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Target analysis of the turkey erythrocyte adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] system showed that the molecular weight of the ground state enzyme increases from 92,000 with MnATP as substrate and no stimulatory ligands to 226,000 when activated by fluoride ion or by 5'-guanyl imidodiphosphate (p[NH]ppG) subsequent to clearance of previously bound GDP. The identical increment in size (130,000) suggests that the same regulatory unit is involved in the activation by both effectors. When assayed with isoproterenol and p[NH]ppG, the enzyme system displayed a further increment in size of 90,000 daltons. Based on binding of the antagonist 125I-labeled hydroxybenzylpindolol, the beta-adrenergic receptor is about 90,000 daltons or the same as that seen for activation of the enzyme by isoproterenol through the beta-adrenergic-receptor. Because single targets were seen for the ground state enzyme system under all conditions, it would appear that the various regulatory and catalytic components are structurally linked prior to activation by hormone, guanine nucleotides, and fluoride ion. Furthermore, based on reported subunit sizes of the nucleotide regulatory and receptor components are composed of multiple subunits, either homologous or heterologous in structure.

Previous studies have shown that guanine nucleotides, acting at a site termed nucleotide regulatory site, are required for activation of hepatic adenylate cyclase and that glucagon facilitates this process. This study shows that only guanine nucleotides containing triphosphate groups at the 5' position of ribose (or 3'-deoxyribose) are capable of activating the enzyme. The terminal phosphate is not utilized in the activation process since 5'-guanylylimidodiphosphate (Gpp(NH)p and 5'-guanylyl methylenediphosphonate, analogues of GTP that are not utilized in transferase or hydrolase reactions, stimulate enzyme activity. The nucleotides bind in their free form at the regulatory site; chelation by magnesium ion shifts the apparent concentration dependence for activation by Gpp(nh)p. GDP inhibits competitively Gpp(NH)p-stimulated activity and inhibits basal activity and activities stimulated by glucagon. Activation of the enzyme by Gpp(NH)p is a slow process; the length of the lag time increases as an inverse function of nucleotide concentration and is as long as 4 min before onset of increased enzyme activity. Following pretreatment with Gpp(NH)p and extensive washing of hepatic membranes, the enzyme displays immediate increases in activity with rates that are a function of the nucleotide concentration during pretreatment; the rates remain constant for at least 6 min despite the absence of Gpp(NH)p in the medium. Studies with labeled Gpp(NH)p show that the intact nucleotide remains firmly bound to the membranes after extensive washing, suggesting that the persistence of adenylate cyclase activity may be related to slow dissociation of the nucleotide from the regulatory site. Addition of 1 nM glucagon, a submaximal concentration, does not abolish the lag phase of Gpp(NH)p activation even at saturating concentration of the nucleotide (1 muM or higher). The maximal steady state rate is achieved under these conditions. Addition of 2 muM glucagon, a saturating hormone concentration, does not alter the steady state rate but abolishes the lag phase of Gpp(NH)p activation. The transient kinetics of Gpp(NH)p activation and the effects of glucagon thereon are discussed in terms of a three state model in which the guanine nucleotide induces the formation of an intermediate transition state that displays no increase in enzyme activity over the basal state and which slowly isomerizes to a high activity state of the adenylate cyclase system; glucagon acts by accelerating the rate of isomerization.

5'-Guanylylimidodiphosphate (Gpp(NH)-p) stimulates adenylate cyclase [ATP-pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in plasma membranes isolated from frog and salmon erythrocytes, from rat adrenal, hepatic, and fat cells, and from bovine thyroid cells. The nucleotide acts cooperatively with the various hormones (glucagon, secretin, ACTH, thyrotropin, and catecholamines) that stimulate these adenylate cyclase systems with resultant activities that equal or exceed those obtained with hormone plus GTP or with fluoride ion. In the absence of hormones, Gpp(NH)p is a considerably more effective activator than GTP, and, under certain conditions of incubation, stimulates rat fat cell adenylate cyclase to levels of activity (about 20 nmoles of 3',5'-adenosine monophosphate mg protein per min) far higher than reported hitherto for any adenylate cyclase system examined. The nucleotide activates frog erythrocyte adenylate cyclase when the catecholamine receptor is blocked by the competitive antagonist, propranolol, and activates the enzyme from an adrenal tumor cell line which lacks functional ACTH receptors. In contrast, Gpp(NH)p does not stimulate adenylate cyclase in extracts from Escherichia coli B. Gpp(NH)p appears to be a useful probe for investigating the mechanism of hormone and nucleotide action on adenylate cyclase systems in eukaryotic cells.

Rat liver plasma membranes were incubated with phospholipase A2 (purified from snake venom) or with filipin, a polyene antibiotic, followed by analysis of the binding of glucagon to receptors, effects of GTP on the glucagon-receptor complex, and the activity and responses of adenylate cyclase to glucagon + GTP, GTP, Gpp(NH)p, and F-. Phospholipase A2 treatment resulted in concomitant lossess of glucagon binding and of activation of cyclase by glucagon + GTP. Greater than 85% of maximal hydrolysis of membrane phospholipids was required before significant effects of phospholipase A2 on receptor binding and activity response to glucagon were observed. The stimulatory effects of Gpp(NH)p or F- remained essentially unaffected even at maximal hydrolysis of phospholipids, whereas the stimulatory effect of GTP was reduced. Detailed analysis of receptor binding indicates that phospholipase A2 treatment affected the affinity but not the number of glucagon receptors. The receptors remain sensitive to the effects of GTP on hormone binding. Filipin also caused marked reduction in activation by glucagon + GTP. However, in contrast to phospholipase A2 treatment, the binding of glucagon to receptors was unaffected. The effect of GTP on the binding process was also not affected. The most sensitive parameter of activity altered by filipin was stimulation by GTP or Gpp(NH)p; basal and fluoride-stimulated activities were least affected. It is concluded from these findings that phospholipase A2 and filipin, as was previously shown with phospholipase C, are valuable tools for differentially affecting the components involved in hormone, guanyl nucleotide, and fluoride action on hepatic adenylate cyclase.

Adenylate cyclase in liver membranes was solubilized with Lubrol PX and partially purified by gel filtration. The partially purified enzyme was susceptible to activation by guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Studies on the binding of [3H]Gpp(NH)p to various fractions eluted from the gels revealed that an upper limit of 1% of the Gpp(NH)p binding sites is associated with adenylate cyclase activity stimulated by the nucleotide. The glucagon receptor, pretagged with 125I-glucagon in the membranes, solubilized with Lubrol PX, and fractionated on the same gel columns, eluted in a peak fraction that overlaps with, but is separate from, adenylate cyclase in its Gpp(NH)p-stimulated form. Addition of GTP to the solubilized glucagon-receptor complex caused complete dissociation of the complex, as has been shown with the membrane-bound form of the complex. Since the GTP-sensitive form of the glucagon receptor complex separates from the Gpp(NH)p-sensitive form of adenylate cyclase, it is concluded that the receptor and the enzyme are separate molecules, each associated with a distinct nucleotide regulatory site or component. These findings are discussed in terms of the possible structure of the hormone-sensitive state of adenylate cyclase.

This study probes the structure and mutual interactions of the components of adenylate cyclase. We use a complementation assay which involves the addition of an adenylate cyclase-related guanine nucleotide-binding protein component to a membrane lacking this component to measure guanine nucleotide-stimulated-adenylate cyclase. Instead of using detergent extracts we were able to achieve full complementation by mixing intact membrane preparations in the presence of the nucleotide component. Of particular interest was the human erythrocyte membrane which contains very low amounts of catalytic activity and no measurable beta-adrenergic receptor but has normal amounts of the nucleotide component. This component appears to be the same, by several criteria, as components found in pigeon and turkey erythrocytes and in rat liver plasma membrane. The component confers Gpp(NH)p, fluoride, and GTP stimulation of adenylate cyclase along a single reconstitution curve. It is labeled with NAD by cholera toxin, and has an apparent molecular weight of 39 000 upon sodium dodecyl sulfate gel electrophoresis. The presence of the nucleotide unit in the virtual absence of the active catalytic unit allowed us to determine those properties intrinsic to each unit and those conferred by the association of the units. The nucleotide component binds guanine nucleotides weakly in the human erythrocyte membrane, yet produces persistent activation of adenylate cyclase and tight binding (of Gpp(NH)p) upon combination with the catalytic unit. Treatment of the human erythrocyte membrane with N-ethylmaleimide causes a simultaneous diminution in both Gpp(NH)p and fluoride stimulation in reconstituted activities, suggesting that both activities are conferred by the same component.

Guanyl-5'-yl-(alpha,beta-methylene)di[gamma-32P]phosphonate (Gp-(CH2)pp) is not hydrolyzed by rat liver membranes under conditions in which GTP and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) are hydrolyzed. Gp(CH2)pp activates adenylate cyclase in hepatic membranes with characteristics similar to those of Gpp(NH)p activation but with lower potency and effectiveness. The analogs, although with lower potency than GTP, also share the ability to change the glucagon receptor from a high to a low affinity state. Both Gp(CH2)pp and Gpp(NH)p stimulate adenylate cyclase activity following a lag period of about 1 min addition of GTP after steady state rates are achieved results in reduction in the rate following a lag period of 6 min from the time of addition of GTP. Pretreatment of the enzyme with Gpp(NH)p or Gp(CH2)pp, followed by washing the membranes, leads to a high activity state of the enzyme which slowly decays in rate unless the analogs are continuously present in the medium. These data suggest that the guanosyl nucleotide analogs act on the enzyme system by a slowly reversible process that possibly reflects slow binding and dissociation from different transition states of the enzyme system and suggest that activation of adenylate cyclase by GTP, Gpp(NH)p, and Gp(CH2)pp does not involve covalent modification of the enzyme.

The glucagon-sensitive adenylate cyclase system, viewed from the perspective of its behavior with isolated membrane preparations, displays far more complex regulatory characteristics than could have been envisioned from its behavior in the intact cell. What has emerged from our studies with isolated hepatic membranes is that glucagon can exert at least three actions which we believe are interdependent: desentization of the receptor, activation of adenylate cyclase, and promotion of adenosine inhibition of adenylate cyclase activity. Although the molecular basis remains unknown, GTP is intimately involved in the three processes. Undoubtedly, further levels of complexity will develop when the enzyme system is dissected and its components become amenable to study at the molecular level. At the moment, it is clear that adenylate cyclase systems are provided with a plethora of regulatory processes for controlling cyclic AMP production both in the absence and presence of hormones.

A three-state model developed originally from analysis of the steady state kinetics of hepatic adenylate cyclase has been extended to account for the transient kinetics of activation by guanyl-5'-yl imidodiphosphate (Gpp(NH)p). In contrast to activation by Gpp(NH)p, activation of the enzyme by GTP proceeds not only without a lag phase but is of considerably lower magnitude. These differences between Gpp(NH)p and GTP can be explained by the hypothesis that GTP is hydrolyzed at the nucleotide regulatory site(s) associated with adenylate cyclase and that GTPase activity is revealed uniquely when the enzyme system is in its state of highest adenylate cyclase activity. With this hypothesis, the characteristics of activation by GTP could be simulated. The implications of this model are discussed with respect to the actions of hormones and cholera toxin on adenylate cyclase activity.

Hormone-sensitive adenylate cyclase systems are composed of hormone-recognition units (R), a nucleotide-regulatory unit (N) for reaction with GTP and divalent cations, and the catalytic unit (C). From the reported sizes of purified R and N subunits and target analysis of functional sizes of these units, the functions of the components for the binding and actions of hormones and GTP require minimally dimers, homologous or heterologous. It is proposed that the catalytic unit exists in the membrane also as a dimer and that its transition to the active state with MgATP as substrate involves corresponding transitions in linked dimers of the hormone-recognition and nucleotide-regulatory units. It is postulated that hormones trigger the activation process by inducing in concert with GTP and divalent cations the appropriate dimer structure of the holoenzyme. In large aggregates of such structures, realignment of only a few occupied holoenzyme units may be sufficient to induce activation of the total aggregate enzyme. This theory serves to explain the synergistic actions of hormones, and how several hormones can activate a common enzyme. It also provides an explanation for 'spare' receptors, and for the efficacy of hormone action.

Adenylate cyclase in particulate fractions from rat adrenal glands is subject to regulation by purine nucleotides, particularly guanine nucleotides. While GTP activates the enzyme, this effect is not evident in all particulate fractions. Following dialysis of the refractory fractions activation by GTP is observed, an indication that endogenous nucleotides may obscure the effects of added GTP. The analog, guanyl-5'-yl imidodiphosphate (Gpp(NH)p gives considerable more activity than does GTP. GDP, on the other hand, is inhibitory, an effect revealed only in the absence of a nucleotide-regenerating solution. GDP blocks the action of both GTP and Gpp(NH)p. These results show that the gamma-phosphate of the nucleotide is required for but need not be metabolized in the activation process. At low substrate concentration (0.1 mM ATP or adenyl-5'-yl imidodiphosphate) stimulation of the enzyme by ACTH occurs only in the presence of added guanine nucleotide (GTP or Gpp(NH)p); the hormone and nucleotide act synergistically. While both GTP and Gpp(NH)p inhibit fluoride-stimulated activity, the level of fluoride required to demonstrate such inhibition appears not to be related to the level of fluoride required for activation of the enzyme. In the presence of GTP, or GTP plus ACTH, the enzyme exhibits normal Michaelis-Menten kinetics with respect to substrate utilization (K-m equal to 0.16 mM). In the activated state, produced with ACTH plus GTP, the enzyme is less susceptible to inhibition by a species of ATP uncomplexed with Mg2+, but is more susceptible to inhibition by Mg2+. These results demonstrate that fundamental differences exist between different states of the adenylate cyclase. The difficulties in describing kinetically the regulation of adenylate cyclase systems in view of the multiple actions of nucleotides and magnesium are discussed.

Isoproterenol plus guanylyl imidodiphosphate (Gpp(NH)p) activate frog erythrocyte adenylate cyclase to a level much higher than the sum of the activities produced by the catecholamine and the synthetic nucleotide tested separately. Propranolol, the beta-receptor blocking agent, failed to inhibit activity when added after the enzyme system had been preincubated with both isoproterenol and Gpp(NH)p. However, if propranolol was added after only one of the two components had been added, it inhibited the effect of isoproterenol. Production of the propranolol-resistant state by treatment with isoproterenol and Gpp(NH)p did not require the presence of the productive substrate (MgATP). The activated propranolol-resistant state persisted following various treatments of the enzyme preparation including extensive washings of the membranes; considerable activity was retained even after sonication or treatment with the detergent Lubrol-PX, treatments which caused inactivation of the native enzyme. Extensive dilution of the membranes following pretreatment with isoproterenol and Gpp(NH)p did not significantly reduce to the activity of the enzyme. Readdition of isoproterenol after dilution caused some inhibition of adenylate cyclase activity, indicating apparently that the beta-receptor has not become inaccessible. In contrast, preincubation with isoproterenol alone failed to render the enzyme system refractive to propranolol, and dilution readily reduced the activity to negligibly low values. Preincubation with Gpp(NH)p alone also produced a persistent active state but the activity was much lower than that obtained throught the combined action of isoproterenol and Gpp(NH)p. The findings suggest that the hormone may be required only to facilitate the initial interaction of the enzyme with Gpp(NH)p. The differences, in this respect, between Gpp(NH)p and the more labile natural nucleotide, GTP, are discussed.

Cell membrane receptors for hormones and neurotransmitters form oligomeric complexes with GTP-regulatory proteins and inhibit the latter from reacting with GTP. Hormones and neurotransmitters act by releasing the inhibitory constraints imposed by the receptors, thus allowing the GTP-regulatory proteins to interact with and control the activity of enzymes such as adenylate cyclase. This theory may apply generally to membrane signal transduction involving surface receptors.

Several changes were noted in the characteristics of the turkey erythrocyte beta-adrenergic receptor and in the kinetic properties of adenylate cyclase following pretreatment of erythrocyte membranes with isoproterenol and GMP, and thorough washing to remove these agents. The changes include modifications in the binding of agonist (isoproterenol) and in revelation of marked effects of GTP on agonist binding; reduction in the lag in Gpp(NH)p activation of adenylate cyclase; short lived activation by GTP which is lengthened by treatment with cholera toxin and NAD prior to pretreatment with isoproterenol and GMP. Treatment with cholera toxin also shortened the lag in activation by Gpp(NH)p and increased the steady state levels of activation by both Gpp(NH)p and GTP. The following conclusions can be drawn: (i) catecholamines, in the presence of a guanine nucleotide, stimulate the exchange of bound and exogenous nucleotide; (ii) the exchange reaction is involved in both the activation of adenylate cyclase and in the reciprocal effects of hormone and guanine nucleotides on each other's binding: (iii) the beta-adrenergic receptor and nucleotide regulatory components are linked in turkey erythrocyte membranes; (iv) both cholera toxin and catecholamines, although by different mechanisms, stimulate the exchange reaction at the nucleotide regulatory sites.

A fragment of glucagon encompassing its first six NH2-terminal residues (His-Ser-Gln-Gly-Thr-Phe) binds to the glucagon receptor and stimulates adenylate cyclase activity in rat liver plasma membranes. Glucagon1-6 is a partial agonist since it stimulates, at saturating concentrations, to the extent of 75% of the maximal activity given by the native hormone. The binding affinity and potency of glucagon1-6 are 0.001% the native hormone. Discussed are the implications of these findings on the structure-function relationships required for the action of glucagon and for preparing clinically useful analogs of the hormone.

GTP and hormones activate, synergistically, adenylate cyclase in purified plasma membranes from rat adipocytes. Addition of chelating reagents (EDTA or ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) or thiol-reducing reagents (dithiothreitol or 2-mercaptoethanol) results in marked inhibition of enzyme activity without altering the synergistic stimulatory effects of GTP and hormones. The inhibitory effects of the reagents required the presence of GTP, indicating that inhibition involves a GTP-dependent process. This process is separate from the GTP-dependent process responsible for activation of the enzyme since it is selectively abolished by pretreatment of fat cell membranes with trypsin. It is suggested that inhibition and activation of fat cell adenylate cyclase by GTP occur through distinct regulatory processes.

The synthesis of 2-thioltryptophan-glucagon is described. Oxidation of this compound gives the dimer (Trp-S-glucagon)2. Both monomer and dimer are equi-potent on a molar basis with native glucagon as activators of adenylate cyclase in hepatic plasma membranes. However, from the ability to compete with 125I-glucagon for binding at the glucagon receptor, the dimer has one-fourth the binding affinity for the receptor as does native glucagon, 2-thiol-Trp-glucagon, and Trp-(2,4-dinitrophenylsulfenyl)-glucagon which have equal affinities for the receptor. Addition of GTP, which converts the receptor from a tight-binding to a lower affinity form and which activates adenylate cyclase in the presence of glucagon, allows (Trp-S-glucagon)2 to bind equally with native glucagon and the other thiol derivatives of the hormone. This effect of GTP on the binding of the glucagon dimer and the uses of 2-thiol-Trp-glucagon in the semisynthesis of new glucagon derivatives are discussed.

A simple model is developed to explain the activation of rat liver plasma membrane adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by guanosine nucleotides and glucagon and the dependence of the cATALYTIC RATE ON Mg2+, H+, and substrate concentrations. The basic model proposes that the adenylate cyclase system can exist in two states, A and B; that activating ligands bind preferentially to the B state; and that only the B state is active. Kinetic data are quantitatively fit to this model, and the binding constants for the interaction of the A and B states with glucagon, GTP, and guanyl-5'-ylimidodiphosphate are obtinaed. The substrates ATP and adenyl-5'-ylimidodiphosphate appear to show little preference between the A and B states, and simple Michaelis-Menten kinetics are sufficient to describe the dependence of the catalytic rate on substrate concentration under optimal conditions. The dependence of the rate on pH can be explained by postulating that one ionizable group in its acid form and one ionizable group in its basic form must be present at the active site in order for catalysis to occur. The activation and inhibition of the activity by Mg2+ can be explained by a similar mechanism with Mg2+ binding to activating and inhibiting sites. Glucagon and guanosine nucleotides appear to influence the dependence of the rate on Mg2+ and glucagon. The Mg2+ also may display some preference for the B state. A comparison of this model with others that have been proposed is given. The proposed model appears to provide a simple conceptual frame-work that is applicable to many adenylate cyclase systems.

The kinetic characteristics of substrate utilization by hepatic adenylate cyclase were investigated under a variety of incubation conditions, including veriations in pH, [substrate], [Mg2+], and in the absence or presence of glucagon. Activities were compared with ATP and 5'-adenylylimidodiphosphate (App(NH)p) as substrates. The Km for both substrates was about 50 muM; Vmax given with App(NH)p was about 40% lower than obtained with ATP as substrate. In the presence of a saturating concentration of substrate (1 mM), basal activity was increased 4-fold by increasing [Mg2+] from 5 to 50 mM. The stimulatory effect of Mg2+ was not due to an allosteric action since basal activity was only marginally enhanced (40%) when the substrate concentration was reduced to 10 muM. As suggested by deHaen ((1974 J. Biol. Chem. 249, 2756), it is likely that Mg2+ increases enzyme activity by decreasing the concentration of an inhibitory, unchelated form of substrate that competes with the productive magnesium-substrate complex at the active site. Activity-pH profiles differed with ATP and App(NH)p as substrates; a shift in pH optimum was observed which correlated with the different pKa of the terminal phosphate groups of ATP and App(nh)p, and which reflect the concentration of protonated substrate (ATPH-3 minus) present in the incubation medium. Accordingly, protonated substrate is the predominant inhibitory species of unchelated substrate and probably has a considerably higher affinity for the active site than does the magnesium-substrate complex. Glucagon-stimulated activity was less susceptible to inhibition by protonated substrate than is the basal state as evidenced by lower stimulatory effect when the [Mg2+] was increased from 5 to 20 mM. However, increasing the [Mg2+] from 20 to 50 mM resulted in marked inhibition of glucagon-stimulated activity, particularly in the presence of 10 muM substrate. Conversely, at a fixed [Mg2+], concentrations of substrate at least 20-fold higher than the Km were required to achieve maximal hormone-stimulated activity. These findings suggest that the unchelated, fully ionized form of substrate serves as an activating ligand, as has been observed with guanine nucleotides at considerably lower concentrations. Thus, Mg2+ affects adenylate cyclase activity by forming the productive substrate complex and by titrating the inhibitory protonated and activating free forms of substrate. As a result of these effects of unchelated substrate, it proved difficult to evaluate the kinetic parameters involved in substrate binding and utilization and the effects of hormone thereon when substrate was added as the only source of activating ligand. However, linear Michaelis kinetic data were obtained by adding the activating ligand 5'-guanylylimidodiphosphate with glucagon and by making appropriate adjustments of pH and [Mg2+]. Vmax was increased 4-fold without changes in Km by the actions of 5'-guanylylimidodiphosphate and glucagon.

GTP evoked both an activatory and an inhibitory response from adipocyte adenylate cyclase. This paper describes the persistence of the bimodal response under a variety of assay conditions. Additionally, manipulations are described which eliminate one or other of these actions. Treatment of adipocyte plasma membranes with cholera toxin A1 peptide and NAD+ abolishes the inhibitory phase of GTP action while preserving the activating phase. Treatment of the membranes with p-hydroxymercuriphenylsulfonic acid eliminates the activatory phase while maintaining the inhibitory processes mediated by GTP in adipocytes normally coexist and operate through different pathways since either phase can be abolished leaving the other intact. Adenosine and its purine-modified analogs inhibit fat cell adenylate cyclase in the GTP inhibitory phase (Londos, C., Cooper, D. M. F., Schlegel, W., and Rodbell, M. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 5362-5366). When this effect of GTP is abolished by either cholera toxin or Gpp(NH)p pretreatment, the inhibitory action of adenosine analogs is also lost. These data suggest a central role for GTP in mediating both activation and inhibition of adenylate cyclase by agents which act through cell surface receptors.

Treatment of membranes from HeLa cells, rat adipocytes, and rat liver with organic mercurials results in complex effects on adenylate cyclase activity that are not mimicked by the reversible sulfhydryl reagent, tetrathionate. At low concentrations (0.1 mM or less 1 mercurials inactivate the enzyme; inactivation is reversed by the thiol-reducing agent, dithiothreitol. Treatment with higher concentrations of organic mercurials (1 mM and above) results in a time-dependent, irreversible change in the ability of guanine nucleotides and fluoride ion to stimulate adenylate cyclase activity. The irreversible changes are blocked by treatment of membranes with cholera toxin and NAD, suggesting that the GTP-regulatory component is the site of mercurial action. This is further suggested by the lack of irreversible effects of mercurials on adenylate cyclase activity in membranes from mouse lymphoma cells that lack this component. Irreversible effects of mercurials on the adipocyte cyclase system also include enhancement of basal activity and potentiation of the inhibitory effects of GTP on cyclase activity; the latter effects of GTP are mediated through a process independent from that mediating stimulation of activity by GTP. It is concluded that the GTP-regulatory proteins responsible for the modulation of adenylate cyclase activity by hormones and neurotransmitters contain the sites of action of organic mercurials. Their possible mode of action is discussed.

This paper presents a steady state kinetic model for hepatic adenylate cyclase. The activity of the enzyme has been assayed in the presence of a range of concentrations of magnesium, adenylylimidodiphosphate (App(NH)p), 5'-guanylylimidodiphosphate (Gpp(NH)p), and in the presence and absence of saturating concentrations of glucagon. The data were tested against proposed models using an iterative least squares curve fitting program (SAAM25) and confidence estimates for the model parameters were obtained. Hepatic adenylate cyclase is viewed as an enzyme having three characteristic states of catalytic function (E, E', E''). Each state has its own intrinsic activity in carrying out the catalysis of MgApp(NH)p-3 minus to form cyclic adenosine 3':5'-monophosphate. It is shown, in agreement with a proposal by de Haën, that unchelated substrate can inhibit adenylate cyclase activity. It is further concluded that this inhibition is principally due to App(NH)pH-3 minus. The three catalytic states differ markedly in their susceptibility to inhibition as well as in their Vmax, but the Km for MgApp(NH)p-2 minus is essentially the same for all states. The state transitions induced by Gpp(NH)p and by hormone are considered. Gpp(NH)p binding to state E causes transformation to state E'. State E' undergoes spontaneous transformation to state E''. Glucagon augments the transition from E' to E''. We conclude that the activating species of Gpp(NH)p is an unchelated form, most probably Gpp(NH)p-4 minus. Our results indicate that state E' is significantly more susceptible to inhibition by App(NH)pH-3 minus than the other two states. Certain phenomena occurring in fat cell adenylate cyclase are discussed in light of our findings in hepatic adenylate cyclase.

Adenylate cyclase activity in purified plasma membranes from rat fat cells displays transient kinetic characteristics in the absence and presence of guanyl=5'=yl imidodiphosphate (Gpp(NH)p). Gpp(NH)p causes immediate inhibition of enzyme activity; the inhibitory phase is followed by a slow increase in activity which, depending on incubation temperature, exceeds activity stimulated in the presence of hormones (glucagon, secretin, epinephrine, or adrenocorticotropin). Basal activity displays an initial high rate of activity which decays to a low state of activity within 2 min of incubation. Hormones do not alter the initial rate but prevent the decay in enzyme activity. The inhibitory phase of Gpp(NH)p action and the previously reported (Harwood, J.P., Low, H., and Rodbell, M. (1973) J. Biol. Chem. 248, 6239-6245) inhibitory effects of GTP are abolished by increasing (Mg2+) and pH to 50 mM and 8.5, respectively. Under these conditions, Gpp(NH)p and GTP cause marked stimulation of activity, the stimulatory effect of Gpp(NH)p being greater than that of GTP both in the absence and presence of hormones...