Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.6.1.1 (adenylate cyclase)
 19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The activation energy of adenylate cyclase by p[NH]ppG in rat pancreatic plasma membranes was estimated to be 141-189 kj/mol. When a high concentration of secretin or CCK-8 (C-terminal octopeptide of cholecystokinin-pancreozimin) was added to the assay medium, the activation energy was reduced to 73 kj/mol. This hormone effect was exerted on the activation energy of the activation process of adenylate cyclase by p[NH]ppG. Indeed, when plasma membranes were preactivated with p[NH]ppG alone or with p[NH]ppG and CCK-8 and then washed, there resulted a persistent activation with low activation energy (65 and 48 kj/mol, respectively). A similar low activation energy was observed in membranes preincubated with GMP and CCK-8. The latter treatment could not induce persistent activation but facilitated the activation by p[NH]ppG, suggesting that the step of p[NP]ppG activation requiring a high activation energy in the absence of hormone had developed during preincubation with GMP and CCK-8, and had not been reversed by membrane washing. By contrast, EDTA pretreatment did not influence p[NH]ppG activation while provoking a reversible deactivation of persistently activated adenylate cyclase.
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PMID:Effect of hormone and guanyl nucleotide pretreatment on the activation energy of pancreatic adenylate cyclase. 11 1

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.
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PMID:Multiple inhibitory and activating effects of nucleotides and magnesium on adrenal adenylate cyclase. 16 69

The ability of 5'-guanylylimidodiphosphate (Gpp(NH)p) to stimulate irreversibly the adenylate cyclease activity of fat cell membranes has been studied by preincubating the membranes with this or related analogs followed by assaying after thoroughly washing the membranes. Activation can occur in a simple Tris-HCl buffer, in the absence of added divalent cations and in the presence of EDTA. Dithiothreitol enhances the apparent degree of activation, perhaps by stabilization. The importance of utilizing optimal conditions for stabilizing enzyme activity, and of measuring the simultaneous changes in the control enzyme, is illustrated. The organomercurial, p-aminophenylmercuric acetate, inhibits profoundly the activity of the native as well as the Gpp(NH)p-stimulated adenylate cyclase, but in both cases subsequent exposure to dithiothreitol restores fully the original enzyme activity. However, the mercurial-inactivated enzyme does not react with Gpp(NP)p, as evidenced by the subsequent restoration of only the control enzyme activity upon exposure to dithiothreitol. Thus, reaction with Gpp(NH)p requires intact sulfhydryl groups, but the activated state is not irreversibly destroyed by the inactivation caused by sulfhydryl blockade. GTP and, less effectively, GDP and ATP inhibit activation by Gpp(NH)p, but interpretations are complicated by the facts that this inhibition is overcome with time and that GTP and ATP can protect potently from spontaneous inactivation. These two nucleotides can be used in the Gpp(NH)p preincubation to stabilize the enzyme. The Gpp(NH)p-activated enzyme cannot be reversed spontaneously during prolonged incubation at 30 degrees C in the absence or presence of GTP, ATP, MgCl2, glycine, dithiothreitol, NaF or EDTA. The strong nucleophile, neutral hydroxylamine, decreases the Gpp(NH)p-activated enzyme activity and no subsequent activation is detected upon re-exposure to the nucleotide.
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PMID:Irreversible stimulation of adenylate cyclase activity of fat cell membranes of phosphoramidate and phosphonate analogs of GTP. 17 35

1. Adenylate cyclase in plasma membranes from rat liver was stimulated by prostaglandin E1, and to a lesser extent by prostaglandin E2. Prostaglandin F1alpha and A1 did not stimulate the cyclase. The prostaglandin E1-mediated activation was found to require GTP when the substrate ATP concentration was reduced from 3 mM to 0.3 mM in the reaction mixture. Adenylate cyclase of the plasma membranes from rat ascites hepatomas AH-130 and AH-7974 was not stimulated by prostaglandin E1 in the presence or the absence of GTP, although the basal activity of adenylate cyclase as well as its stimulation by GTP alone were similar to normal liver plasma membranes. 2. Liver plasma membranes were found to have two specific binders for [3H] prostaglandin E1 with dissociation constants of 17.6-10(-9) M and 13.6-10(8) M (37 degrees C) and one specific binder for [3H]prostaglandin F2alpha with a dissociation constant of 2.31-10(8) M (37 degrees C). The specific binders for prostaglandin E1 could not be detected in the hepatoma plasma membranes. 3. Binding of [3H] prostaglandin E1 to the liver plasma membranes was exchange by, GTP dGPT, GDP, ATP and GMP-P(N)P, but not by GMP, CGMP, DTTP, UTP or CTP. The increase in the binding of [3H] prostaglandin E1 was found to be due to the increased affinity of the specific binders to prostaglandin F2alpha was not affected by GTP. 4. GTP alone was found to increase V of adenylate cyclase of liver plasma membranes, while GTP plus prostaglandin E1 was found to decrease Km of adenylate cyclase in addition to the increase of V to a further extent.
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PMID:Prostaglandin receptor-adenylate cyclase system in plasma membranes of rat liver and ascites hepatomas, and the effect of GTP upon it. 18 13

Epinephrine increased adenylate cyclase activity 10 to 15 fold in lysates of the cultured human astrocytoma cell line 132-1N1. GTP had little effect on adenylate cyclase activity of lysed cell preparations either with or without added epinephrine. However, the epinephrine stimulation of adenylate cyclase was essentially lost (less than 90%) when a washed nuclei-free membrane preparation of the cyclase was assayed. A 10 to 15 fold epinephrine stimulation of the membrane adenylate cyclase could be demonstrated if cytosol of GTP were added to the assay with the hormone. The criteria of anion exchange, cation exchange, gel exclusion and paper chromatography indicated that the cytosolic agents which acted synergistically with hormones were GTP and GDP. The apparent Kact's for the synergistic action of GDP and GTP were essentially identical (1.0 muM) and of all the other nucleotides examined only GDP had a potency similar to GTP. However, the effect of GDP was apparently due to its rapid conversion to GTP even in the absence of a regenerating system. With epinephrine pretreatment of the intact 132-1N1 cells there was a specific loss of epinephrine stimulation of adenylate cyclase activity. The hormone pretreatment did not alter the capacity of the cytosol from these desensitized cells to potentiate epinephrine stimulation of the cyclase. Rather, the alteration was in the particulate fraction of the lysate. The desensitization of the membranous cyclase was stable and not reversed by GTP.
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PMID:Endogenous GTP and the regulation of epinephrine stimulation of adenylate cyclase. 20 54

The fate of the guanyl nucleotide bound to the regulatory site of adenylate cyclase was studied on a preparation of turkey erythrocyte membranes that was incubated with [3H]GTP plus isoproterenol and subsequently washed to remove hormone and free guanyl nucleotide. Further incubation of this preparation in the presence of beta-adrenergic agonists resulted in the release from the membrane of tritiated nucleotide, identified as [3H]GDP. The catecholamine-induced release of [3H]GDP was increased 2 to 3 times in the presence of the unlabeled guanyl nucleotides GTP, guanosine 5'-(beta,gamma-imino)triphosphate [gpp(NH)p], GDP, and GMP, whereas adenine nucleotides had little effect. In the presence of Gpp(NH)p, isoproterenol induced the release of [3H]GDP and the activation of adenylate cyclase, both effects following similar time courses. The findings indicate that the inactive adenylate cyclase possesses tightly bound (GDP, produced by the hydrolysis of GTP at the regulatory site. The hormone stimulates adenylate cyclase activity by inducing an "opening" of the guanyl nucleotide site, resulting in dissociation of the bound GDP and binding of the activating guanosine triphosphate.
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PMID:Mechanism of adenylate cyclase activation through the beta-adrenergic receptor: catecholamine-induced displacement of bound GDP by GTP. 21 37

A comparison was made of the effects of cholera toxin and p[NH]ppG on the binding affinity of beta-adrenergic receptors in toad erythrocyte membranes. This was determined by studying the ability of isoproterenol and propranolol to compete for the receptor with (-)-[3H]dihydroalprenolol. p[NH]ppG decreased the receptor affinity for the agonist isoproterenol (i.e. a 'right' shift in the displacement-concentration curve), but was without effect on the affinity for the antagonist propranolol. Toad erythrocyte membranes after treatment with cholera toxin exhibited increased receptor affinity for isoproterenol (i.e. a 'left' shift in the displacement curve), but did not affect the affinity for propranolol. p[NH[ppG was able to exert its right shift even in cholera-toxin treated membranes. The ability of cholera toxin to alter beta-adrenergic-receptor affinity is interpreted as further evidence that the toxin affects the nucleotide-regulatory component of adenylate cyclase. The regulatory component affected may be the catecholamine-sensitive guanosine triphosphatase.
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PMID:Effects of cholera toxin and guanosine 5'-[betagamma-imido]triphosphate on beta-adrenergic-receptor affinity. 21 63

The beta 1-adrenergic receptors of turkey erythrocyte membranes have been identified by binding of the radioactively labeled antagonist (--)-[3H]dihydroalprenolol, solubilized by treatment of the membranes with the detergent digitonin, and purified by affinity chromatography. Binding of (--)-[3H]dihydroalprenolol to the membranes occurred to a single class of non-cooperative binding sites (0.2--0.3 pmol/mg protein) with a equilibrium dissociation constant (Kd) of 8 (+/- 2) nM. These sites were identified as the functional, adenylate-cyclase-linked beta 1-adrenergic receptors on the basis of: firstly, the fast association and dissociation binding kinetics at 30 degrees C; secondly, the stereospecific displacement of bound (--)-[3H]dihydroalprenolol by beta-adrenergic agonists and antagonists; and thirdly, the order of potencies for agonists to displace bound tracer (isoproterenol congruent to protokylol greater than norepinephrine congruent to epinephrine) similar to the one found for adenylate cyclase activation, and typical for beta 1-adrenergic receptors. Treatment of the membranes with the detergent digitonin solubilized 30% of the receptors in an active form. Digitonin solubilized also adenylate cyclase activity with a yield of 20 to 30%, provided the membranes were first treated with an effector known to produce a persistent active state of the enzyme: e.g. sodium fluoride. Binding sites for guanine nucleotides ([3H]p[NH]ppG) were solubilized as well. Their concentration (24 pmol/mg protein) was in large excess over the concentration of solubilized receptors (0.30--0.45 pmol/mg protein). Solubilized receptors were purified 500--2000-fold by affinity chromatography with a 25 to 35% yield, using an alprenolol-agarose affinity matrix. Affinity purified receptors were devoid of measurable adenylate cyclase activity and guanine nucleotide binding sites, thus showing that receptors and adenylate cyclase are distinct membrane constituents, and that guanine nucleotides apparently do not bind directly to the receptor molecules. Membrane-bound, solubilized and purified receptors were sensitive to inactivation by dithiothreitol, but not by N-ethylmaleimide, suggesting that receptors are at least partly constituted of protein molecules, with essential disulfide bonds.
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PMID:Affinity chromatography of the beta-adrenergic receptor from turkey erythrocytes. 22 63

In rat liver plasma membranes preactivated with guanosine 5'-[beta,gamma-imido[triphosphate (GuoPP[NH]P), GDP promoted coupling of occupied glucagon receptor to adenylyl cyclase [adenylate cyclase; ATP, pyrophosphate-lyase (cyclizing), EC 4.6.1.1] with an apparent association constant Ka of 0.1-0.15 microM. The apparent Ka for the same effect of GTP was 0.2 microM. The effect of GDP was shown not to be due to GTP formed by putative transphosphorylation reaction(s) when ATP was present in the assay as substrate. In membranes not preactivated with GuoPP[NH]P, GDP both competitively inhibited GuoPP[NH]P stimulation of adenylyl cyclase (Ki 0.10 microM) and supported stimulation of cyclizing activity (apparent Ka 0.10 microM) by glucagon. These effects of GDP occurred in the absence of added GTP and in the absence of sufficient formation of GTP by putative transphosphorylation reaction(s) to account for them. It is concluded that two levels of regulation of liver adenylyl cyclase (cyclizing) activity must exit. One level is termed "receptor regulation"; it depends on occupancy of a receptor-related R site by nucleotide and is specific for either GDP or GTP. The second level of regulation is termed "GTPase regulation"; it is inhibited by GDP, depends on both GTP and GTPase, and accounts for activation of cyclizing activity by nonhydrolyzable analogs of GTP. The data suggest that both levels of regulation coexist and may synergize, one mediating responses to stimuli external to the cell (receptor regulation) and the other mediating stimuli of intracellular origin (GTPase regulation).
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PMID:Coupling of the glucagon receptor to adenylyl cyclase by GDP: evidence for two levels of regulation of adenylyl cyclase. 22 58

When adipocyte membranes are successively exposed to (-)-propranolol or (+/- alprenolol at 25 or 4 degrees C, repeatedly washed and then assayed for (-)-[3H]dihydroalprenolol binding, the apparent number of beta-adrenergic binding sites is markedly decreased. Induction of this peculiar type of receptor desensitization does not require prolonged exposure of the membranes to the beta-adrenergic antagonists (half-time: 1 min), is stereospecific, concentration-dependent and almost complete with high concentrations of antagonists. p[NH]ppG, which reduces the affinity of fat cell beta-adrenergic receptors for agonists, does not prevent the antagonist-induced decrease in the receptor number. The magnitude of the desensitizating effect induced separately by (-)-isoproterenol and (-)-propranolol is not additive in membranes exposed to both drugs, suggesting that the receptors lost after exposure to agonists are the same sites as part of those lost after exposure to antagonists. However, contrary to the results found in membranes desensitized by agonists, adenylate cyclase activity remained fully responsive to catecholamines in membranes exposed to beta-antagonists. As shown by kinetic studies on (-)-[3H]dihydroalprenolol binding, this beta-antagonist-induced receptor desensitization is reversible after prolonged incubation. These data which have never yet been described in the other reported desensitizable beta-adrenergic systems, suggest that, when exposed to beta-antagonists, the fat cell beta-adrenergic receptors undergo a conformational change leading to a peculiar state which has low affinity for antagonists but behaves towards agonists as does the receptor in its resting state.
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PMID:Evidence for a second desensitized state of beta-adrenergic receptor with low affinity for beta-antagonists and normal reactivity towards beta-agonists in adipocyte membranes previously exposed to beta-antagonists. 22 82


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