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 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

Binding of cAMP to cell surface receptors evokes the transient activation of of adenylate cyclase in Dictyostelium discoideum. Dithiothreitol is also known as an activator of this enzyme. We found that the dithiothreitol-induced activation was specifically enhanced by extracellular polyamines or divalent cations. Furthermore, EDTA, a chelating agent of divalent cations, completely inhibited the dithiothreitol-induced activation of adenylate cyclase while EDTA did not inhibit the cAMP-induced activation. The inhibition was nullified by addition of polyamines or divalent cations. These results suggest that extracellular polyamines and divalent cations play a specific role in the dithiothreitol-induced activation of adenylate cyclase.
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PMID:Inhibition by EDTA and enhancement by divalent cations or polyamines of the dithiothreitol-induced activation of adenylate cyclase in the cellular slime mold, Dictyostelium discoideum. 184 20

6-Cloro-9-beta-d-ribofuranosylpurine 5'-triphosphate (CIRTP) and 6-mercapto-9-beta-d-ribofuranosylpurine 5'-triphosphate (SRTP) irreversibly inhibit adenylate cyclase from rat brain. Adenosine 5'-[beta, gamma -imido] triphosphate protects the enzyme against inactivation by CIRTP and SRTP and acts as a competitive inhibitor with respect to ATP with the Ki value 2 X 10(-4) M. Study of the pH-dependence of the rate of the enzyme inactivation by CIRTP showed that pK for the group modified by this compound is equal to 7.45. Inactivation is first order with respect to the enzyme; the saturation effect is observed at the increased concentration of CIRTP. The k2 and KI values for irreversible inhibition of brain adenylate cyclase by CIRTP were 0.25 min-1 and 1.9 X 10(-4) M, respectively. Adenylate cyclase inhibition by SRTP is also time-dependent. Partial protection against the enzyme inactivation was observed. Dithiothreitol restores the activity of SRTP-inactivated adenylate cyclase. The results obtained indicate the presence of an -SH group in the purine amino group binding area of the enzyme active site.
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PMID:Evidence for the existence of a sulfhydryl group in the adenylate cyclase active site. 401 68

Dithiothreitol (DTT) treatment of WT or cyc- lymphoma membranes resulted in the simultaneous loss of epinephrine-stimulated adenylate cyclase activity and beta-adrenergic antagonist binding. The treatment produced no significant decrease in NaF-stimulated activity and only a partial loss of the PGE1 stimulation. Epinephrine partially protected against the loss of epinephrine-stimulated cyclase activity. The decrease in beta-adrenergic stimulation of adenylate cyclase was characterized by over a 100-fold increase in Kact for epinephrine stimulation of adenylate cyclase (10 mM DTT for 30 min) with no effect on the Vmax. We have previously shown that two polypeptides (Mr = 55,000 and 65,000 daltons) are specifically labeled by [125I]iodoazidobenzylpindolol (IABP) in WT and cyc-. The IABP photolabeling of the 55,000 dalton beta-receptor polypeptide was preferentially reduced by 1.0 mM DTT, whereas 10 mM DTT eliminated the photolabeling of both polypeptides. The effects of DTT were not due to either scavenging of the nitrene or reduction of the azide. A reduction in epinephrine stimulation of adenylate cyclase was also observed after treatment of intact WT, and the effects were readily reversed in the cells by washout of the DTT. The DTT effects on membranes were not reversed by washout. Our results demonstrate that the oxidized state of the lymphoma beta-receptor is necessary for maximum sensitivity to agonist stimulation of adenylate cyclase and that low concentrations of reducing agent selectively decrease specific photo-labeling of the 55,000 dalton beta-receptor polypeptide.
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PMID:Effect of dithiothreitol on the beta-adrenergic receptor of S49 wild type and cyc- lymphoma cells: decreased affinity of the ligand-receptor interaction. 632 73

Radioligand binding studies identified two classes of 125I-angiotensin II-binding sites in rat liver membranes. High affinity binding sites (Kd = 0.35 +/- 0.13 nM, N = 372 +/- 69 fmol/mg of protein) were inactivated by dithiothreitol (0.1-10 mM) without any apparent change in low affinity binding sites (Kd = 3.1 +/- 0.8 nM, N = 658 +/- 112 fmol/mg of protein). Dithiothreitol inactivation was readily reversible but could be made permanent by alkylation of membrane proteins with iodoacetamide. Angiotensin II stimulation of glycogen phosphorylase in isolated rat hepatocytes (maximal stimulation 780%, EC50 = 0.4 nM) was completely inhibited by 10 mM dithiothreitol, a concentration which also abolished high affinity site binding; phosphorylase stimulation by glucagon and norepinephrine under these conditions was unaltered. Angiotensin II inhibition of glucagon-stimulated adenylate cyclase activity in hepatocytes required higher angiotensin II concentrations (EC50 = 3 nM) than phosphorylase stimulation and was not affected by dithiothreitol. Fractional occupancy of high affinity binding sites by 125I-angiotensin II correlated closely with angiotensin II-mediated phosphorylase stimulation, whereas occupancy of low affinity sites paralleled inhibition of adenylate cyclase activity. These data indicate that the physiologic effects of angiotensin II in rat liver are mediated by two distinct receptors, apparently not interconvertible, and provide the first evidence for angiotensin II receptor subtypes with differing biochemical features and mechanisms of action.
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PMID:Characterization of angiotensin II receptor subtypes in rat liver. 633 67

The heat-labile enterotoxin of Escherichia coli, like cholera toxin, activates adenylate cyclase by catalyzing the transfer of adenosine diphosphate-ribose from HAD+ (oxidized nicotinamide adenine dinucleotide) to the guanyl nucleotide-dependent regulatory component of the cyclase. A preparation of enterotoxin that had been released from E. coli following exposure to polymyxin B and then partially purified was found to contain two enzymatically active peptides, one of about 29,000 and the other of about 24,000 daltons, which correspond in molecular size to the enzymatically active subunit A and fragment A1 of cholera toxin, respectively. As with cholera toxin, the enzymatic activity of E. coli enterotoxin was elevated by incubation with sodium dodecyl sulfate to release active peptides. Treatment with dithiothreitol, however, had no effect. Dithiothreitol activates subunit A of cholera toxin by reducing an internal disulfide bond, but no corresponding bond appears to be present in the partially purified E. coli enterotoxin.
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PMID:Adenosine diphosphate-ribosylation of adenylate cyclase catalyzed by heat-labile enterotoxin of Escherichia coli: comparison with cholera toxin. 698 18