND CHANGEUX, J.-P. (1965) J. Mol. Biol. 12, 88-118. 38. WANG, J. H., TEO, T. S., Ho, H. C., AND STEVENS, F. C. (1975) Advan. Cyclic Nucleotide Res. 5, 179-194. 39. KAKIUCHI, S., YAMAZAKI, R., TEBHIMA, Y., MENISI, K., AND MIYAMOTO, E. (1975) Aduan. Cyclic Nucleotide Res. 5, 163-178.
OF
BIOCkEYISTRY
AND
BIOPHYSICS
182,
124-133 (I9771
Rat Brain Adsnylate Further THOMAS Department
Studies
on Its Stimulation
J. LYNCH,
of Biochemistr$,
E. ANN
Cyclase by a Ca2+-Binding
TALLANT,
AND
WA1 YIU
St. Jude Children’s Research Hospital and University the Health Sciences, Memphis, Tennessee 38101 Received January
Protein* CHEUNG of Tennessee Center for
5, 1977
Bovine or rat brain adenylate cyclase (EC 4.6.1.1) solubilized by Lubrol-PX, a nonionic detergent, requires a Ca*+-binding protein activator for full activity (Cheung et al., 1975, Biochem. Biophys. Res. Commun. 66,1055-1062). We now show that particulate rat brain adenylate cyclase also required the activator for maximum activity. A brain particulate fraction was extracted with a hypertonic NaCl solution containing [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. This procedure removed preferentially the activator, making adenylate cyclase activator deficient and, consequently, dependent on an exogenous activator for maximum activity. The activator increased the V of adenylate cyclase without affecting’its apparent K, for ATP. In the presence of the activator, the enzyme was more stable against thermal inactivation, suggesting that the activator probably induced a conformational change to the enzyme. F- and 5’-guanylylimidodiphosphate [GMP-p(NH)p] greatly stimulated brain adenylate cyclase. Adenylate cyclase activity obtained in the presence of the activator and F- was comparable to the summed activities of the two agents assayed separately, indicating that their effects were additive. Similarly, the effects of the activator and GMP-p(NH)p were additive. These results suggest that the action of the activator is independent of the other two ligands. Since the activator is present in excess over adenylate cyclase, the cellular flux of CaZ+ is believed to be important in modulating the enzyme activity. The role of the Cal+/ activator is discussed with respect to cyclic AMP metabolism in brain.
Although CAMP’ is known to mediate the action of many polypeptide hormones and catecholamines, the mechanism by which its intracellular level is regulated on a moment-by-moment basis is not yet understood. The effect of CAMP is rapid and the increase of CAMP in target cells in response to a stimulus is usually shortlived, often appearing as a peak response (1). There are two enzymes or enzyme sys-
terns that control the tissue level of the cyclic nucleotide. Adenylate cyclase (EC 4.6.1.1), associated with the plasma membrane, catalyzes the formation of CAMP, and cyclic 3’,5’-nucleotide phosphodiesterase (EC 3.1.4.17), partly particulate and partly soluble, catalyzes its degradation. An endogenous Ca2+-binding protein activator first discovered for bovine brain phosphodiesterase (2) also stimulates brain adenylate cyclase (3, 4). In the presence of Ca2+, the activator assumes a more helical structure and combines with the apoenzyme to form an enzyme * activator complex (5). Lowering the Ca2+ concentration dissociates the two proteins and returns the enzyme activity to its basal level. Stimulation of adenylate cyclase by the activator is immediate and reversible and appears similar to that found with phosphodiesterase (6-8). The sequence of
t This work was supported by a grant from the U.S. Public Health Service (NS080591 and by a grant-in-aid from the American Cancer Society (ACS IN 990 1 Abbreviations used: CAMP, cyclic AMP; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; DEAE, diethylaminoethyl; DTT, dithiothreitol; GMP-p(NH)p, 5’guanylylimidodiphosphate; ECTEOLA-cellulose, epichlorohydrin triethanolamine cellulose. 124 Copyright
8 1977 by Academic
Press, Inc.
All rights of reproduction in any form reserved.
ISSN 0003-9861
ACTIVATOR.Ca2+-DEPENDENT reactions leading to activation picted as follows. (Activator)
+ Ca*+ inactive
(Activator*.
ADENYLATE
may be deCa*+) active
(Enzyme) + (Activator*. Ca*+) + active less active (Enzyme*. Activator*. Ca*+) active
HI
M
The asterisk depicts a different conformation. The stoichiometry of the components in these equations has not been established; the equations simply designate the interaction of these components. Stimulation of adenylate cyclase by the activator was demonstrated with a LubrolPX-solubilized preparation (3, 4). Since adenylate cyclase is associated with the plasma membrane, and the detergent-solubilized enzyme may have properties different from those of the membrane enzyme, it is important to demonstrate the same effect directly on the membrane enzyme in the absence of the detergent. In this communication we describe the preparation of a particulate adenylate cyclase without the use of a detergent and largely free of its endogenous activator, the stimulation of the particulate enzyme by the activator, and its effect on the stimulation of adenylate cyclase by F- or by 5’guanylylimidodiphosphate. A preliminary note has appeared (9). EXPERIMENTAL
PROCEDURES
Chemicals and reagents. [3H]ATP (specific activity, 18 Ci/mmol) and r3H]cAMP (specific activity, 27 Ci/mmol) were obtained from Schwarz/Mann. Amberlite IRP-58 (260-400 mesh), a phenolic polyamine, was a generous gift from Dr. Anthony Pitochelli of Rohm and Haas. The resin was washed sequentially with 0.25 N NaOH, H20, 0.5 N HCl, and then exhaustively with H,O. The resin was suspended in H,O to give a 33% slurry (1 vol of resin to 2 vol of H,O). Lubrol-PX, pyruvate kinase, phosphoenolpyruvate, and ECTEOLA-cellulose were obtained from Sigma. Dowex 5OW-X8 (ZOO-400 mesh) was obtained from either Sigma or Bio-Rad. It was washed with 0.5 N NaOH, H20, 0.5 N HCl, and then exhaustively with H,O. The resin was suspended in 1 mM HCl. 5’-Guanylylimidodiphosphate [GMPp(NH)p] was obtained from ICN and 3-isobutyl-lmethylxanthine was from Aldrich Chemical Company. All chemicals were of reagent grade. Preparation of activator-deficient particulate ade-
CYCLASE
125
nylate cyclase. Step A: Sprague-Dawley rata weighing 160-180 g were decapitated and the whole brains were removed. They were either used fresh or stored at -90°C. The tissue was homogenized in a glass homogenizer with a Teflon pestle in 10 v01 of 20 mM Tris-HCl, pH 7.5 (Buffer A). The homogenate was centrifuged at 1OOOgfor 15 min. The precipitate was homogenized in 10 vol of Buffer A and was collected by centrifugation at 1OOOgfor 15 min. This step was repeated twice. The preparation was designated as unwashed particles. Step B: The sediment from Step A was further washed in 10 vol of 20 mM Tris-HCl (pH 7.5) containing 1 M NaCl and 2 mM EGTA (Buffer B). The suspension was centrifuged at 15,000g for 10 min. The washing was repeated twice. Step C: To remove the NaCl and EGTA, the pellet was then washed twice with 20 mM Tris-HCl (pH 7.5) containing 3 mM DTT (Buffer C). After the second wash, the pellet was resuspended in Buffer C to a final protein concentration of 5-10 mg/ml. The yield of washed particles from 1 g of brain tissue was lo-20 mg of protein. The sample was divided into small fractions which were stored at -90°C. Adenylate cyclase in this preparation was activator delicient and required an exogenous activator for maximum activity. The extent of stimulation was two- to threefold. Unless otherwise stated, all experiments were performed using this preparation. Alternatively, the sediment from Step A was washed four times in 10 vol of 20 mM Tris-HCl (pH 7.5), 3 mM DTT, 1 mM EGTA, and 0.05% Lubrol-PX (Buffer D). The rest of the procedure was as described in the preceding paragraph. Buffer D was more effective in removing the activator; the stimulation of detergent-washed adenylate cyclase by an exogenous activator was usually more than fivefold. Assay of adenylate cyclase. Crude preparations of adenylate cyclase were contaminated with CAMP phosphodiesterase and ATPase. The assay system, therefore, contained 3-isobutyl-1-methylxanthine and caffeine to inhibit phosphodiesterase, carrier CAMP to protect the newly synthesized [3H]cAMP from degradation, and an ATP regeneration system to counteract the effect of ATPas’e. The reaction mixture in a final volume of 0.1 ml contained 40 mM Tris-HCl (pH 7.5), 2 mM 3-isobutyl-l-methylxanthine, 40 mM caffeine, 5 mM MgC12, 2 to 4 mM CAMP, 1 mM 13HlATP (500,000 cpm/tube), 80-140 pg of protein, 4 mM phosphoenolpyruvate, and 40 pg/ml of pyruvate kinase. The final concentration of D’lT in the incubation ranged from 0.6 to 1.5 mM. When present, the concentration of the exogenous activator was 5 pg of protein/tube. Other additions are indicated in the legends. The reaction was usually started by the addition of the enzyme. At the end of the incubation at 37”C, usually lasting 10 min, the reaction was terminated by adding 50 ~1 of 1 N HCl. The reaction mixture was neutralized with 300 ~1 of
126
LYNCH,
TALLANT,
0.233 M Tris. Unreacted ATP was precipitated by the sequential additions of 80 ~1 each of 0.25 M ZnSOl and 0.25 M Ba(OH), (10). The reaction mixture was quantitatively transferred to a Dowex BOW-X8 cationic exchange resin column (6.5 x 6 cm), which was washed with 6 ml of 1 mM HCl. The eluant was discarded. The column was eluted with 3.5 ml of H,O and the eluant was collected into a scintillation vial. Cyclic AMP recovery was monitored photometrically at 256 nm; the recovery was 60-70%. The eluant was evaporated to dryness at 80°C. The dried sample was taken up in 0.4 ml of H,O. Seven milliliters of Bray’s solution was added for counting. All data were corrected for their CAMP recoveries. The background count was less than 0.1% of the amount of radioisotope added initially. The specific activity of adenylate cyclase is defined as picomoles of CAMP formed per milligram of protein per minute at 37°C under the assay conditions. All determinations were done in duplicate and were validated several times with different enzyme preparations. Assay of cyclic nucleotide phosphodiesterase. Phosphodiesterase was assayed by a two-stage procedure (11) modified after Thompson and Appleman (12). The reaction mixture contained 40 mM TrisHCl (pH 8.0), 5 mr+i MgCIZ, 50 PM CaCl,, 1 mM 13H]cAMP, and an appropriate amount of enzyme in a total volume of 0.1 ml. The reaction was initiated by the addition of substrate. After 10 min at 3O”C, the reaction was terminated by placing the tubes in a boiling water bath for 30 s. The tubes were cooled to 30°C and 20 ~1 of snake venom (Crotalus atrox, 1.0 mg/ml), as a source of 5’-nucleotidase to convert 5’AMP to adenosine, was added to start the second incubation. After 10 min, 1 ml of an IRP-58 slurry was added to terminate the reaction and the tubes were centrifuged. The resin binds the unreacted CAMP and little or no adenosine. A fraction of the supernatant fluid, which contained 13H1adenosine, was counted in a liquid scintillation spectrometer. Preparation of activator. A Ca2+-binding protein activator was purified from bovine brain to the stage of DEAE-cellulose column chromatography (13). Assay of activator. The activator was measured by its ability to stimulate an activator-deficient phosphodiesterase purified from bovine brain (14). Briefly, a sample to be tested was heated at 95°C for 1 min to inactivate phosphodiesterase and then assayed for the activator in a reaction mixture containing 40 mM Tris-HCl (pH 8.0), 5 mM MgC12, 50 PM CaCl,, 7 pg of activator-deficient phosphodiesterase, the heated sample, and 1 mM 13H1cAMP. The remaining procedure and assay were identical to that of phosphodiesterase in the preceding section. Determination ofprotein. Protein was determined according to Lowry et al. (15) with bovine serum albumin as a standard or according to Warburg and Christian (16).
AND
CHEUNG RESULTS
Preparation of an Activator-Deficient ticulate Adenylate Cyclase
Par-
Although stimulation of brain adenylate cyclase by the Ca2+-binding protein, was previously demonstrated with a detergentsolubilized preparation, it was deemed important to show the same activator response on the membrane enzyme prepared without the use of a detergent. Adenylate cyclase is associated with the plasma enmembrane; a detergent-solubilized zyme may have properties quite different from those of the enzyme associated with the membrane. Moreover, detergents affect adenylate cyclase activity in a variable manner (17-221, which may mask or distort the real effect of the activator. Therefore, we first set out to prepare a particulate adenylate cyclase that is responsive to the activator. Two observations are relevant in this regard. First, solubilization of adenylate cyclase by a detergent facilitates the dissociation of the endogenous activator from the enzyme in an anionic exchange column. The activator, being an acidic protein, was retained by the column and was clearly separated from the enzyme, which emerged with the void volume of the column virtually free of the activator (3, 4). Separation of the activator from phosphodiesterase was achieved similarly (14). Second, EGTA suppressed adenylate cyclase and phosphodiesterase activity to a basal level by dissociating the enzymeactivator complex (6-8). A hypertonic salt solution also depressed phosphodiesterase to its basal level, presumably by dissociating the enzyme *activator complex (23). Preliminary experiments suggested that brain adenylate cyclase was more tightly associated with a membranous fraction than was the activator. We reasoned that washing the membranes with a hypertonic salt solution would remove preferentially the activator. A brain particulate fraction was washed with 20 mM Tris-HCl (pH 7.5) containing 1 M NaCl and 2 mM EGTA. After two to three washes, all removable activator appeared to have been extracted from the membranes. The removal of the activator made the washed particulate en-
ACTIVATOR.
Ca2+-DEPENDENT
zyme activator deficient and dependent on an exogenous activator for full activity (Table I). However, the extent of stimulation was relatively small compared to that obtained with the detergent-solubilized enzyme. The apparently smaller stimulation was presumably due to a less effective removal of the endogenous activator from the particles by the hypertonic solution, as there was some residual activator in the washed particles but not in the detergentsolubilized enzyme. Alternatively, brain particles were washed with a buffer solution containing 20 mM Tris-HCl (pH 7.51, 3.0 mM DTT, 1 mM EGTA, and 0.05% Lubrol:PX. This medium removed essentially all of the endogenous activator, as the final washed particles showed no detectable activator activity. Adenylate cyclase activity in this preparation was higher than that of the detergent-solubilized enzyme and the extent of stimulation was comparable. The specific activities of both the detergentsolubilized and the detergent-washed enzymes were lower than the specific activity of the NaCl-washed particles. These results demonstrated that the endogenous activator could be removed from membrane adenylate cyclase without the use of a detergent and that the activatordeficient membrane enzyme was responsive to an exogenous activator. Although the extent of stimulation was relatively small, this was not an inherent characteristic of the particulate enzyme, since it could be made to respond as avidly as the detergent-solubilized enzyme, provided that the endogenous activator was effectively removed, for example, by washing the enzyme with a low concentration of Lubrol-PX. Differential Effect of EGTA on Adenylate Cyclase Activity of Holoenzyme or Apoenzyme EGTA, which has a greater affinity for Ca2+ than for Mg2+ (24), inhibits markedly adenylate cyclase activity; the inhibition is fully reversed by a molar excess of Ca2+ (25). EGTA inhibits the enzyme presumably by chelating the Ca2+ required for the formation of the active enzyme. activator
ADENYLATE
127
CYCLASE TABLE
I
STIMULATION OF RAT BRAIN ADENYLATE CYCLASE BY A C$+-BINDING PROTEIN ACTIVATORS Preparation
Addition
Adenylate CYclase (P,mgoil/
Residual activator
min) NaCl-washed partitles Detergent-washed particles Detergent-solubilized enzyme
None Activator None Activator None Activator
579 1602 173 1073 76 485
(+I (-) (-)
a Adenylate cyclase activity was determined in the presence or absence of an exogenous activator purified from bovine brain (13). It has been established that the activator lacks species specificity and that the activator from bovine brain cross-reacts with adenylate cyclase from rat brain, and vice versa (14). The NaCl- or detergent-washed particles were prepared as described under Experimental Procedures. The detergent-solubilized enzyme was prepared from an ECTEOLA-cellulose column (3). The concentration of the exogenous activator was 5 pgltube. Ten micrograms of each enzyme preparation was heated for 1 min and then assayed for the activator activity with the phosphodiesterase system as described in the text. The NaCl-washed particulate enzyme after heat denaturation stimulated phosphodiesterase twofold; no activator activity was detected in the other two preparations.
complex (9). However, the possibility that EGTA directly inhibits the enzyme was not excluded. The availability of an apparently activator-free adenylate cyclase offers an experimental system to examine this. As shown in Table II, EGTA markedly suppressed the enzyme activity of the unwashed particles, slightly decreased that of the NaCl-washed particles, but did not alter that of the detergent-washed particles. Since the unwashed particles contained the activator (91, and the detergentwashed particles were activator free, these results demonstrated unequivocally that EGTA inhibited the activity of the holoenzyme and not that of the apoenzyme. The slight inhibition of the NaCl-washed particles was due to the presence of some residual activator (see Table I). It should be noted that the amount of residual activator varied from preparation to preparal
128
LYNCH, TABLE
TALLANT,
II
DIFFERENTIAL EFFECT OF EGTA ON ADENYLATE CYCLASE ACTIVITY OF WASHED AND UNWASHED PARTICLES ‘OF RAT BRAINY Preparation
Unwashed
Addition
particles
NaCl-washed
particles
Detergent-washed titles
par-
None EGTA None EGTA None EGTA
Activity (pmoll mglmin) 597 218 377 267 138 138
a Adenylate cyclase was assayed in the presence or absence of 200 PM EGTA as described in the text. Unwashed and washed particles were prepared as described in the text.
tion, as did the extent of EGTA inhibition on the cyclase activity. Sensitivity to ca2+
of Adenylate
Cyclase Activity
Bradham (25) first demonstrated that brain adenylate cyclase required Ca2+ in addition to Mg2+ for maximum activity. Figure 1A shows the titration of adenylate cyclase with EGTA. The enzyme activity diminished with increasing concentrations of EGTA and, at 40 PM EGTA, the activity was reduced to a basal level. Determination of calcium with atomic absorption spectrophatometry indicated that the majority of the Ca2+ was derived from the reagents. Because of the contamination of Ca2+ in the reaction mixture, a molar excess of EGTA was usually added when the true basal adenylate cyclase was to be determined. To test the sensitivity of adenylate cyclase to a change in Ca2+ concentration, enough EGTA (40 PM) was added to the incubation mixture to chelate the endogenous Ca2+.The abscissa in Fig. 1B refers to the concentration of Ca2+ added to the reaction mixture. The concentration of exogenous Ca2+ giving half-maximum stimulation was about 20 PM and that giving maximum stimulation was about 50 PM. A concentration higher than 200 pM caused inhibition. In this experiment, the activator was present in excess over adenylate cyclase, a condition believed to be prevalent in vivo (6).
AND
CHEUN,G
Effect of Activator on the Kinetic stunts of Adenylate Cyclase
Con-
The effect of the activator on the kinetic constants of adenylate cyclase was analyzed with the Lineweaver-Burk plot shown in Fig. 2. In the presence of the activator, the V was increased about threefold, from 615 to 1729 pmol/mg/min. The apparent Km for ATP, however, remained unchanged at 270 PM. Thus, the primary effect of the activator is to increase the velocity of the enzyme rather than its affmity for the substrate. Effect of Activator ity of Adenylate
on the Thermal
Stabil-
Cyclase Adenylate cyclase activity is unstable, especially at elevated temperatures. The thermal stabilities of the holoenzyme (in the presence of Ca2+ and an exogenous’ activator) and the apoenzyme tin the presence of EGTA) are compared in Fig. 3. The enzyme was preincubated at 47°C for various times before being assayed at 37°C. A
1,000 BOO 600. /1 O ,LG--Em
180 Co”
CONCENTRATION (uMl
Fro. 1. Effect of EGTA (A) and Ca2+ (B) on adenylate cyclase activity. The reaction mixture was supplemented with an exogenous protein activator (5 pg/tube). (A) ‘Ike concentration of EGTA was varied as indicated. (Bl EGTA (40 PM) was added to chelate the endogenous Caz+. The concentrations of Cal+ on the abscissa refer to Caz+ added to the reaction mixture.
ACTIVATOR.Ca2+-DEPENDENT
FIG. 2. Double-reciprocal plot of adenylate cyclase in the presence or absence of the activator. The reaction mixture contained 20 PM Ca2+ in the presence of the activator or 60 FM EGTA in the absence of the activator (5 pg/tube). Mg*+ was kept constant at 5 mM; ATP varied from 10 pM to 2 mM.
ADENYLATE
129
CYCLASE
After 2 min, the holoenzyme lost 20%.of its activity, whereas the apoenzyme lost 40%. Although the difference appeared small, it was reproducible; a similar differential was noted in five experiments using different preparations. Parallel controls were run with the enzyme preincubated with Ca2+but no activator and with both activator and EGTA. The cyclase activities were all less stable than the activity’of the holoenzyme (data not shown). This suggested that the holoenzyme was more thermally stable than the apoenzyme. Stability of an enzyme reflects its conformation; the activator. Ca2+ presumably induced a conformational change on the enzyme, making it more thermally stable. Alternatively, the differential thermal stability may result from the action of a Ca2+-dependent proteolytic enzyme on adenylate cyclase. This possibility appears unlikely as adenylate cyclase was more stable in Ca2+ than in EGTA, contrary to the expectation of a Ca2+-dependent proteolytic enzyme. Additive Effect of Activator Adenylate Cyclase
and F- on
One of the salient features of mammalian adenylate cyclase is its stimulation in vitro by F- (1). Table III shows the activity of adenylate cyclase in the presence of the activator, F-, or both. The activity of the enzyme obtained in the presence of the two agents was comparable to the sum of the FIG. 3. Effect of activator on the thermal stability of adenylate cyclase. The experiment was done in two stages. During the preincubation at 47”C, the reaction mixture (1.0 ml) contained 20 mM Tris-HCl (pH 7.5), 3 mg of protein, and, when present, 100 pg of the activator. In the tube containing the activator, 80 PM Cal+ was present (holoenzyme); in that containing no activator, 400 PM EGTA was present (apoenzyme). At the times indicated, 50 ~1 was transferred to a clean glass tube kept at 0°C. At the end of the thermal treatment, the chilled tubes were transferred to a water bath kept at 37°C and 50 ~1 of the substrate and other reaction components was added to initiate the enzyme reaction. Adenylate cyclase was assayed with 5 fig of activator/tube and 40 pM Ca2+ or with 200 PM EGTA. The data are representative of five experiments using different enzyme preparations.
TABLE ADDITIVE CYCLASE
Additions
None Activator FActivator
III
STIMULATION OF RAT BRAIN ADENYLATE BY THE PROTEIN ACTIVATOR AND BY F-”
+ F-
Adenylate cyclase activity (pmoll mglmin) 436 1602 2302 3435
Increase over basal activity (Pm-$Vwl 1166 1866 2999
c1Adenylate cyclase was assayed in the presence of its activator, F-, or both. Where indicated, NaF was 10 mhi. Exogenous Ca*+ (20 PM) was included when the activator was present; in its absence, EGTA was added to give a final concentration of 100 WM.
130
LYNCH,
TALLANT,
two activities obtained individually with each agent, indicating that the effect of the two agents was additive. This was not unexpected because the mode of action of the two agents appears different: The activator interacts with the enzyme to form an active enzyme. activator complex, whereas F- is believed to act directly on the catalytic subunit (26). In the experiment summarized in Table III, optimal concentrations of F- and the activator were used. In other experiments with a suboptimal concentration of the two ligands, additive effects were also observed (data not shown). Additive Effect of Activator and 5’-Guanylylimidodiphosphate on Adenylate Cyclase
Rodbell et al. (27) first reported that low concentrations of GTP (0.05 to 50 pM) augmented the effect of glucagon on hepatic adenylate cyclase activity. 5’-Guanylylimidodiphosphate, GMP-p(NH)p, a derivative of GTP, stimulated the enzyme even more effectively (28). Londos et al. (29) further showed that the derivative stimulates adenylate cyclase of all mammalian tissues examined. Rat brain adenylate cyclase is also markedly stimulated by low concentrations of the nucleotide; maximum stimulation was obtained at 10 pM GMP-p(NH)p, with 50% stimulation at about 3 FM (data not shown). GTP also stimulated the enzyme, but much less effectively. Table IV compares the effect of GMPp(NH)p with that of the activator. The stimulated activity of adenylate cyclase by GMP-p(NH)p was usually greater than that by the activator. The activity in the presence of optimal concentrations of the two agents was comparable to the summed activities of the two agents assayed individually, indicating that the effect of the two agents was additive. An additive effect was also seen with a suboptimal concentration of the two ligands (data not shown). Lack of Effect of Activator on Brain Particulate Phosphodiesterase
Rat or bovine brain phosphodiesterase is partly soluble and partly particulate (30,
AND
CHEUNG
31). Table V compares the effect of EGTA or the activator on the soluble and particulate brain phosphodiesterase. As shown previously (14), the activator markedly increased the activity of the soluble enzyme; TABLE
IV
ADDITIVE EFFECT OF PROTEIN ACTIVATOR AND GMPp(NH)p ON RAT BRAIN ADENYLATE CYCLAMEN Addition
None Activator GMP-p(NH)p Activator + p(NH)p
Adenylate cyclase activity (pmol/mg/ min)
Increase over basal activity (pmol/mg/ min)
357 730 1426 1919
373 1068 1562
GMP-
a Adenylate cyclase was assayed in the absence or presence of its activator, GMP-p(NH)p, or both. The concentration of the protein activator was 5 pg/ tube and that of GMP-p(NH)p was 100 PM. Exogenous Ca2+ (50 PM) was included when the activator was present. However, EGTA was not added to the control tube because preliminary experiments showed that this enzyme preparation contained very little residual activator, and the cyclase activity was essentially notkhibited by EGTA. TABLE
V
LACK OF EFFECT OF PROTEIN ACTIVATOR ON BRAIN PARTICULATE PHOSPHODIESTERASE~ Experiment
Additions
Soluble phosphodiesterase activity (nmol/fractionlmin)
Particulate phosphodiesterase activity (nmol/fractionlmin)
1
None EGTA Activator
17 ND 49
20 22 21
2
None EGTA Activator
73 77 298
140 129 142
a A particulate and a soluble phosphodiesterase from rat or bovine brain were assayed in the presence or absence of an exogenous protein activator or EGTA. The concentration of the activator was 5 pg/ tube and that of EGTA was 200 PM. The soluble enzyme was purified to the stage of the DEAEcellulose column chromatography and was activator deficient (14). The particulate phosphodiesterase is the same.NaCl-washed particles used as a source of activator-deficient aclenylate cyclase. Experiment 1 and Experiment 2 used rat and bovine brain, respectively. ND, not determined.
ACTIVATOR.Ca2+-DEPENDENT
ADENYLATE
CYCLASE
131
probably more Ca2+is available to adenylate cyclase in the plasma membrane than to phosphodiesterase in the cytoplasm. Since the activator confers stability to adenylate cyclase against thermal inactivation, it is believed that the apoenzyme, when it forms an enzyme *activator complex, assumes a different conformation. Based on the results here and elsewhere, we may conclude that the sequence of reactions depicting the stimulation of brain cytoplasmic phosphodiesterase by the protein activator, shown by Eqs. 111and [2], applies also to membrane adenylate cyclase. Stimulation of adenylate cyclase by Fand by GMP-p(NH)p is comparable in several respects. First, the two ligands actiDISCUSSION vate adenylate cyclase. in all mammalian Previous studies showed that a Ca2+- tissues (26, 29); second, stimulation is greater in broken cell preparations than in binding protein activator stimulated brain intact cells (26, 35); third, under certain adenylate cyclase which was solubilized by Lubrol-PX (3, 4). The present studies conditions, stimulation appears irreversible (26, 35). On the other hand, F- appears demonstrate that the activator also stimulates the enzyme associated with the mem- to act directly on the catalytic subunit of branes. A detergent-solubilized adenylate adenylate cyclase, whereas GMP-p(NH)p cyclase is apparently uncoupled from its interacts with a specific regulatory subhormone receptor (32); since the activator unit distinct from the hormone receptor stimulates both the particulate and the (36). The additive effect of the activator detergent-solubilized enzyme, it follows and F- or GMP-p(NH)p suggests that the that the activator interacts with the apo- action of the activator is independent of enzyme independent of the hormone re- that of the two ligands. ceptor. It does not necessarily follow, howIn comparing the stimulation of adenylever, that the effect of the activator is ate cyclase by the activator with that of divorced from that of the hormone. Brain cytoplasmic phosphodiesterase, several saadenylate cyclase is usually not very re- lient features may be noted. First, formasponsive to neurohormones (33). tion of the enzyme *activator complex is The mechanism of stimulation of ade- Ca2+ dependent, although the two enzyme nylate cyclase and that of phosphodiestersystems may display different sensitivities ase by the protein activator appear to be toward Ca2+. Second, the primary effect of qualitatively identical: In the presence of the activator was on the V of the reaction Ca2+, the activator interacts with the and not on the apparent K, of the subapoenzyme to form an active en- strate. Thus, both enzymes may be classizyme *activator complex, and removal of fied as “V type” (37). Third, although the Ca2+ dissociates the complex into its com- activator affects the thermal stability of ponents. With a saturating concentration the holoenzyme, the effects on the two sysof the activator, the concentrations of Ca2+ tems were opposite: Adenylate cyclase was needed to give half-maximum stimulation more stable whereas phosphodiesterase of adenylate cyclase and phosphodiesterwas less stable in the presence of the actiase were about 20 (Fig. 1B) and 5 ,UM (13), vator (5, 38, 39). respectively. The concentration of free The stimulation of adenylate cyclase Ca2+ in the cytoplasm is about lO-'j M. and cytoplasmic phosphodiesterase by the Following depolarization, there is an in- same Ca2+-binding protein raises the flux of Ca2+ (34) into the nerve cell and question of its biological utility. In analogy
in contrast, it did not alter the activity of the particulate enzyme. The particulate phosphodiesterase had been washed exhaustively with a hypertonic NaCl-EGTA solution to remove the endogenous activator and was the same preparation used for adenylate cyclase, which was stimulated by an exogenous activator. Moreover, EGTA did not inhibit the activity of particulate phosphodiesterase. These results show that the particulate phosphodiesterase is activator independent. The response of the soluble and not the particulate phosphodiesterase to the activator is of particular interest in considering the brain metabolism of CAMP relative to the cellular flux of Ca2+ (see Discussion).
132
LYNCH,
TALLANT,
to the scheme depicted for phosphodiesterase, the activity of adenylate cyclase may be regulated by the cellular flux of Ca2+ through the plasma membrane or the release of membrane-associated Ca2+ in response to stimuli, resulting in an increase in intracellular CAMP. Ca2+ subsequently arriving in the cytosol stimulates the cytoplasmic phosphodiesterase, which then returns the elevated intracellular CAMP level to its prestimulated level. The sequential stimulation of adenylate cyclase and cytoplasmic phosphodiesterase would allow a transient elevation of cellular CAMP in response to stimuli. On the other hand, the cytoplasmic phosphodiesterase also catalyzes the hydrolysis of cGMP; in fact, at a micromolar concentration of the substrates, the rate of cGMP hydrolysis is greater than that of CAMP. The cellular flux of Ca2+, therefore, could cause an increase of CAMP or a concomitant decrease of cGMP. Since the cytoplasmic but not the membrane phosphodiesterase is responsive to the Ca2+/activator, the two enzymes may play complimentary roles. The membrane enzyme in close proximity to adenylate cyclase may be important in preventing an excessive accumulation of CAMP in or adjacent to the plasma membrane, whereas the cytoplasmic enzyme may be particularly crucial when the level of CAMP is elevated following stimulation of the nerve cells. REFERENCES 1. ROBISON, G. A., BUTCHER, R. W., AND SUTHERLAND, E. W. (1971) Cyclic AMP, Academic Press, New York. 2. CHEUNG, W. Y. (1967) B&hem. Biophys. Res. Commun. 29, 478-482. 3. CHEUNG, W. Y., BRADHAM, L. S., LYNCH, T. J., LIN, Y. M., AND TALLANT, E. A. (1975) Biothem. Biophys. Res. Conmun. 66, 1055-1062. 4. BROBTROM, C. O., HUANG, Y. C., BRECKENRIDGE, B. McL., AND WOLFF, D. J. (1975)Proc. Nat. Acad. Sci. USA 72, 64-68. 5. LIU, Y. P., AND CHEUNG, W. Y. (1976) J. Biol. Chem. 251, 4193-4198. 6. LYNCH, T. J., TALLANT, E. A., AND CHEUNG, W. Y. (1976) Biochem. Biophys. Res. Commun. 68, 616-625. 7. LIN, Y. M., LIU, Y. P., AND CHEUNG, W. Y. (1975) FEBS Lett. 49, 356-360.
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8. TESHIMA, Y., AND KAKIUCHI, S. (1974) Biochem. Biophys. Res. Commun. 56, 489-495. 9. LYNCH, T. J., TALLANT, E. A., AND CHEUNG, W. Y. (1976) Fed. Proc. 35, 1633 (abstr.). 10. KRISHNA, G., WEISS, B., AND BRODIE, G. J. (1968) Pharmacol. Exp. Ther. 163, 379-385. 11. LYNCH, T. J., TALLANT, E. A., ANDCHEUNG, W. Y. (1975) Biochem. Biophys. Res. Commun. 65, 1115-1122. 12. THOMPSON, W. J., AND APPLEMAN, M. J. (1971) Biochemistry 10, 311-316. 13. LIN, Y. M., LIU, Y. P., AND CHEUNG, W. Y. (1974) J. Biol. Chem. 249, 4943-4954. 14. CHEUNG, W. Y. (1971) J. Biol. Chem. 246, 28592869. 15. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 16. WARBURG, O., AND CHRISTIAN, W. (1941) Bio&em. Z. 310, 384-421. 17. NEER, E. J. (1973) J. BioZ. Chem. 248,3742-3744. 18. MIDDLEMIBS, D. N., AND FRANKLIN, T. J. (1975) FEBS Lett. 55, 225-228. 19. PERKINS, J. P., AND MOORE, M. M. (1971) J. Biol. Chem. 246,62-68. 20. DYE, I., ANDSUTHERLAND, E. W. (1966)Bioehim. Biophys. Acta 127, 347-354. 21. JOHNSON, R. A., AND SUTHERLAND, E. W. (1973) J. Biol. Chem. 248, 5114-5121. 22. BROSTROM, M. A., BROBTROM, C. A., BRECKENRIDGE, B. M., AND WOLFF, D. J. (1976) J. Biol. Chem. 251, 4744-4750. 23. LIN, Y. M. (1973) Doctoral dissertation, The University of Tennessee Center for the Health Sciences, Memphis, Tennessee. 24. HOLLOWAY, J. H., AND REILLEY, C. N. (1960) Anal. Chem. 32, 249-256. 25. BRADHAM, L. S. (1972) Biochim. Biophys. Acta 276, 434-443. 26. PERKINS, J. P. (1973) Advan. Cyclic Nucleotide Res. 3, l-64. 27. RODBELL, M., KRANS, H. M. J., POHL, S. L., AND BIRNBAUMER, L. (1971) J. Biol. Chem. 246, 1872-1876. 28. HARWOOD, J. P., Liiw, H., AND RODBELL, M. (1973) J. Biol. Chem. 248, 6239-6245. 29. LONDOS, C., SOLOMON, Y., LIN, M. C., HARWOOD, J. P., SCHRAMM, M., WOLFF, J., AND RODBELL, M. (1974) Proc. Nat. Acad. Sci. USA, 71, 3087-3090. 30. CHEUNG, W. Y., AND SALGANICOFF, L. (1967) Nature (London) 214, 90-91. 31. DE ROBERTIS, E., ARNAIZ, G. R. D. L., ALBERICI, A., BUTCHER, R. W., AND SUTHERLAND, E. W. (1967) J. Biol. Chem. 242, 3487-3493. 32. PUCHWEIN, G.‘, PFEUFFER, T., AND HELMREICH, E. J. M. (1974) J. Biol. Chem. 249, 3232-3240. 33. KLAINER, L. M., CHI, Y. M., FRIEDBERG, S. L.,
ACTIVATOR*Ca2+-DEPENDENT ROLL, T. W., AND SUTHERLAND, E. W. (1962) J. Biol. Chem. 237, 1239-1243. 34. BAKER, P. F., HODGKIN, A. L., ANDRIDGWAY, E. B. (1971) J. Physiol. 218, 709-755. 35. LEFKOWITZ, R. J., AND CARON, M. G. (1975) J. Biol. Chem. 205, 4418-4422. 36. PFEUFFER, T., AND HELMREICH, E. J. M. (1975) J. Biol. Chem. 250, 867-876.
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37. MONOD, J., WYMAN, J., AND CHANGEUX, J.-P. (1965) J. Mol. Biol. 12, 88-118. 38. WANG, J. H., TEO, T. S., Ho, H. C., AND STEVENS, F. C. (1975) Advan. Cyclic Nucleotide Res. 5, 179-194. 39. KAKIUCHI, S., YAMAZAKI, R., TEBHIMA, Y., MENISI, K., AND MIYAMOTO, E. (1975) Aduan. Cyclic Nucleotide Res. 5, 163-178.
