Properties of the adenylate cyclase catalytic unit from caudate nucleus.

The solubilized catalytic unit (C) of adenylate cyclase from bovine caudate nucleus was separated from the component which mediates activation by guanine nucleotides (G/F) by the method of Strittmatter and Neer (Strittmatter, and Neer, E. J. (1980) Proc. Natl. Acad Sci U. S. A. 77,6344-6348). The separated catalytic unit is much more active in the presence of Mn2+ than when M&+ is the only divalent cation. The affinity of the catalytic unit for its substrate, Me. ATP, is the same with either cation. Free Mn2+ activates the enzyme at concentrations greater than that required to convert all the ATP to MnATP. Mn2+ stabilized the catalytic unit even in the absence of ATP. These observations are consistent with the idea that Mn2+ can interact with C both at the active site (with ATP) and at a separate divalent cation site. Occupancy of this site by Mn2+ does not block its activation by guanine nucleotide-activated G/F (G/F*). The separated catalytic unit from caudate nucleus can be activated by the plant diterpene, forskolin. Forskolin does not significantly affect the apparent K, of the enzyme for ATP. With forskolin, the catalytic unit is active even with M&+ as the divalent cation. However, activation by forskolin is synergistic with that of Mn2+ and free Mn2+ still activates the enzyme. Forskolin also potentiates the activation of C by G/F*, but this depends critically on the order in which activators are added to the catalytic unit. Forskolin must be added before the catalytic unit is activated by G/F*, otherwise there is no potentiation. The highly active state induced by forskolin and G/F* persists after the removal of forskolin and decays with a half-time of 20 min at 23 “C. This state can be further activated by Mn2+ so that a 1000-fold activation of the catalytic unit can be produced by forskolin, G/F*, and Mn2+. Forskolin had no effect on the rate of activation of the G/F unit by guanosine 5’-@,y-imino)triphosphate or on the amount of G/F* needed to activate C. G/F is known to mediate the activation of adenylate cyclase by hormone receptors. The activity of the catalytic unit has been considered to reflect passively the activity state of the G/F unit. The studies with forskolin suggest that this may not always be the case and that the state of the catalytic

The solubilized catalytic unit (C) of adenylate cyclase from bovine caudate nucleus was separated from the component which mediates activation by guanine nucleotides (G/F) by the method of Strittmatter and Neer (Strittmatter,  is much more active in the presence of Mn2+ than when M&+ is the only divalent cation. The affinity of the catalytic unit for its substrate, Me. ATP, is the same with either cation. Free Mn2+ activates the enzyme at concentrations greater than that required to convert all the ATP to Mn-ATP. Mn2+ stabilized the catalytic unit even in the absence of ATP. These observations are consistent with the idea that Mn2+ can interact with C both at the active site (with ATP) and at a separate divalent cation site. Occupancy of this site by Mn2+ does not block its activation by guanine nucleotide-activated G/F (G/F*).
The separated catalytic unit from caudate nucleus can be activated by the plant diterpene, forskolin. Forskolin does not significantly affect the apparent K , of the enzyme for ATP. With forskolin, the catalytic unit is active even with M&+ as the divalent cation. However, activation by forskolin is synergistic with that of Mn2+ and free Mn2+ still activates the enzyme. Forskolin also potentiates the activation of C by G/F*, but this depends critically on the order in which activators are added to the catalytic unit. Forskolin must be added before the catalytic unit is activated by G/F*, otherwise there is no potentiation. The highly active state induced by forskolin and G/F* persists after the removal of forskolin and decays with a half-time of 20 min at 23 "C. This state can be further activated by Mn2+ so that a 1000-fold activation of the catalytic unit can be produced by forskolin, G/F*, and Mn2+. Forskolin had no effect on the rate of activation of the G/F unit by guanosine 5'-@,y-imino)triphosphate or on the amount of G/F* needed to activate C. G/F is known to mediate the activation of adenylate cyclase by hormone receptors. The activity of the catalytic unit has been considered to reflect passively the activity state of the G/F unit. The studies with forskolin suggest that this may not always be the case and that the state of the catalytic unit itself may be important in determining the overall activity of the adenylate cyclase system.
The hormone-stimulated adenylate cyclase system is made up of at least three separable components: the hormone receptor, the catalytic unit, and the guanine nucleotide binding regulatory unit which couples the receptor to the catalytic unit (for a review, see Ref. 1). Each of these components is probably made up of more than one polypeptide subunit.
Adenylate cyclase can be solubilized from the plasma membrane by detergents. Nonionic detergents, such as Lubrol 12A9 or Triton X-100, solubilize the catalytic and G/F' units as a complex, but hormone responsiveness is lost (1, 2). Recently, this laboratory developed a method for separating the catalytic and G/F units from bovine cerebral cortex by gel filtration in ammonium sulfate and cholate, an ionic detergent (3). A very similar method was independently described for liver adenylate cyclase by Ross (4). The separate G/F unit could then be activated by the nonhydrolyzable GTP analogue, Gpp(NH)p, in the absence of C and the two components could be subsequently reconstituted into an active complex. This gave the possibility of studying the function of the brain catalytic and G/F units separately.
The separated catalytic unit from brain is activated by at least two known proteins, the G/F unit and calmodulin (3,5). Its activity is also greatly enhanced by a divalent cation, Mn2+ (3, 6), and, as this study shows, by the plant diterpene, forskolin. The work described in this paper defines some of the interactions of Mn", forskolin, and the G/F unit in controlling the activity of the catalytic unit.
Kinetic studies of membrane-bound or soluble adenylate cyclase have suggested that the enzyme is regulated by metal ions at sites distinct from the active site, which requires Me. ATP as a substrate (7,8). The initial studies could not distinguish sites on the G/F unit from sites on the catalytic unit. Using separated components, it has now been possible to show that the function of the G/F unit is modulated by Mg2+ (9, 10). The catalytic unit has a different metal ion requirement. When it is separated from the G/F unit, it is only slightly active with M$+ as the only divalent cation but ' The abbreviations used are: G/F, the component of adenylate cyclase which mediates activation of the enzyme by guanine nucleotides. C, the catalytic component of adenylate cyclase, this component may be made of more than one polypeptide; G/F*, the guanine nucleotide regulatory component which has been fully activated by Gpp(NH)p as described in the under "Materials and Methods"; C .
G/F, the complex of catalytic and G/F units; Gpp(NH)p, guanosine 5'-(P,yimino)triphosphate; App(NH)p, adenosine 5'-(P,y-imino)triphosphate; EGTA, ethylene is stimulated 7-20-fold by Mn'+ (3, 6). There are two possible explanations for this Mn2+ dependence: either C specifically requires Mn. ATP as substrate or it has a regulatory metal ion binding site, distinct from the active site, which must be filled by Mn2+ for the enzyme to be active. Previous studies from this laboratory suggested, on the basis of indirect experiments with the intact enzyme, that adenylate cyclase from brain might indeed have a regulatory Mn2+ site (11). Studies with the isolated, soluble components now allow us to define the Mn2+ site on the brain catalytic unit and to investigate its function. The conclusion that the catalytic unit itself has a binding site for metal ions which is distinct from the active site agrees with the studies reported by Cech and Maguire (12) and by Somkuti et al. (13) in membranes of the cycvariant of S49 lymphoma cells which lack a functional G/F unit.
The plant diterpene, forskolin, derived from the root of Coleus forskholii, has recently been shown to be an activator of adenylate cyclase (14). It not only stimulates basal activity but modifies the response of membrane-bound adenylate cyclase to hormones. Seamon and Daly (15) also showed that forskolin could activate adenylate cyclase in the cyc-mutant of S49 lymphoma cells suggesting it acts directly on the catalytic unit. We now report that forskolin activates the isolated catalytic unit from bovine cerebral cortex and caudate nucleus. In saying this, we do not mean to imply that forskolin acts on the polypeptide which bears the active site. The separated catalytic unit may be multimeric and we cannot pinpoint effects to possible subunits of the catalytic component. In the presence of forskolin, the catalytic unit is active even with Mg2+ as the divalent cation. However, the activation by forskolin is synergistic with that of Mn2+. Forskolin also potentiates the activation of the catalytic unit by the G/F unit, but this depends critically on the order in which activators are added to the catalytic unit. Forskolin must be added before the catalytic unit is activated by the G/F unit; otherwise there is no potentiation. G/F is known to mediate the activation of adenylate cyclase by hormone receptors, by GTP and its nonhydrolyzable analogues, and by fluoride. It is known to be the site of action of cholera toxin in stimulating the system (1, 16,17). The activity of the catalytic unit has been considered to reflect passively the activity state of the G/F unit. The studies with forskolin suggest that this may not always be the case and that the state of the catalytic unit itself may be important in determining the overall activity of the adenylate cyclase system.

MATERIALS AND METHODS'
Preparation o f I s o l a t e d C a t a l y t i c U m t C a t a l y t i c u n l t was prepared from frozen bovlile cerebral Cortex   and c e n t r l f u g e d a t 45.000 x g f o r 45 m w t e 6 a t 4OC. P e l l e t s were resuspended In 50 nM g l y c y l g l y c l n e (Signal. 50 aH maleate ISlgnaI; pho5phol?pid 13.5 ms/n11; 0.1% Lubrol 1ZA9

RESULTS3
Interaction of the Catalytic Unit with Mn2+-The activity of the catalytic unit from caudate nucleus is much greater with Mn2+ than it is with Mg2+, but the activity with Mg is still easily measurable. As shown in Fig. 1, the affinity of the catalytic unit for Me. ATP is the same whether the divalent cation is Mg2+ or Mn2+ (0.11 f 0.02 II~M Mg. ATP, 0.14 f 0.02 mM Mn ATP, n = 3).
Since the greater activity with Mn2+ than with Mg'+ could not be explained as a difference in affinity for substrate, we wished to determine whether the catalytic unit has a regulatory metal ion binding site. A test for such a site would be to demonstrate that free Mn2+ stimulates the activity of the catalytic unit even after all the ATP is in the Mn. ATP form. Fig. 2 shows than Mn2+ does, indeed, do this. The ATP concentration in the lower curve is 0.06 mM. When Mn2+ is equimolar with ATP, essentially all the ATP is in the Mn. ATP form. Nevertheless, the enzyme is further activated by the metal ion with a half-maximal activation at 0.5 mM Mn2+. Fig. 2 also shows that the total MnZ+ concentration for halfmaximal activation does not depend on the ATP concentration. Similar results were obtained when 15 mM MgC12 was present in addition to MnC12 (not shown), suggesting that the putative divalent cation site on the catalytic unit has a higher affinity for Mn2+ than for Mg2+. Mn2+ (5 mM) could stabilize the catalytic unit against inactivation a t 30 "C even in the absence of substrate: the halftime for inactivation of C in the presence of Mg2+ was 5 min, but increased to 8 min in the presence of Mn2+ alone or to 35 min in the presence of Mn2+ and the nonhydrolyzable ATP analogue, App(NH)p ( Fig. 3; the stabilization by forskolin The experiments presented here were performed with catalytic unit from the caudate nucleus but similar results were obtained with catalytic unit from cerebra1 cortex. The catalytic unit from cerebral cortex had an apparent K , for ATP which was similar to that from caudate nucleus. It displayed the same evidence for a regulatory Mn2+ binding site and was synergistically activated by forskolin, Mn2+, and G/F in the same way as the caudate nucleus. Thus, the properties of the catalytic unit described here are not unique to the caudate nucleus enzyme.  Fig. 3 will be discussed later). App(NH)p was used instead of ATP to minimize hydrolysis of the nucleotide during the inactivation. The rate of inactivation with 0.25 mM App(NH)p and Mg2+ was only slightly slower than with Mg2+ alone. The decay curves obtained from these experiments do not obey simple f i s t order kinetics. However, the observation that Mn2+ can stabilize the enzyme is consistent with the idea that free Mn" can interact with C both at the active site (with ATP) and at a separate divalent cation site.
Occupancy of the Mn2+ site on the catalytic unit does not block its activation by G/F*. As shown in Fig. 4, G/F* can activate the catalytic unit even in the presence of 20 mM MnC12. The experiments shown were done in glycylglycine/ maleate buffer to avoid oxidation of Mn2+ which is catalyzed by Tris buffers. However, similar results were obtained when Tris buffers were used. We have previously reported that Lubrol 12A9-solubilized cerebral cortical adenylate cyclase can be activated by Gpp(NH)p in the presence of 5 mM Mn2+ (11). Gpp(NH)p can stimulate adenylate cyclase in membrane preparations from cerebral cortex in the presence of 20 mM Mn2+ whether Tris or glycylglycine/maleate buffer is used,  half-time for inactivation at 30 "C increased from 5 min in the presence of Mg2+ alone to 17 min in the presence of forskolin and Mg2+ (Fig. 3).
Forskolin may activate the catalytic unit by dissociating an inhibitory subunit of C. T o test for this possibility, we incubated the catalytic unit with 100 ,UM forskolin at 23 "C for 20 min, then passed it over a Sepharose 6B column equilibrated with Buffer 5 (see "Materials and Methods") containing 0.1% Lubrol12A9 and 50 PM forskolin. The apparent Stokes radius of the isolated catalytic unit (relative to marker enzymes of known Stokes radius) was 70 A whether or not the gel fitration was performed with forskolin in the buffer. This suggests that forskolin does not cause the dissociation of a large enzyme component. Dissociation of a small enzyme component might not affect the elution position of the catalytic unit, but a small component would be separated from C by the gel fitration. Therefore, we took the fractions containing the peak of enzymatic activity from Sepharose 6B and separated away the forskolin by passing the enzyme over a column of Sephadex G-50 equilibrated in Buffer 5 with 0.1% Lubrol 12A9 but without forskolin. If an inhibitory unit had been separated by the original Sepharose 6B gel filtration, we would expect the enzyme to be activated persistently and no longer responsive to forskolin. This was not the observed result as shown in Table I, Activation by forskolin was reversed by removal of forskolin on Sephadex G-50. The enzyme was then again sensitive to stimulation by forskolin.
Synergistic Activation of the Catalytic Unit by Mn2+, G/F, and Forskolin"Mn*+ (5 n " ) and forskolin (100 p~) together activate the catalytic unit 2.6 +-0.1-fold (n = 9) over the sum of the individual activities (Table 11, lines 2,3, and 5). Forskolin also slightly increases the affinity of the catalytic unit for free Mn". The half-maximal stimulation occurs at 0.25 mM Mn with forskolin (Fig. 7 ) compared with 0.5 mM Mn without forskolin (Fig. 2). However, this small difference in affinity does not explain the synergism between Mn2+ and forskolin which occurs at saturating concentrations of both ligands and substrate.

TABLE I
Reversibitity of forskolin activation Catalytic unit was passed over a column of Sepharose 6B in Buffer 5 containing 0.1% Lubrol 12A9,50 p~ forskolin. A 350-pl sample from the peak of activity was then passed over a 1 0 -4 column of Sephadex G-50 in Buffer 5 with 0.1% Lubrol 12A9 to remove the forskolin. Ten pl of a solution of human hemoglobin was added to the sample before gel filtration as a marker. The hemoglobin-containing effluent was collected as one fraction. Dilution on the column was measured by comparing the optical density at 418 nm of the applied and collected samples. The activity reported below was corrected for this dilution. Values are mean S.E. for three experiments each assayed in duplicate. Activation of the catalytic unit by Mnz+, forskolin, and G/F* Twenty pl of caudate nucleus catalytic unit (C) diluted 1:5 in Buffer 1 was incubated for 30 min at 23 "C with 5 mM MnClz (Mnzt), 100 p~ forskolin (F), or 20 p1 of G/F* (G/F*) as indicated. 5 mM MnC12 or 100 pM forskolin was also added after incubation, to the assay, as indicated. Twenty p1 of diluted catalytic unit contained 1 pg of protein and 20pl of G/F* contained 2 pg of protein. Assays without G/F' (1-3 and 5) did not lose more than 20% activity during incubation (controls not shown). Incubation with forskolin for an additional 30 min at 23 "C had no effect on activity in assays 8, 10, 11, and 12 (controls not shown). Note that final compositions of assays 6 and 7, of assays 8 and 9, and of assays 10 to 13 are the same. This experiment is representative of four similar ones. Activation by forskolin was also synergistic with that produced by interaction of the catalytic unit with G/F*. However, this was critically dependent on the time when forskolin was added. It was essential that forskolin be present in the 30-min incubation at 23 "C during which G/F* combined with the catalytic unit. When the reconstitution took place with forskolin, the activity of the stimulated complex was 2.7 & 0.2fold greater than the sum of activity with forskolin or G/F (Table 11, lines 3,4, and 9). On the other hand, if the catalytic unit and G/F were allowed to combine and forskolin was added afterwards, the final activity was only equal to the sum of the individual ones (Table 11, lines 3,4, and 8). The results are the same regardless of the concentration of forskolin (Fig.  8). Stabilization of the catalytic unit by forskolin would not explain these results since assays without G/F or forskolin did not lose more than 20% of their activity during the incubation at 23 "C.
Since forskolin has to be present during the activation of Buffer 5. Catalytic unit from caudate nucleus was diluted 5-fold into Buffer 5. Equal volumes of diluted G/F and catalytic unit were mixed and incubated at 23 "C for 30 min with (Reaction I, 0,O) or without (Reaction 11, A, A) 50 ~L M forskolin. After the incubation, 50 p~ forskolin was added to Reaction 11. A small amount of human hemoglobin was added as a marker and samples were removed to measure starting activities. Three hundred pl of each set were applied to 10-ml Sephadex G-50 columns equilibrated with Buffer 5 containing 2 ~L M Gpp(NH)p and 0.1% Lubrol 12A9 in order to remove forskolin. The portion of the effluent containing the hemoglobin was collected into one fraction. Comparing the optical density at 418 nm of the applied sample and the fraction collected allowed us to correct for dilution of the enzyme on the columns. In separate experiments, we had shown that hemoglobin has no effect on enzymatic activity. The gel-filtered samples were incubated at 23 "C. At the times indicated, aliquots from each set were removed and assayed for adenylate cyclase activity with (closed symbols) or without (open symbols) 50 WM forskolin. The activities shown are corrected for dilution on the columns in order to compare the recovered activities with the initial ones. The data are given as the mean f S.E. for three independent experiments each assayed in duplicate. The starting activities in picomoles of cAMP/25 pl X 10 min were: C. G/F*, no forskolin, 23 f 5; Reaction I, 81 -C 30; Reaction 11, 242 f 38. The scatter in the data is due to variations in absolute activity among the preparations used and not to variability in the curve obtained. When the recovery of activity was calculated as a per cent of the starting activity in paired analysis, at time = 0 (no incubation at 23 "C), recovery for Reaction I was 97 -C 5% and for Reaction I1 98 f 20%. the catalytic unit by G/F, it is possible that it has some effect on G/F as well as on the catalytic unit. It is difficult to rule out such a possibility. However, we could find no effect of forskolin on the rate of activation of the isolated G/F unit by Gpp(NH)p (Fig. 9). We could also find no effect of forskolin on the amount of G/F* required to activate the catalytic unit (Fig. 10). In any event, a change in the affinity of the catalytic unit for G/F* would not explain the synergism when all the components are saturating. Since we know that forskolin does act on the catalytic unit, the simplest explanation of the results is that the synergism is a consequence of the state of the catalytic unit.4 A clue to the mechanism of forskolin-G/F* synergism may be found in the behavior of one unusual preparation of caudate nucleus catalytic unit which had a very high activity. In this preparation, The activity of the catalytic unit stimulated synergistically by G/F* and forskolin can be increased still further by the addition of Mn". This final activity is again greater than the sums of the individual stimulations (Table 11). It is only the "superactive" state of the enzyme which can respond to Mn2+ in this way. Once the C . G/F* complex has formed, activation by Mn2+ only produces the increment in activity that it would have, had it been added to the catalytic unit with forskolin (compare lines 10 and 11 with the sum of 4 and 5).
Reversal of the Forskolin-induced Active State-Our previous experiments ( Table I) as well as those of others (14, 25) showed that the action of forskolin is reversible. We wished to determine whether the "superactive" state induced by forskolin persists even in its absence. To answer this question, we activated the catalytic unit with G/F* without or with forskolin generating the states shown in lines 8 and 9 of Table 11. In the former situation, we added forskolin to the sample after activation by G/F*. We then passed the two preparations over Sephadex G-50 to remove free forskolin. The stripped enzyme samples were incubated at 23 "C for the times noted in Fig.  11. They were assayed with or without readdition of forskolin. Fig. 11 shows that the "superactive" state induced by forskolin does persist for a measurable time even in the absence of forskolin. When forskolin is removed from the "superactive" enzyme, the activity drops to a level similar to that of a C-G/F* complex. This indicates that the columns do indeed remove forskolin from the sample. However, readdition of forskolin fully restores the original, synergistic, forskolin-induced activity. In contrast, the control C.G/F* complex to which forskolin was added after activation of the catalytic unit by G/F*, can only be reactivated to its initial, lower level. As can be seen from the figure, the capacity of the C.G/F* complex to respond to forskolin in a synergistic fashion decays at 23 "C with a half-time of about 20 min.

DISCUSSION
In this study, we have investigated several aspects of the function of the separated catalytic unit of adenylate cyclase from bovine caudate nucleus: the interaction with Mn2+, activation by forskolin, and the role of both of these activators in modulating the response of the catalytic unit to activation by G/F*.
Separation of the catalytic unit from the G/F unit (3) and, in the case of brain tissue, from calmodulin (5) changes its metal ion requirement. However, the affinity of the enzyme for its substrate, Me. ATP, is the same whether activity is measured with Mg2+ or Mn2+. The apparent K , of the isolated catalytic unit for ATP is similar to values determined for the holoenzyme (1) or membrane-bound C from cyc-S49 cells (12) suggesting that neither the interaction of the catalytic unit with the G/F unit nor association with a membrane affects this parameter. The activation of the enzyme by Mn2' could be because Mn.ATP is a better substrate than Mg. ATP. Our studies do not rule out this possibility but they demonstrate that in addition a large part of the activation of the catalytic unit by Mn2+ is due to the interaction of this metal ion with a regulatory site on the enzyme. The evidence for this is that free Mn2+ continues to activate the enzyme at synergism increased as the preparation was diluted. At a 50-fold dilution, the results were the same as with standard preparations diluted 5-fold. We could not explain the exceptionally high activity of this single preparation, but we speculated that it might contain aggregates of catalytic unit which dispersed on dilution. The possibility is consistent with the idea that catalytic unit and G/F must be unhindered before synergistic activation by forskolin and G/F is possible. The affinity of the isolated catalytic unit for Mn2+ is not high (0.5 mM). However, it is hard to know what the affinity of the site might be in the membrane. Adenylate cyclase from various regions of the brain is inhibited by EGTA (11,(26)(27)(28)(29). This laboratory has shown that EGTA inhibition can be partially reversed by Ca2+ but virtually entirely by Mn2+, suggesting that EGTA may inhibit by chelating bound Mn2+ (11). Studies with membrane-bound brain enzyme suggested that membrane structure must be somewhat disrupted to allow access of EGTA to the metal ion (30). If a metal ion binding site is indeed sequestered in the membrane, its affinity for metal in the native enzyme might be quite different from that observed in a solubilized, separated component.
The isolated catalytic unit is stabilized by Mn2+ even in the presence of M e and in the absence of substrate. However, addition of the substrate analogue, App(NH)p, enhances the stabilization. Stabilization by App(NH)p is minimal in the presence of M e alone. The observation that free Mn" stabilizes the enzyme is consistent with the idea that there is a regulatory Mn2+ site on the enzyme. We cannot, at present, say whether the regulatory metal ion site is on the polypeptide bearing the active site or on another component of the catalytic unit.
It has been reported by several laboratories that Mn2+ uncouples the catalytic unit from the G/F unit preventing activation of the enzyme by Gpp(NH)p (14,24). We have not been able to uncouple the enzyme with Mn2+ up to 20 mM either in studies with isolated components or in membrane preparations. Mn2+ activates the C.G/F* complex equally well whether added before or during reconstitution. At present, we have no explanation for the difference between our results and those of others.
The plant diterpene, forskolin, is a recently discovered activator of adenylate cyclase (14). Forskolin increases the V,,, of the catalytic unit without affecting the apparent K,,, for Mg. ATP or Mn. ATP. It slightly increases the affinity of the Mn2+ site for the Mn2+. Forskolin stabilizes the catalytic unit against inactivation at 30 "C. Although forskolin clearly affects the function of the component which we have separated from the G/F unit and from calmodulin, we cannot say that it acts on the catalytic polypeptide itself since we do not yet know the polypeptide composition of the catalytic unit.
The most interesting and potentially instructive aspect of the interaction of forskolin with the catalytic unit is the synergistic activation which we observe among forskolin, Mn2+, and G/F*. Forskolin and Mn2+ together activate the catalytic unit 2.6 times more than the sum of the individual activities. Forskolin also potentiates the activation of the catalytic unit by G/F, but this depends on forskolin's being present during the enzyme reconstitution. If forskolin is added after the C . G/F complex has formed, then the effects are only approximately additive. A simple interpretation would be that association of the catalytic unit with the G/F unit blocks the access of forskolin to a site which must be occupied for potentiation to occur. This notion is consistent with the observation that synergism is not observed in Lubrol 12A9solubilized bovine brain enzyme even when forskolin is present during the activation by Gpp(NH)p (data not shown). In Lubrol 12A9-solubilized enzyme, the components are not dissociated and therefore the site may not be accessible.
The state of the reconstituted enzyme induced by forskolin persists for a time even in the absence of the diterpene and relaxes back to the conformation of the C. G/F complex obtained when the reconstitution takes place without forskolin. Like other conformational changes in the adenylate cy-clase system, the process is rather slow with an approximate half-time of 20 min at 23 "C.
The "superactive" state induced by forskolin and G/F* allows a further activation by Mn2+ (compare lines 10 and 11 with 12 and 13 in Table 11). Overall, we can activate the catalytic unit about 1000-fold by adding forskolin, Mn2+, and G/F* in the right order. The specific activity of the fully activated enzyme (based on the amount of protein in the catalytic unit preparation) is 0.1 pmol of cAMP/mg/min.
We do not yet know the mechanism of the forskolin-induced potentiation of the G/F activation of the catalytic unit. Seamon and Daly have recently observed synergistic activation of adenylate cyclase by Gpp(NH)p and forskolin in membranes from tissues which lack a mechanism for hormonally induced inhibition of adenylate cyclase (31). One could postulate that we have removed or inactivated an inhibitory guanine nucleotide regulatory unit in the course of preparing G/F. However, the fact that we can see either additive or synergistic effects depending on the order of addition of the components argues against this interpretation. Other possibilities are that forskolin might act by removing an inhibitory peptide from the catalytic unit or that it might increase activity by recruiting a population of silent catalytic units. We could find no evidence of the former possibility in direct experiments and the arguments outlined below suggest this is not the explanation. Either of the proposals could explain synergism of forskolin and Mn2+ or forskolin and G/F. However, both seem unlikely in view of the finding that the combination of Mn2', G/F, and forskolin during enzyme reconstitution gives activity which is again a multiple of the individual synergistic activations. Thus, if forskolin and Mn2+ specifically recruited one population of catalytic units and forskolin plus G/F recruited a second population, one would expect that all three together would only activate those two populations. To account for the multiplicative effect when all three activators are present together, one would have to postulate a third population of catalytic units which are only active with all three stimulators. A similar argument applies to putative inhibitors. Although such theories cannot be ruled out, they do not seem, at present, to be the simplest interpretation of the data. We conclude therefore that forskolin induces a variety of conformational states of the enzyme which are reflected in different activities and different responsiveness to the G/F unit (the physiological activator) and to metal ions.
The overall activity of the adenylate cyclase system has been thought to reflect the activation state of the G/F unit which is in turn controlled by the hormone receptor. In this view, the control goes only from outside the cell to inside. If the forskolin site should, in fact, turn out to be the site for an as yet unrecognized intracellular modulator of adenylate cy-