Human fat cell adenylate cyclase. Enzyme characterization and guanine nucleotide effects on epinephrine responsiveness in cell membranes.

Human adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) has been studied in preparations of fat cell membranes ("ghosts"). As reported earlier, under ordinary assay conditions (1.0 mM ATP, 5 mM Mg2+, 30 degrees C, 10 min incubation) the enzyme was activated 6-fold by epinephrine in the presence of the GTP analog, 5'-guanylyl-imidodiphosphate [GMP-P(NH)P] (Cooper, B. et al. (1975) J. Clin. Invest. 56, 1350-1353). Basal activity was highest during the first 2 min of incubation then slowed and was linear for at least the next 18 min. Epinephrine, added alone, was often without effect. but sometimes maintained the initial high rate of basal activity. GMP-P(NH)P alone produced inhibition ("lag") of basal enzyme early in the incubation periods. Augmentation of epinephrine effect by GMP-P(NH)P, which also proceeded after a brief (2 min) lag period, was noted over a wide range of substrate (ATP) concentrations. GTP inhibited basal levels of the enzyme by about 50%. GTP also allowed expression of an epinephrine effect, but only in the sense that the hormone abolished the inhibition by GTP. Occasionally a slight stimulatory effect on epinephrine action was seen with GTP. At high Mg2+ concentration (greater than 10 mM) or elevated temperatures (greater than 30 degrees C) GMP-P(NH)P alone activated the enzyme. Maximal activity of human fat cell adenylate cyclase was seen at 50 mM Mg2+, 1.0 mM ATP, pH 8.2, and 37 degrees C in the presence of 10(-4) M GMP-P(NH)P; under these conditions addition of epinephrine did not further enhance activity. Human fat cell adenylate cyclase of adults was insensitive to ACTH and glucagon even in the presence of GMP-P(NH)P.     

Calcitonin-sensitive adenylate cyclase in rat renal tubular membranes.

1. Renal tubular membranes from rat kidneys were prepared, and adenylate cyclase activity was measured under basal conditions, after stimulation by NaF or salmon calcitonin. Apparent Km value of the enzyme for hormone-linked receptor was close to 1 x 10(-8) M. 2. The system was sensitive to temperature and pH. pH was found to act both on affinity for salmon calcitonin-linked receptor and maximum stimulation, suggesting an effect of pH on hormone-receptor binding and on a subsequent step. 3. KCl was without effect areas whereas CoCl and CaCl2 above 100 muM and MnCl2 above 1 muM inhibited F- -and salmon calcitonin-sensitive adenylate cyclase activities. The Ca2+ inhibition of the response reflected a fall in maximum stimulation and not a loss of affinity of salmon calcitonin-linked receptor for the enzyme. 4. The measurement of salmon calcitonin-sensitive adenylate cyclase activity as a function of ATP concentration showed that the hormone increases the maximum velocity of the adenylate cyclase. GTP, ITP and XTP at 200 muM did not modify basal, salmon calcitonin- and parathyroid hormone-sensitive adenylate cyclase activities. 5. Basal, salmon calcitonin- and F- -sensitive adenylate cyclase activities decreased at Mg2+ concentrations below 10 mM. High concentrations of Mg2+ (100 mM) led to an inhibition of the F- -stimulated enzyme. 6. Salmon calcitonin-linked receptor had a greater affinity for adenylate cyclase than human or porcine calcitonin-linked receptors. There was no additive effect of these three calcitonin peptides whereas parathyroid hormone added to salmon calcitonin increased adenylate cyclase activity, thus showing that both hormones bound to different membrane receptors. Human calcitonin fragments had no effect on adenylate cyclase activity. 7. Salmon calcitonin-stimulated adenylate cyclase activity decreased with the preincubation time. This was due to progressive degradation of the hormone and not to the rate of binding to membrane receptors.     

Activation of adenylate cyclase by molybdate.

The effect of molybdate on adenylate cyclase (EC 4.6.1.1) in rat liver plasma membranes has been examined. The apparent K alpha for molybdate activation of the enzyme is 4.5 mM, and maximal, 7-fold stimulation is achieved at 50 mM. The observed increase in cAMP formation in the adenylate cyclase assay is not due to: (a) an inhibition of ATP hydrolysis; (b) a molybdate-catalyzed conversion of ATP to cAMP; (c) an inhibition of cAMP hydrolysis; or (d) an artifact in the isolation of cAMP formed in the reaction. Molybdate activation of adenylate cyclase is a general phenomenon exhibited by the enzyme in brain, cardiac, and renal tissue homogenates and in erythrocyte ghosts. However, like fluoride and guanyl-5'-yl imidodiphosphate (Gpp(NH)p), molybdate does not activate the soluble rat testicular adenylate cyclase. Molybdate is a reversible activator of adenylate cyclase. Activation is not due to an increase in ionic strength and is independent of the salt used to introduce molybdate. Molybdate does not activate adenylate cyclase previously stimulated with Gpp(NH)p or fluoride. At concentration greater than 20 mM, molybdate inhibits fluoride-stimulated adenylate cyclase, and at concentrations greater than 100 mM, molybdate stimulation of basal adenylate cyclase activity is diminished.     

Adenylate cyclase in bloodstream forms of Trypanosoma (Trypanozoon) brucei sp.

1. The adenylate cyclase in Trypanosoma brucei is located in the plasma membrane. 2. A partial kinetic analysis of the properties of the enzyme revealed a Km for ATP of 1.75 mM and a Km for Mg2+ of 4mM. 3. At low concentrations, Mg2+ activated the enzyme directly in addition to its effect of lowering the concentration of inhibitory free ATP species. 4. At high concentrations, Mg2+ inhibited the enzyme. Furthermore, the enzyme was inhibited at any Mg2+ concentration if the concentration of ATP exceeded that of Mg2+. 5. The opposing effects of Mg2+ at low and high concentrations would be consistent with more than one binding site for Mg2+ on the enzyme. 6. A study of the patterns of product inhibition revealed little or no effect of 3':5'-cyclic AMP, but a profound inhibition by pyrophosphate, which was competitive with respect to ATP (Ki 0.135 mM). This result suggests that the substrate-binding domain on T. brucei adenylate cyclase interacts mainly with the triphosphate portion of the ATP molecule. 7. The enzyme activity was unaffected by the usual mammalian enzyme effectors glucagon, adrenaline, adenosine, GTP and guanyl-5'-yl imidodiphosphate. 8. The enzyme was not activated by fluoride, instead a powerful inhibition was found. The enzyme was also inhibited by relatively high concentrations of Ca2+ (1 mM).     

Effects of glucose, glucose metabolites and calcium ions on adenylate cyclase activity in homogenates of mouse pancreatic islets.

The effects of glucose, a series of glucose metabolites, nicotinamide nucleotides, Ca2+ and p-chloromercuribenzenesulphonate on adenylate cyclase activity in homogenates of mouse pancreatic islets were studied. The basal activity of the adenylate cyclase was approx. 6 pmol of cyclic AMP formed/30 min per microng of DNA at 30 degrees C. The enzyme activity was stimulated by some 150% by fluoride. Starvation of the animals for 48h had no effect on either the basal or the fluoride-stimulated activity. The adenylate cyclase activity was increased by 40-50% when 17 mM-glucose, 10 micronM-phosphoenolpyruvate or 10 micronM-pyruvate was added to the assay medium. The effect of glucose was unchanged in the presence of 17 mM-mannoheptulose, and mannoheptulose alone had no effect. The other glycolytic intermediates, and the coenzymes NAD+, NADH and NADPH, at concentrations up to 1 mM were without any detectable effect on the rate of formation of cyclic AMP. The insulin secretagogue p-chloromercuribenzenesulphonate inhibited the adenylate cyclase markedly even at a concentration of 10 micronM. Calculated concentrations of free Ca2+ of 10 micronM and 0.1 mM inhibited adenylate cyclase by 29 and 71% respectively. It is concluded that both glucose itself and phosphoenolpyruvate and/or pyruvate are true activating ligands for islet and adenylate cyclase and that inhibition of the cyclase by Ca2+ may be of physiological significance.     

Effect of temperature on the adenylate cyclase activity of Mytilus galloprovincialis mantle tissue.

1. The basal and NaF-stimulated adenylate cyclase activities of Mytilus galloprovincialis mantle tissue were studied at different temperatures. 2. There are no significant differences in the Km for ATP at 13 degrees C and 20 degrees C in both basal and NaF-stimulated conditions. 3. NaF increases the Vmax of the enzyme (5-fold) and decreases about 50% the Km for ATP at both temperatures assayed. 4. Activation energy of the enzyme reaction is 33.4 kJ/mol. K in basal conditions and 29.4 kJ/mol. K when NaF is present. The Q10, at saturating substrate concentrations, is approximately 1.5 and this value is constant in the temperature range studied, 10-30 degrees C. 5. The adenylate cyclase starts being inactivated from 30 degrees C. The enzyme shows greater sensitivity to denaturalization by temperature in NaF-stimulated than in basal conditions.     

Inhibition of hepatic adenylate cyclase by NADH

Adenylate cyclase activity in isolated rat liver plasma membranes was inhibited by NADH in a concentration-dependent manner. Half-maximal inhibition of adenylate cyclase was observed at 120 microM concentration of NADH. The effect of NADH was specific since adenylate cyclase activity was not altered by NAD+, NADP+, NADPH, and nicotinic acid. The ability of NADH to inhibit adenylate cyclase was not altered when the enzyme was stimulated by activating the cyclase was not altered when the enzyme was stimulated by activating the Gs regulatory element with either glucagon or cholera toxin. Similarly, inhibition of Gi function by pertussis toxin treatment of membranes did not attenuate the ability of NADH to inhibit adenylate cyclase activity. Inhibition of adenylate cyclase activity to the same extent in the presence and absence of the Gpp (NH) p suggested that NADH directly affects the catalytic subunit. This notion was confirmed by the finding that NADH also inhibited solubilized adenylate cyclase in the absence of Gpp (NH)p. Kinetic analysis of the NADH-mediated inhibition suggested that NADH competes with ATP to inhibit adenylate cyclase; in the presence of NADH (1 mM) the Km for ATP was increased from 0.24 +/- 0.02 mM to 0.44 +/- 0.08 mM with no change in Vmax. This observation and the inability of high NADH concentrations to completely inhibit the enzyme suggest that NADH interacts at a site(s) on the enzyme to increase the Km for ATP by 2-fold and this inhibitory effect is overcome at high ATP concentrations.     

Stimulation of rat liver adenylate cyclase by halothane.

Rat liver membranes prepared by a modification of the procedure of Neville were exposed to clinical and toxic concentrations of the general anesthetic, halothane, for 10 minutes. Basal, glucagon (5 × 10⁻⁵M) and sodium fluoride (20 mM) stimulated adenylate cyclase activity was assayed. Clinical and toxic concentrations of halothane augmented basal adenylate cyclase activity. Glucagon and sodium fluoride stimulated adenylate cyclase activity was enhanced at greater than clinically useful halothane concentrations only. The study provides direct evidence that halothane stimulates adenylate cyclase, the extent of augmentation of enzyme activity is halothane concentration dependent, and modified by other drugs.     

The role of surface charge and hydrophobic interaction in the activation of rat liver adenylate cyclase

Rat liver plasma membranes were incubated either with procaine, lidocaine or tetracaine to study the binding of glucagon to receptors and the responses of adenylate cyclase to glucagon or fluoride. Procaine treatment increased the glucagon and fluoride activation of the cyclase and the stimulation was concentration-dependent; this compound seemed to act at the G/F unit level since changes in the glucagon binding were not observed and the basal activity was not modified. Tetracaine inhibited the adenylate cyclase activity in the order glucagon greater than basal greater than fluoride; it seems that tetracaine acted at the receptor unit level since it reduced the binding affinity. Tetracaine at high concentration (10 mM) also inhibited the fluoride stimulation of the Lubrol PX-solubilized enzyme; apparently the anesthetic acts on the G/F unit and this would indicate the component is still bound to the catalytic unit. The solubilized enzyme is not longer stimulated by procaine. These data suggested that the F component site of the G/F units is in some aspects different to the G component and more resistant to the detergent. The results of this work allowed a clear distinction among the different components of the glucagon-stimulated adenylate cyclase system and showed the importance of surface charge and hydrophobic interactions as regulatory mechanisms.     

Activation of rat adipocyte plasma membrane adenylate cyclase by sodium azide

The adenylate cyclase of rat adipocyte plasma membrane is stimulated by sodium azide with a half maximal activation of 100–150% occuring at 50 mM NaN₃. Studies of the effects of azide and fluoride indicate different mechanisms of stimulation of the enzyme by these ions. Comparable stimulation of the activity is obtained by 100 mM NaN₃ or 10 mM NaF but unlike azide, higher concentrations of fluoride cause inhibition of the enzyme. Fluoride activated adenylate cyclase is further stimulated by azide. Epinephrine stimulation of the enzyme is absent in the presence of fluoride but the hormone enhances the activity in the presence of azide. Reversal of the inhibitory action of GTP on adenylate cyclase by epinephrine is demonstrated even in the presence of azide but not in the presence of fluoride.     

Inhibition of a parathyroid hormone-stimulated renal adenylate cyclase by cyclic AMP.

Plasma membranes were isolated from bovine renal cortex. This particulate, adenylate cyclase-containing fraction was stimulated to produce cyclic AMP by parathyroid hormone and fluoride. When the time-course of adenylate cyclase activity was investigated, it was found that while PTH-stimulated cyclic AMP production comes to a halt in about 15 minutes after the initiation of the reaction, fluoride-stimulated activity continues unabated for at least an hour. Experiments to determine the cause of this showed that the cyclase enzyme is not degraded under our experimental conditions, but is inhibited by a soluble, unbound product of the reaction which requires ATP for its synthesis. In our experiments degradation of parathyroid hormone was relatively slow and could not account for the rapid inhibition of PTH-stimulated cyclase activity. Of the various agents tested, cyclic AMP was found capable of inhibiting PTH-stimulated cyclic AMP production by our purified membrane preparation. Half-maximal inhibition was observed at around 10(-6) M concentrations of the nucleotide. Pyrophosphate, adenosine, 5'-AMP and ADP had no effects. The significance of these results in relation to the regulation of adenylate cyclase activity is discussed.     

Modulation of the response of bovine adrenocortical adenylate cyclase to corticotropin.

An assessment was made of some of the basic parameters responsible for the modulation of adenylate cyclase activity in a bovine adrenocortical plasma-membrane preparation. When determined at 0.1 mM-ATP, basal adenylate cyclase activity increased with increasing MgCl2 concentrations, whereas in the presence of corticotropin activity was essentially maximal at 10mM-MgCl2; high concentrations (25mM) of MgCl2 inhibited adenylate cyclase activity determined in the presence of both corticotropin and GTP. At all MgCl2 concentrations, corticotropin and GTP activated the enzyme in a synergistic fashion. The magnitude of the stimulation of basal activity produced by corticotropin was a function of Mg2+ concentration, whereas that produced by GTP appeared largely independent of Mg2+ concentration. Adenylate cyclase activity in the bovine adrenal membrane was half-maximally stimulated by corticotropin concentrations in the range 0.3--1.0 nM. The concentration of corticotropin evoking half-maximum response was not significantly affected by raising the free Mg2+ concentration from 0.4 to 4.9 mM, nor by the presence of GTP. In the presence of GTP, high concentrations (over 1 micrometer) of corticotropin inhibited adenylate cyclase activity, although no inhibition was apparent in the absence of guanine nucleotide.     

Regulation by calcium and calmodulin of adenylate cyclase from rabbit intestinal epithelium

The effect of calcium on adenylate cyclase from rabbit small intestine has been studied using a particulate preparation obtained from isolated epithelial cells. Both basal and vasoactive intestinal peptide-stimulated activities were inhibited by calcium concentrations in the micromolar range. In the presence of calmodulin, a biphasic response was obtained. At low calcium concentration (4 X 10(-9)-6 X 10(-8) M) the enzyme was activated up to 50%. As the Ca2+ concentration was increased, the enzyme was concomitantly inhibited. Half-maximal inhibition of calmodulin-dependent activity was obtained at 1 microM free Ca2+. The activation of the enzyme was also dependent on the concentration of Mg2+. At less than 1 microM Ca2+, the enzyme exhibited a biphasic response, being activated at below 3 mM Mg2+ and inhibited at higher concentrations. At Ca2+ concentrations that were inhibitory, the enzyme did not show the biphasic response to Mg2+. At concentrations above 3 mM, the maximal rate (Vmax) remained constant. Vmax was inversely proportional to the concentration of Ca2+ present. Calmodulin altered Vmax when acting on vasoactive intestinal peptide-stimulated enzyme. Calmodulin had no effect on the Km for hormone activation. The calmodulin-dependent activity was inhibited by incubation with trifluoperazine.     

Effect of proteases and protease inhibitors on adenylate cyclase activity in rat cerebral cortical membranes

Mild proteolysis of membrane preparations from rat cerebral cortex with low concentrations of endopeptidases such as trypsin or chymotrypsin caused a 50–400% increase in the basal adenylate cyclase activity. Maximal activation of adenylate cyclase was obtained by including the protease in the adenylate cyclase assay, although an activated preparation could be obtained by pretreatment of the membranes with proteolytic enzymes. The proteolytically activated enzyme showed an increased V, with very little change in the K_m for the substrate, ATP. The proteolytically activated enzyme retained responsiveness to activation by sodium fluoride and 5′-guanylylimidodiphosphate (GppNHp), but was no longer activated by gangliosides or calcium-dependent activator protein. Activation by alcohols and detergent was lost or reduced in magnitude. The activity of adenylate cyclase after protease treatment showed a very marked temperature dependence, with maximal activity expressed in the 30–40 °C range and no activation due to the prior protease treatment expressed at either 10 or 50 °C. Basal adenylate cyclase activity was usually slightly inhibited in the presence of various protease inhibitors. Activation by fluoride, gangliosides, or GppNHp was little affected by protease inhibitors although one inhibitor, N-α-tosyl-l-lysine chloromethyl ketone, caused an inhibition of the ganglioside and GppNHp responses, slightly inhibited the fluoride response, and blocked the norepinephrine response normally seen in the presence of gangliosides or GppNHp. This inhibitor caused a loss of β-adrenergic binding sites for dihydroalprenolol in rat cortical membranes which paralleled the loss of the responsiveness of adenylate cyclase to a GppNHp-norepinephrine combination.     

Inhibition of adenylate cyclase from luteinized rat ovary by monovalent cations: roles of the stimulatory guanine nucleotide-binding regulatory component and stimulatory hormone receptor

Sodium and other monovalent cations (added as chloride salts) inhibited adenylate cyclase of luteinized rat ovary. Sodium chloride (150 mM) inhibited basal enzyme activity by 20%. Sodium chloride inhibition was enhanced to 34-54% under conditions of enzyme stimulation by guanine nucleotides (GTP and its nonhydrolyzable analog 5'-guanylyl imidodiphosphate), fluoride anion, and agonists (ovine luteinizing hormone (oLH) and the beta-adrenergic catecholamine isoproterenol) acting at stimulatory receptors linked to adenylate cyclase. Sodium chloride inhibition was dependent on salt concentration over a wide range (25-800 mM) as well as the concentrations of GTP and oLH. Inhibition by NaCl was of rapid onset and appeared to be reversible. The order of inhibitory potency of monovalent cations was Li+ greater than Na+ greater than K+. The role of individual components of adenylate cyclase in the inhibitory action of monovalent cations was examined. Exotoxins of Vibrio cholerae and Bordetella pertussis were used to determine respectively the involvement of the stimulatory and inhibitory guanine nucleotide-binding regulatory components (Ns and Ni) in NaCl inhibition. Sodium chloride inhibited cholera toxin-activated adenylate cyclase activity by 29%. Ni did not appear to mediate cation inhibition of adenylate cyclase because pertussis toxin did not attenuate inhibition by NaCl. Enzyme stimulation by agents (forskolin and Mn2+) thought to activate the catalytic component directly was not inhibited by NaCl but was instead significantly enhanced. Sodium chloride (150 mM) increased both the Kd for high-affinity binding of oLH to 125I-human chorionic gonadotropin binding sites and the Kact for oLH stimulation of adenylate cyclase by sevenfold. In contrast, NaCl had no appreciable effect on either isoproterenol binding to (-)-[125I]iodopindolol binding sites or the Kact for isoproterenol stimulation of adenylate cyclase. The results suggest that in luteinized rat ovary monovalent cations uncouple, or dissociate, Ns from the catalytic component and, in a distinct action, reduce gonadotropin receptor affinity for hormone. Dissociation of the inhibitory influence of Ni from direct catalytic activation could account for NaCl enhancement of forskolin- and Mn2+-associated activities. On the basis of these results, the spectrum of divergent stimulatory and inhibitory effects of monovalent cations on adenylate cyclase activities in a variety of tissues may be interpreted in terms of differential enzyme susceptibilities to cation-induced uncoupling of N and catalytic component functions.     

Multiple inhibitory and activating effects of nucleotides and magnesium on adrenal adenylate cyclase.

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.     

Insulin release from mouse islets. Effect of glucose and hormones on adenylate cyclase

The adenylate cyclase system of normal mouse islets was characterized. The pH optimum of the system was 7.6. The enzyme preparation contained particulate phosphodiesterase activity. This could be removed by treatment with 0.4% (v/v) Triton X-100 or inhibited by 8mm-theophylline in the presence of 2mm-cyclic AMP (adenosine 3':5'-cyclic monophosphate). ATP at 0.32mm produced one-half maximal enzyme activity. The enzyme was stimulated in the presence of F(-) and strongly inhibited by Ca(2+). The isolated enzyme retained hormonal sensitivity and was stimulated by glucagon, pancreozymin and secretin at physiological concentrations. Glucose at 17mm, 8mm and 2mm had no direct effect on the activity of the enzyme; neither did galactose at the same concentrations. Groups of islets incubated in 17mm- or 2mm-glucose for 5 or 15min and then homogenized and assayed for adenylate cyclase activity showed no differences in adenylate cyclase activity. The results suggest that the mechanism of glucose-mediated insulin release is not via the adenylate cyclase system. Hormones, however, could mediate insulin secretion via their effects on the adenylate cyclase system.     

The hamster adipocyte adenylate cyclase system I. Regulation of enzyme stimulation and inhibition by manganese and magnesium ions

In hamster adipocyte ghosts, ACTH stimulates adenylate cyclase by a GTP-dependent process, whereas prostaglandin E E₁, α-adrenergic agonists and nicotinic acid inhibit the enzyme by a mechanism which is both GTP- and sodium-dependent. The influence of the divalent cations Mn²⁺ and Mg²⁺, was studied on these two different, apparently receptor-mediated effects on the adipocyte adenylate cyclase. At low Mn²⁺ concentrations, GTP (1 μM) decreased enzyme activity by about 80%. Under this condition, ACTH (0.1 μM) stimulated the cyclase by 6- to 8-fold, and NaCl (100 mM) caused a similar activation. In the presence of both GTP and NaCl, prostaglandin E₁ (1 or 10 μM) and nicotinic acid (30 μM) inhibited the enzyme by about 70–80% and epinephrine (300 μM, added in combination with a β-adrenergic blocking agent) by 40–50%. With increasing concentrations of Mn²⁺, the GTP-induced decrease and the NaCl-induced increase in activity diminished, with a concomitant decrease in prostaglandin E_1^?, nicotinic acid- and epinephrine-induced inhibitions as well as in ACTH-induced stimulation. At 1 mM Mn²⁺, inhibition of the enzyme was almost abolished and stimulation by ACTH was largely reduced, whereas activation of the enzyme by KF (10 mM) was only partially impaired. The uncoupling action of Mn²⁺ on hormone-induced inhibition was half-maximal at 100–200 μM and appeared not to be due to increased formation of the enzyme substrate, Mn · ATP. It occurred without apparent lag phase and could not be overcome by increasing the concentration of GTP. Similar but not identical findings with regard to adenylate cyclase stimulation and inhibition by hormonal factors were obtained with Mg²⁺, although about 100-fold higher concentrations of Mg²⁺ than of Mn²⁺ were required. The data indicate that Mn²⁺at low concentrations functionally uncouples inhibitory and stimulatory hormone receptors from adenylate adenylate cyclase in membrane preparations of hamster adipocytes, and they suggest that the mechanism leading to uncoupling involves an action of Mn²⁺ on the functions of the guanine nucleotide site(s) in the system.     

Proteolysis of skeletal muscle adenylate cyclase. Destruction and reconstitution of fluoride and guanylnucleotide sensitivity

Adenylate cyclase was measured in skeletal muscle plasma membranes incubated with subtilisin. Under specific conditions the protease preferentially inactivated fluoride and guanylnucleotide sensitivity. Following protease treatment, membranes were solubilized with Lubrol 12A9 and subjected to ion-exchange chromatography. Adenylate cyclase was eluted with 200 mM NaCl; the enzyme recovered was completely unresponsive to either NaF or guanylyl imidodiphosphate. Responsiveness to the two ligands was restored by adding a heart fraction in which basal activity had been destroyed by heating at 40 degrees C or by adding a soluble skeletal muscle fraction in which basal activity had been largely destroyed by N-ethylmaleimide. The solubilized subtilisin-treated skeletal muscle preparation may serve as a source of catalytic activity for the study and purification of regulatory factors for adenylate cyclase.