Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

We have characterized the ANF-R2 receptor-mediated inhibition of adenylate cyclase with respect to its modulation by several regulators. ANF (99-126) inhibits adenylate cyclase activity only in the presence of guanine nucleotides. The maximal inhibition (approximately 45%) was observed in the presence of 10-30 microM GTP gamma S, and at higher concentrations, the inhibitory effect of ANF was completely abolished. ANF-mediated inhibition was not dependent on the presence of monovalent cations, however Na+ enhanced the degree of inhibition by about 60%, whereas K+ and Li+ suppressed the extent of inhibition by about 50%. On the other hand, divalent cation, such as Mn2+ decreased the degree of inhibition in a concentration dependent manner, with an apparent Ki of about 0.7 mM, and at 2 mM; the inhibition was completely abolished. In addition, proteolytic digestion of the membranes with trypsin (40 ng/ml) resulted in the attenuation of ANF-mediated inhibition of adenylate cyclase. Other membrane disrupting agents such as neuraminidase and phospholipase A2 treatments also inhibited completely, the ANF-mediated inhibition of enzyme activity. N-Ethylmaleimide (NEM), phorbol ester and Ca(2+)-phospholipid dependent protein kinase (C-kinase) which have been shown to interact with inhibitory guanine nucleotide regulating protein (Gi) also resulted in the attenuation of ANF-mediated inhibition of adenylate cyclase activity. These results indicate that in addition to the Gi, the phospholipids and glycoproteins may also play an important role in the expression of ANF-R2 receptor-mediated inhibition of adenylate cyclase.
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PMID:Characterization of ANF-R2 receptor-mediated inhibition of adenylate cyclase. 132 94

Regulation of GTP and GDP binding and GTPase activity of cardiac sarcolemmal guanine nucleotide-binding proteins was investigated. In purified sarcolemmal membranes, carbachol and a variety of other muscarinic receptor (MR) agonists induced increases in [3H]GTP, [gamma-32P]GTP, and [3H]GDP binding to relatively high affinity sites. Carbachol-dependent GTP and GDP binding changes were maximal within 5 sec at 30 degrees and thereafter remained at steady state. Carbachol increased GTP binding to two sites with apparent Kapp values of 50 nM and 250 nM and GDP binding to a single site with a Kapp of 100 nM. N-Ethylmaleimide attenuated carbachol-dependent GDP and GTP binding, tentatively identifying the binding sites as Gi and/or Go. Further studies showed that [3H]GDP and [3H]GTP bound to Gi/Go in the presence of carbachol rapidly exchanged with GTP and GDP in the medium. In membranes preincubated with carbachol and [gamma-32P]GTP or carbachol and [3H]GDP, postaddition of atropine resulted in complete hydrolysis of [gamma-32P]GTP bound to Gi/Go, to unlabeled GDP and 32Pi, by GTPase, within 10 sec, whereas [3H]GDP remained bound. This study also showed that bound [3H]GDP did not exchange with GDP or GTP in the absence of an MR agonist. Under identical conditions, atropine reversed adenylate cyclase (AC) inhibition by carbachol and GTP or GDP in 5-10 sec. MR agonists appear to increase the rate of dissociation of GDP from Gi/Go, which results in rapid GTP turnover on these sites by a combination of GTPase and GDP/GTP exchange reactions. Furthermore, MR-Gi/Go may be tightly coupled during AC inhibition, so that GTP hydrolysis as well as MR-Gi/Go uncoupling may be required to reverse AC inhibition.
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PMID:Regulation of GDP and GTP binding in cardiac sarcolemma by muscarinic receptor agonists. 173 18

In freshly collagenase-isolated rat pancreatic islets and in islets cultured for 72 hours, the effects of thiol reagents on glucagon (5 micrograms/ml) and/or glucose (16.7 mM)-mediated increases in cAMP formation as well as on clonidine (10 microM)-induced inhibition of these actions were studied. In freshly isolated islets and to a more pronounced degree in islets cultured for 72 hours glucagon (5 micrograms/ml) increased the cAMP content above the basal value. Clonidine (0.1-100 microM) had no significant effect on the basal cAMP formation, but inhibited the glucagon-mediated effect. The thiol reagents diamide (10-100 microM) and NEM affected neither the basal nor the glucagon-mediated effect, but abolished the inhibitory action of clonidine on cAMP formation. In freshly isolated islets, high glucose concentrations (8.3-16.7 mM) increased the cAMP formation. Diamide (100 microM) and NEM (100 microM) attenuated the stimulatory effect of 16.7 mM glucose. It is suggested that these selective effects of the thiol reagents on glucagon-mediated increase in cAMP formation in the presence of substimulatory concentration of glucose may be due to the differences in the sensitivity of the sulfhydryl groups of the G-proteins to thiol reagents i.e. Gi or proteins closely related to Gi being more sensitive than Gs. The data further suggest that glucose acts on the cAMP cascade at a step distinct from Rs. Since both glucose and glucagon effects were influenced by the addition of clonidine, it is possible to interpret the data as indicating that the effects of both stimulators eventually converge at some common step in the adenylate cyclase cascade.
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PMID:Thiol reagents (diamide and N-ethylmaleimide) inhibit increase in cAMP in response to glucose and abolish the clonidine-mediated attenuation of glucagon-induced cAMP formation in isolated rat pancreatic islets. 196 19

An iodinated photosensitive derivative of norepinephine, N-(p-azido-m-iodophenethylamidoisobutyl)-norepinephrine (NAIN), has been synthesized and characterized. NAIN stimulated adenylate cyclase activity in guinea pig lung membranes in a manner similar to (-)-isoproterenol and was inhibited by (-)-alprenolol. NAIN was shown to compete with [125I]iodocyanobenzylpindolol for the beta-adrenergic receptor in guinea pig lung membranes with an affinity which was dependent on the presence of guanyl nucleotides. Carrier-free radioiodinated NAIN ([125I]NAIN) was used at 2 nM to photoaffinity label the beta-adrenergic receptor in guinea pig lung membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of (-)-alprenolol (1 microM) protectable [125I]NAIN labeling showed the same molecular mass polypeptide (65 kDa) that was specifically derivatized with the antagonist photolabel [125I]iodoazidobenzylpindolol. Specific labeling of the beta-adrenergic receptor with [125I]NAIN was dependent on the presence of MgCl2 and the absence of guanyl nucleotide. Guanosine-5'-O-(3-thiotriphosphate (100 microM) abolished specific labeling by [125I]NAIN. N-Ethylmaleimide (2 mM) in the presence of [125I]NAIN protected against the magnesium and guanyl nucleotide effect. These data show that NAIN is an agonist photolabel for the beta-adrenergic receptor.
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PMID:Photoaffinity labeling the beta-adrenergic receptor with an iodoazido derivative of norepinephrine. 284 70

Agents that react chemically with sulfhydryl groups of proteins modify the response of adenylate cyclase to stimulation by beta-adrenergic agonists. N-Ethylmaleimide, an agent that alkylates sulfhydryl groups, inactivates both the catalytic moiety of adenylate cyclase and the stimulatory, regulatory guanine nucleotide binding protein Ns of rat fat cells but fails to affect binding of antagonists to the beta-adrenergic receptor [Malbon, C. C., Graziano, M. P., & Johnson, G. L. (1984) J. Biol. Chem. 259, 3254-3260]. Treating membranes of rat fat cells with dithiothreitol or beta-mercaptoethanol, agents that reduce disulfide bridges of proteins, results in a loss of binding of beta-adrenergic radioligands to the receptor. The specific binding of radioligands to beta-adrenergic receptors that are solubilized in digitonin is affected similarly by treatment with disulfide bridge reducing agents. beta-Adrenergic receptor purified from rat fat cells and treated with beta-mercaptoethanol (10%) and then subjected to gel electrophoresis in the presence of sodium dodecyl sulfate migrates as a Mr 67 000 peptide [Cubero, A., & Malbon, C. C. (1984) J. Biol. Chem. 259, 1344-1350]. In the absence of disulfide bridge reducing agents, however, the purified receptor exhibits greater electrophoretic mobility, migrating as a peptide with Mr 54 000. Treating the native form of the purified receptor with beta-mercaptoethanol (0.1-10%) or dithiothreitol (0.1-10 mM) decreases the ability of the receptor to bind beta-adrenergic ligands, decreases the electrophoretic mobility of the receptor, and results in receptor peptides migrating with molecular weight ranging from 54 000 to 67 000 when subjected to gel electrophoresis in the presence of sodium dodecyl sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fat cell beta 1-adrenergic receptor: structural evidence for existence of disulfide bridges essential for ligand binding. 300 32

In crude membrane fractions of rat pancreatic islets and of RIN-A2-cells, forskolin and NaF stimulated adenylate cyclase activity. Basal and stimulated enzyme activity was approximately 3 to 6 fold higher in membranes of RIN-A2-cells than in membranes of islet cells. In RIN-A2-cells GppNHp and NEM inhibited forskolin-stimulated enzyme activity. The inhibitory effect of GppNHp could be reduced by NEM. It is suggested that the adenylate cyclase system of RIN-A2-cells contains inhibitory and stimulatory N-proteins and that there are critical thiols related to Ni, Ns and/or the catalytic unit. Thus, membrane fractions of RIN-A2-cells may be an appropriate model for studies on the adenylate cyclase system of insulin-producing cells.
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PMID:Adenylate cyclase in membrane fractions of RIN-A2-cells: studies with forskolin, NaF, GppNHp and NEM. 337 16

Gold (III) (Au(III) up to 0.25 microM increased prostaglandin E2-sensitive chick osteoblast and fibroblast adenylate cyclase activity without affecting 5'-guanylylimidodiphosphate-stimulated enzyme activity. Au (III) at 5-50 microM inhibited hormone- and nucleotide-mediated activation of adenylate cyclase. This process is time-dependent, irreversible and can not be explained by desensitivation of the PGE2-receptor. Basal adenylate cyclase activity was not influenced by Au (III) in the given concentrations. Pretreatment of membranes with 5'-guanylylimidodiphosphate prior to incubation with Au (III) prevented the inhibitory effect of Au (III) on adenylate cyclase, similar to the incubation experiments with NEM instead of Au (III). Our data suggest that Au (III) at low concentrations unspecifically stimulates PGE2-sensitive adenylate cyclase by binding to sulfhydryl groups of the receptor protein and that Au (III) at high concentrations specifically inhibits PGE2-sensitive adenylate cyclase by binding to sulfhydryl groups of the N-Protein and/or of the catalytic unit.
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PMID:[Effect of gold (III) on prostaglandin E2-stimulated adenyl cyclase in chick fibroblasts and osteoblasts]. 382 21

N-Ethylmaleimide (NEM) differentially modified Ri adenosine receptors in rat fat cells and Ra adenosine receptors in human platelets. Pretreatment of rat fat cell membranes with NEM inhibited the binding of the agonist (-)N6-phenylisopropyl[3H]adenosine [( 3H]PIA), but did not affect the binding of the antagonist 1,3-diethyl-8-[3H]phenylxanthine [( 3H]DPX). The IC50-value for inhibition of [3H]PIA binding was 0.067 mM. Saturation of [3H]PIA binding revealed that NEM converts the high affinity form of the Ri receptor into a low affinity form. NEM also decreased the potency of agonists to displace [3H]DPX binding, as shown by a 74-fold shift of the Ki-value for (-)PIA, whereas antagonist-induced displacement remained unchanged. In addition, low concentrations of NEM (0.01-0.1 mM) attenuated the (-)PIA-induced inhibition of adenylate cyclase activity of rat fat cells. At higher concentrations (0.1-1 mM) NEM reduced basal and stimulated adenylate cyclase activities in rat fat cells and human platelets, presumably by inactivation of the catalytic unit. Radioligand binding of 5'-N-ethylcarboxamido[3H]-adenosine [( 3H]NECA) to Ra adenosine receptors of human platelet membranes was not changed by NEM at low radioligand concentrations. Saturation analysis of [3H]-NECA binding showed that NEM led to an apparent increase of agonist affinity with a concomitant decrease in total [3H]NECA binding sites. These results suggest that NEM reduces the affinity of Ri adenosine receptors, probably by affecting the inhibitory guanine nucleotide binding protein (Ni), whereas [3H]NECA binding sites are inversely affected.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of N-ethylmaleimide on adenosine receptors of rat fat cells and human platelets. 609 76

N-Ethylmaleimide treatment of rat liver plasma membranes results in an adenylyl cyclase (EC 4.6.1.1) system that shows no measurable cyclizing activity but retains both an active glucagon receptor and a receptor-sensitive regulatory component N as assessed by reconstitution into cyclase-negative (cyc-) membranes from S49 murine lymphoma. Treatment of such N-ethylmaleimide-treated membranes, termed C- liver membranes, with guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] ) and Mg2+, followed by the removal of GTP[gamma S] by washing, yields an activated N which upon mixing with cyc- S49 membranes reconstitutes the cyc- S49 membrane adenylyl cyclase in the absence of added GTP[gamma S]. It was found that GTP[gamma S] activation of the N at saturating concentrations of GTP[gamma S] is slow at low Mg2+ concentration and accelerated by increasing Mg2+ concentrations. Addition of glucagon during the activation results in a lowering of the Mg2+ requirement for full activation from 25 mM to around 10 muM and in concomitant increases in both the rate and the extent of N activation. In contrast to its dramatic effect on Mg2+ requirement, glucagon has little (less than 2-fold) effect on the GTP[gamma S] requirement of N activation. These experiments indicate that the glucagon receptor facilitates activation of N by: (i) decreasing the apparent Km of N for Mg2+, and (ii) increasing the extent of activation that can be elicited by saturating concentrations of guanine nucleotide. It is postulated that the mechanism by which Mg2+ and receptors facilitate N activation involves dissociation of n alpha activated ADP-ribosylatable subunits (with guanine nucleotide bound to them) from n beta non-ADP-ribosylatable subunits (with receptor and Mg2+ bound to them).
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PMID:Hormone receptor modulates the regulatory component of adenylyl cyclase by reducing its requirement for Mg2+ and enhancing its extent of activation by guanine nucleotides. 629 Oct 28

Soluble Mn2+-dependent adenylate cyclase (MnAC) activity was found in testicular cytosol from blue foxes castrated during the breeding season. The rate of MnAC activity was approximately constant for 30 min at 35 degrees C and for 2 hr after storage at 25 degrees C. Activity was directly proportional to cytosol protein concentration and was optimal in the physiological pH range. Enzyme activity declined in the presence of an alkylating agent (N-ethyl maleimide, NEM) and was eliminated at a concentration of 1 mM NEM. Low concentrations (0.1-10 mM) of a reducing agent (beta-mercapto ethanol, beta ME) did not increase MnAC activity, whereas a high concentration (100 mM) led to a significant reduction (p less than 0.01) in activity. Substitution of Mn2+ in the assay medium with Mg2+ led to a total loss of enzyme activity, which could not be regained by adding hormones or by preincubation of cytosol for 60 min. The Km for Mn2+ was estimated to be 3.5 mM. The affinity of the enzyme for Mn2+ was not altered by varying the concentration of ATP. In contrast, increasing concentrations of Mn2+ appeared to increase the affinity of the enzyme for MnATP2-. The Km for MnATP2- thus varied from 6 to 18 mM.
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PMID:Soluble Mn2+-dependent adenylate cyclase activity in the testis of the blue fox (Alopex lagopus). 650 30


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