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)

In enzymatically dispersed enriched rat parietal cells we studied the effect of pertussis toxin on prostaglandin E2 (PGE2)- or somatostatin-induced inhibition of H(+)-production. Parietal cells were incubated in parallel in the absence (control cells) and presence of pertussis toxin (250 ng/ml; 4 h). [14C]Aminopyrine accumulation by both pertussis toxin-treated and control cells was used as an indirect measure of H(+)-production after stimulation with either histamine, forskolin or dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) alone and in the presence of PGE2 (10(-9)-10(-7) M) or somatostatin (10(-9)-10(-6) M). PGE2 inhibited histamine- and forskolin-stimulated [14C]aminopyrine accumulation but failed to alter the response to dbcAMP. Somatostatin was less effective and less potent than PGE2 in inhibiting stimulation by histamine or forskolin and reduced the response to dbcAMP. Pertussis toxin completely reversed inhibition by both PGE2 and somatostatin on histamine- and forskolin-stimulated H(+)-production but failed to affect inhibition by somatostatin of the response to dbcAMP. After incubation of crude control cell membranes with [32P]NAD+, pertussis toxin catalysed the incorporation of [32P]adenosine diphosphate (ADP)-ribose into a membrane protein of molecular weight of 41,000, the known molecular weight of the inhibitory subunit of adenylate cyclase (Gi alpha). Pertussis toxin treatment of parietal cells prior to the preparation of crude membranes almost completely prevented subsequent pertussis toxin-catalysed [32P]ADP ribosylation of the 41,000 molecular weight protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Pertussis toxin reverses prostaglandin E2- and somatostatin-induced inhibition of rat parietal cell H(+)-production. 135 83

Somatostatin (SS) receptors in membranes from ovine retinas were examined using 125I-Tyr11-SS as a ligand. Receptor binding was rapid, specific, saturable, reversible and dependent on temperature and membrane concentration. Conditions of apparent equilibrium were obtained at 25 degrees C after a 45 min incubation in the presence of about 0.25 mg membrane protein/ml. Native SS competitively inhibited the binding of 125I-Tyr11-SS in the range of 0.01-10 nM, and half-maximal inhibition was observed at 0.2 nM SS. Scatchard analysis of these data suggested the existence of a single population of SS receptors with a dissociation constant of 0.23 +/- 0.03 nM and a maximum binding capacity of 84 +/- 6 fmol/mg protein. The binding of 125I-Tyr11-SS was inhibited by various synthetic SS analogs in a dose-dependent manner whereas peptides unrelated to SS did not show practically any effect even at concentrations as high as 10(-6) M. SS receptor occupancy appears to be coupled to inhibition of adenylate cyclase activity by a guanine nucleotide-binding regulatory protein, as suggested by the facts that: (a) SS noncompetitively inhibited the stimulatory effect of vasoactive intestinal peptide (VIP) (3 x 10(-7) M) on membrane adenylate cyclase activity but it did not alter basal enzyme activity; and (b) the addition of guanosine 5'-triphosphate (GTP) (10(-5) M) decreased the specific binding of 125I-Tyr11-SS to 26.6% of the control value due to a decrease in SS receptor affinity. The present results support the hypothesis that SS may contribute to the physiological regulation of the functions of the retina.
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PMID:Somatostatin binding and modulation of adenylate cyclase in ovine retina membranes. 136 Sep 27

Neuroblastoma x glioma hybrid NG108-15 cells express a high-affinity IP prostanoid receptor. Saturation binding analysis of this receptor, using [3H]prostaglandin E1 ([3H]PGE1) as ligand, indicated that it was present at some 1.5 pmol/mg of membrane protein and displayed a dissociation constant for this ligand of 30-40 nM. Prolonged exposure of these cells either to PGE1 or to iloprost, which is a stable analogue of prostacyclin, caused a 40-70% decrease in levels of the receptor. The remaining receptors were capable of interacting with the stimulatory G-protein (Gs) of the adenylate cyclase cascade, as saturation analysis of the binding of [3H]PGE1 indicated that they had a similar affinity for the 3H-labelled ligand, and because the specific binding of [3H]PGE1 to these receptors was still sensitive to the presence of poorly hydrolysed analogues of GTP. We have recently demonstrated that prolonged exposure of NG108-15 ells to PGE1 causes a cyclic AMP-independent loss of Gs alpha-subunit (Gs alpha) from these cells [McKenzie & Milligan (1990) J. Biol. Chem. 265, 17084-17093]. Steady-state concentration of the larger 45 kDa form of Gs alpha (which is the predominant form expressed in these cells) was assessed to be 9.6 pmol/mg of membrane protein, and treatment with iloprost decreased levels of this polypeptide to some 3.0 pmol/mg of protein. Time courses of iloprost-mediated down-regulation of the IP prostanoid receptor, loss of Gs alpha protein as assessed by immunoblotting and loss of Gs alpha activity as assessed by the reconstitution of NaF stimulation of adenylate cyclase activity to membranes of S49 cyc- cells by sodium cholate extracts of NG108-15 cells were identical, suggesting that the loss of the IP prostanoid receptor and G-protein occurred in parallel. Each of these effects was half-maximal between 2 and 3 h of exposure to the agonist. Stoichiometry of loss of Gs alpha and IP prostanoid receptor was unchanged by the percentage receptor occupancy, and quantification indicated the loss of some 7-10 mol of Gs alpha/mol of receptor. This is the first report to demonstrate the temporal concurrence of loss of Gs alpha and of a receptor which interacts with this G-protein. Chronic activation of the IP prostanoid receptor on these cells results in the development of a heterologous form of desensitization to agents which function to activate adenylate cyclase [Kelly, Keen, Nobbs & MacDermot (1990) Br. J. Pharmacol. 99, 306-316]. Agonist regulation of Gs alpha levels in these cells may contribute to this process.
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PMID:Concurrent down-regulation of IP prostanoid receptors and the alpha-subunit of the stimulatory guanine-nucleotide-binding protein (Gs) during prolonged exposure of neuroblastoma x glioma cells to prostanoid agonists. Quantification and functional implications. 137 45

It has been documented that the binding activity sites of human chorionic gonadotropin (HCG) receptor and kd in human ovarian tumors are different from those in the normal ovary. And some observations suggest that the HCG receptor depends on adenylate cyclase (AC) for its physiological functions. We studied the activity of AC in normal human ovary and ovarian tumors. Five human ovarian specimens and eighteen ovarian tumor specimens were obtained from women patients undergoing gynecological surgery. Ovaries were homogenized and sonicated. The homogenates were centrifuged at 1000 x g for 15 min. After sucrose density gradient ultracentrifugation (78000 x g, 4 h), the membrane fraction was collected from interface between 33% and 37%. The membrane protein approximately 10 micrograms, ATP 1 mmol/L, 3H-ATP 5 x 10(4) cpm, sulphydryl-ethyl alcohol 10 mmol/L, in a final volume of 200 microliters of Tris-HCl buffer (50 mmol/L), pH7.5, containing MgSO4 5 mmol/L, were incubated at 35 degrees C for 10 min. The reaction was stopped by boiling water bath for 3 min. The AC activity (U/mg): of human normal ovary, 67 +/- 8, of cystadenocarcinoma serous, 146 +/- 70; of cystadenocarcinoma mucinous, 289 +/- 83.
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PMID:[Comparative study on activity of adenylate cyclase in normal human ovary with ovarian tumors]. 145 50

Clopidogrel, like the homologous thienopyridine derivative ticlopidine, selectively inhibits platelet aggregation induced by ADP. We have previously described two nucleotide-binding sites on platelets related to ADP-mediated platelet responses. The first is a high-affinity binding site for 2-methylthio-ADP (2-MeSADP) that is linked to the inhibition of stimulated adenylate cyclase. The second is the 100-kd exofacial membrane protein aggregin, which is labeled by the reactive ADP analogue 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) that is related to shape change and aggregation. We set out to determine if either of these sites is blocked in vivo by clopidogrel or its active metabolite. Six subjects were given clopidogrel (75 mg/day for 10 days) in a double-blind crossover experiment. All of the subjects developed prolonged bleeding times while taking the drug. The rate of onset of the effect on bleeding time varied among subjects. Platelet aggregation induced by ADP or thrombin was significantly impaired by the drug treatment, but no effect was detected on shape change. The incorporation of [3H]FSBA into aggregin was also unaffected. Inhibition of adenylate cyclase by ADP or by 2-MeSADP was greatly reduced in all subjects, and in the case of 2-MeSADP, there was evidence for a noncompetitive effect. Inhibition of adenylate cyclase by epinephrine was unaffected. In the three subjects for whom binding measurements were made, the number of binding sites for [32P]2-MeSADP was reduced from 534 +/- 44 molecules per platelet during control and placebo periods (11 determinations) to 199 +/- 78 molecules per platelet during drug treatment (three determinations). There was no consistent change in the binding affinity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Clopidogrel inhibits the binding of ADP analogues to the receptor mediating inhibition of platelet adenylate cyclase. 155 34

A method for the simultaneous preparation of microvillous and basal membrane vesicles from human placental syncytiotrophoblast is described. Mg2(+)-aggregated basal membranes are separated from microvillous membranes by low-speed centrifugation after initial homogenization and centrifugation steps. Microvillous membranes (MVM) are obtained from the low speed supernatant while basal membranes (BM) contained in the Mg2(+)-aggregated material are resuspended and further purified on a sucrose step gradient. MVM and BM prepared by this method were enriched 20-fold and 11-fold as determined by the membrane marker enzymes, alkaline phosphatase (MVM) and adenylate cyclase (BM). There was minimal cross-contamination of the two isolated plasma membrane fractions and the yields obtained were 26% (MVM) and 21% (BM) compared to the initial homogenate. The MVM and BM fractions were free from contamination by mitochondrial or lysosomal membranes and showed only minor contamination by microsomal membranes. The two membrane fractions were also tested for the presence of non-syncytial plasma membranes by electrophoretic immunoblotting. Contamination of both MVM and BM by fibroblast, endothelial, macrophage and cytotrophoblast plasma membranes amounted to less than 15% of the total membrane protein as determined by immunoblotting. Vesicle orientation, determined from the latency of specific concanavalin A binding, was 88 +/- 4% right-side out for MVM and 73 +/- 12% right-side out for BM. This simple preparative procedure produces a high yield of both MVM and BM from human placenta. The analytical data demonstrates that 'paired' MVM and BM fractions derived from the same placental tissue have a high purity in terms not only of contamination by intracellular membranes, but also in terms of contamination by non-syncytial plasma membranes.
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PMID:Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta. 170 Sep 23

Using retinas prepared from freshly dissected bovine eyes, we have characterized the binding of the A1-selective agonist, [3H]PIA (N6-R-[3H](2-phenylisopropyl)adenosine). Specific binding was linear over a range of membrane protein concentrations from 0.10 to 1.0 mg, and accounted for an average of 80-90% of the total binding. At room temperature (24 degrees C), binding reached equilibrium at 60 min, and was reversible upon addition of an excess of cold ligand. Saturation analysis and Scatchard transformation revealed two apparent populations of receptor binding sites. The higher affinity site exhibited a Kd of 0.134 +/- 0.007 nM and Bmax of 26.18 +/- 3.06 fmol-1 mg protein. The lower affinity site exhibited a Kd of 21.83 +/- 4.39 nM and Bmax of 53.94 +/- 15.80 fmol mg-1 protein. Kinetic analysis of association and dissociation rates, performed at a low concentration of [3H]PIA, yielded a calculated affinity constant for the high affinity site of 0.2 nM, in agreement with saturation studies. Competition experiments with a number of purine nucleoside agonists and antagonists were performed, using radioligand concentrations of 1 nM or less to examine binding at the high affinity site, and revealed a rank order of potency consistent with the reported pharmacology of A1 receptors. We have also assayed for adenylate cyclase activity in this same preparation and determined that PIA inhibited forskolin-activated adenylate cyclase in a dose-dependent manner. Maximum inhibition (40%) was observed with 1 nM PIA, while 10 microM 8-cyclopentyl-1,3-dipropylxanthine, an A1 selective antagonist, completely inhibited this modulation by PIA.
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PMID:Characterization of adenosine A1-receptor binding sites in bovine retinal membranes. 193 68

Estradiol receptors are discovered in nuclei, cytoplasm and plasmatic membranes of the target cells. Estradiol activates the transmembrane polyphosphoinositide system: it stimulates the protein kinase C translocation from cytosol to cell membranes, the membrane protein phosphorylation being elevated. Transmembrane adenylate cyclase system is also activated. The cAMP system stimulation by estradiol is mediated by protein kinase C, phosphorylating a protein of adenylate cyclase complex. Estradiol causes protein kinases A translocation into the cell nuclei and enhances the protein kinase NII activity. The role of protein phosphorylation, activated by steroid hormones, in the transcription and protein synthesis regulation, is discussed.
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PMID:[The participation of transmembrane messenger systems in the action of steroid hormones on target cells]. 196 59

Islet-activating protein (IAP), one of the pertussis toxins, serving [alpha-32P]nicotinamide adenine dinucleotide (NAD) as a substrate for ADP ribosylation, radiolabelled a specific pig epidermal membrane protein. The IAP-specific substrate was detectable by sodium dodecyl sulphate-polyacrylamide gel electrophoresis as a single band corresponding to a molecular weight of 40 kDa. The ADP ribosylation catalysed by IAP was inhibited by the addition of Mg2+ to the reaction mixture. IAP is known to work on intact cell systems resulting in the ADP ribosylation using intracellular NAD as the ADP ribose donor. Following IAP pretreatment of intact pig epidermis, the epidermal receptor adenylate cyclase responses were markedly increased; all the stimulatory receptor adenylate cyclase responses (beta-adrenergic, prostaglandin E, adenosine and histamine responses) were significantly increased. Cholera toxin-induced cyclic AMP accumulation was also significantly increased. Forskolin-induced cyclic AMP accumulation was slightly increased after IAP pretreatment, but this was not statistically significant. The IAP-dependent ADP ribosylation of the epidermal 40 kDa membrane protein, which was prepared from the IAP pretreated epidermis, was significantly decreased. It is known that the tumour promoter, phorbol 12-myristate,13-acetate (PMA), decreases stimulatory receptor adenylate cyclase responses of the epidermis. Following the PMA pretreatment, IAP-dependent ADP ribosylation of the epidermal membrane protein was unaffected. Furthermore, following the PMA pretreatment, the IAP-induced increase in the epidermal receptor adenylate cyclase responses still remained. Our results indicate that pig epidermis contains 40 kDa membrane substrate for IAP-dependent ADP ribosylation, which has an inhibitory tonus on the epidermal adenylate cyclase until its ADP ribosylation by IAP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibitory guanine nucleotide binding protein in pig epidermis: regulation of epidermal adenylate cyclase. 196 35

Three membrane fractions were studied from canine myocardial left ventricle (LV); crude, light vesicle, and enriched sarcolemma. The percent of the total yield of membrane protein was 99.3 +/- 0.2% for the crude fraction, 0.4 +/- 0.1% for the light vesicle fraction, and 0.3 +/- 0.03% for the purified fraction. Sodium, potassium-ATPase activity in the purified fraction (100 +/- 10.8 mumol Pi/h/mg) was five fold more concentrated than in the light vesicle fraction (18.5 +/- 1.85 mumol Pi/h/mg), and nineteen fold more than in the crude fraction (5.29 +/- 0.57 mumol Pi/h/mg). beta-Adrenergic receptors were 8-fold enriched in the purified fraction (1006 +/- 219 vs 132 +/- 13 fmol/mg in the crude fraction) and 4-fold enriched in the light vesicle fraction (497 +/- 152 fmol/mg). Adenylate cyclase activity was enriched by only 13 to 17-fold in the purified fraction, and only 2 to 4-fold in the light vesicle fraction. The percent of the total beta-adrenergic receptors per gram wet weight was 94 +/- 20% for the crude fraction, 2.1 +/- 0.4% for the light vesicle fraction, and 3.8 +/- 0.7% for the purified fraction. When alamethicin was used to uncover latent enzyme activity, beta-adrenergic receptor density was not affected, but Na+,K(+)-ATPase and adenylate cyclase activity were enhanced in each membrane fraction studied. The surprising finding was that Na+,K(+)-ATPase activity was enriched to the same extent as the beta-adrenergic receptor density in the light vesicle fraction. One potential explanation is that the light vesicle fraction is located in a specialized region of the plasma membrane.
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PMID:Characterization of subfractions from purified sarcolemma of canine left ventricle. 196 9


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