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 developed a method to ADP-ribosylate the stimulatory guanine nucleotide-binding protein of adenylate cyclase (GS) in brain membranes by using cholera toxin. In particular, we used isonicotinic acid hydrazide and 3-acetylpyridine adenine dinucleotide to inhibit the potent NAD-glycohydrolase activity of brain membranes, and we used the detergent Triton X-100 (at 0.1%) to improve the accessibility of the toxin and guanine nucleotides used for supporting the ADP-ribosylation. This method reveals that GS is a very abundant protein in membranes derived from calf brain (approximately 30 pmol/mg of protein). In brain, GS exists in large excess over the previously reported amount of the adenylate cyclase catalytic subunit. The modification of GS with an ADP-ribosyl residue (a) elicits a four- to fivefold activation of adenylate cyclase by GTP, (b) increases the stabilization of adenylate cyclase by GTP, and (c) reduces adenylate cyclase activation by fluoride but does not change basal activity, activation by guanosine 5'-(beta, gamma-imido)triphosphate, or the sensitivity of adenylate cyclase to heat-induced denaturation. A correlation between ADP-ribosylation and the alterations in the activation of adenylate cyclase by guanine nucleotides and by fluoride is presented.
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PMID:ADP-ribosylation by cholera toxin of membranes derived from brain modifies the interaction of adenylate cyclase with guanine nucleotides and NaF. 283 59

The number of beta-adrenergic receptors in cardiac myocytes isolated from rats made diabetic with streptozocin (STZ) for 10 wk was measured by use of a hydrophilic nonselective antagonist [3H]CGP 12177 and was found to decrease to 59% of the number in control rats (P less than .05), without any change in affinity. Similarly, using [125I]iodocyanopindolol as a ligand, we found a decrease in the beta-adrenergic-receptor number on cardiac plasma membrane isolated from the diabetic rats [29% decrease (P less than .05) at 1 wk, 50% (P less than .01) at 3 wk, and 49% (P less than .01) at 10 wk compared with control rats]. However, the serum triiodothyronine level that had been known to modulate the beta-adrenergic-receptor-adenylate cyclase system was decreased in the 1-wk-diabetic rats but not in the 10-wk-diabetic rats compared with each control group. Furthermore, there was no difference in urinary catecholamine excretion between diabetic and control groups. In the 10-wk-diabetic rats, the response of adenylate cyclase to isoproterenol was significantly defective (56% decrease compared with control rats; P less than .05), although both the basal and the forskolin-stimulated maximum adenylate cyclase activities and a half-maximum concentration of isoproterenol for the stimulation of adenylate cyclase were similar in control and diabetic rats. On the other hand, both cholera toxin-dependent and islet-activating protein-dependent [32P]NAD incorporations into cardiac plasma membrane were markedly increased in the diabetic rats.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Deficiency of cardiac beta-adrenergic receptor in streptozocin-induced diabetic rats. 284 11

The cellular mechanism of action of the cannabimimetic drugs is examined using cultured cells. In membranes from N18TG2 neuroblastoma cells and the neuroblastoma X glioma hybrid cells, NG108-15, the psychoactive cannabinoid drugs and their nantradol analogs could inhibit adenylate cyclase activity. This response was not observed in either the soluble adenylate cyclase from rat sperm or membrane-bound adenylate cyclases from C6 glioma or S49 lymphoma cells. This cellular selectivity provides further evidence for the existence of specific receptors for the cannabimimetic compounds. Receptor-mediated inhibition of adenylate cyclase requires the presence of a guanine nucleotide-binding protein complex, Gi. Gi can be functionally inactivated as a result of an ADP-ribosylation modification catalyzed by pertussis toxin. The present study demonstrates that pertussis toxin treatment of cells abolished the cannabimimetic response in intact cells and in membranes derived therefrom. The action of pertussis toxin required NAD+ as substrate for in vitro modification of neuroblastoma membranes. Furthermore, pertussis toxin was able to catalyze the labeling of a neuroblastoma membrane protein in vitro using [32P] NAD+ under conditions similar to those by which attenuation of the cannabimimetic inhibition of adenylate cyclase could be demonstrated. This evidence demonstrates the requirement for a functional Gi in the action of cannabimimetic drugs.
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PMID:Involvement of Gi in the inhibition of adenylate cyclase by cannabimimetic drugs. 286 5

Cultured rat glioma C6 cells exfoliate membrane vesicles which have been termed 'exosomes' into the culture medium. The exosomes contained both stimulatory and inhibitory GTP-binding components of adenylate cyclase (the stimulatory, Gs, and the inhibitory, Gi, regulatory components) and beta-adrenergic receptors but were devoid of adenylate cyclase activity. It was therefore apparent that the catalytic component of adenylate cyclase was either not exfoliated or was inactivated during the exfoliation process. The presence of Gs or Gi in the exosomes was detected by ADP ribosylation using [alpha-32P]NAD in the presence of cholera or pertussis toxins, respectively. The exosomal concentration of each of the two components was estimated to be about one fifth of that of the cell membrane when expressed on a per mg protein basis. Exosomal Gs was almost as active as the membrane-derived Gs in its ability to reconstitute NaF- and guanine nucleotide-stimulated adenylate cyclase activity in membranes of S49 cyc- cells, which lack a functional Gs. The ability of exosomal Gs to reconstitute isoproterenol-stimulated activity, however, was much lower than that of membrane Gs. The density of beta-adrenergic receptors in the exosomes was much less than that found in the membranes. Although the exosomal receptors bound the antagonist iodocyanopindolol with the same affinity as receptors from the cell membrane, the affinity for the agonist isoproterenol was 13- to 18-fold lower in the exosomes. In addition, this affinity was not modulated by GTP in the exosomes. Thus, exfoliated beta-adrenergic receptors seem to be impaired in their ability to couple to and activate Gs. This was directly tested by coupling the receptors to a foreign adenylate cyclase using membrane fusion. The fusates were then assayed for agonist-stimulated activity. While significant stimulation of the acceptor adenylate cyclase was obtained using C6 membrane receptors, the exosomal receptors were completely inactive. Thus during exfoliation, there appear to be changes in the components of the beta-adrenergic-sensitive adenylate cyclase that results in a nonfunctional system in the exosomes.
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PMID:Exfoliation of the beta-adrenergic receptor and the regulatory components of adenylate cyclase by cultured rat glioma C6 cells. 287 68

The heat-stable enterotoxin (STa) of E. coli activates intestinal guanylate cyclase and leads to increased cGMP levels by an as yet undetermined mechanism. In comparing this cGMP system to other known toxin-mediated alterations in cAMP metabolism, we observed that pertussis toxin caused lower levels of intestinal cGMP synthesis in response to purified STa. Another participant in ADP-ribosylation reactions, NAD, enhanced the ability of STa to activate guanylate cyclase, yet had no effect on basal enzyme activity. Niacinamide and isoniacinamide also had no effect on basal activity, but attenuated the STa activation. These results are discussed in relation to current models of hormone/toxin-sensitive adenylate cyclase, and may suggest an involvement of guanine-nucleotide-binding proteins in intestinal cGMP metabolism.
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PMID:Activation of guanylate cyclase by E. coli heat-stable enterotoxin (STa). Modulation by NAD and pertussis toxin. 287 59

NAD+ significantly enhances adenylate cyclase activity in crude cardiac membrane preparations. This increase is dose-dependent, does not occur in the presence of nicotinamide, ADP-ribose or NADP+, and can be effected by a 30 min pre-incubation period with NAD+. Time course studies are consistent with an enzymatically mediated modification that used NAD+ as substrate. Furthermore, inhibition of NAD+-mediated activation by arginine suggests that this modulation of cardiac adenylate cyclase is analogous to that catalyzed by endogenous ADP-ribosyl transferases.
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PMID:NAD+-mediated stimulation of adenylate cyclase in cardiac membranes. 295 Aug 54

Activation of muscarinic cholinergic receptors of 1321N1 human astrocytoma cells attenuates cyclic AMP accumulation. This effect results from an activation of phosphodiesterase with no direct inhibition of adenylate cyclase activity. In spite of this lack of coupling of muscarinic receptors to adenylate cyclase, guanine nucleotides reduce the apparent binding affinity of the agonist carbachol in a washed membrane preparation of 1321N1 cells. The order of potency for this effect is guanosine 5'-O-(3-thiotriphosphate) greater than 5'-guanylyl-imidodiphosphate = GTP = GDP; ATP has no effect. The occurrence of a Mr = 41,000 protein labeled in the presence of [32P]NAD and pertussis toxin as well as the occurrence of guanine nucleotide-mediated inhibition of forskolin-stimulated adenylate cyclase activity indicate that the functional inhibitory guanine nucleotide regulatory component of adenylate cyclase (Ni) is present in 1321N1 cells. Pertussis toxin pretreatment of NG108-15 neuroblastoma X glioma cells, which express muscarinic receptors that link through Ni to inhibit adenylate cyclase, blocked the GTP-sensitive, high affinity binding of carbachol. In contrast, pretreatment of 1321N1 cells with a concentration of pertussis toxin that blocked [32P]ADP ribosylation of the Mr = 41,000 substrate and GTP-mediated inhibition of forskolin-stimulated adenylate cyclase activity had no effect on GTP-sensitive high affinity binding of carbachol. These results suggest that muscarinic cholinergic receptors of 1321N1 cells couple to a guanine nucleotide regulatory protein that is distinct from Ni.
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PMID:Guanine nucleotide-sensitive, high affinity binding of carbachol to muscarinic cholinergic receptors of 1321N1 astrocytoma cells is insensitive to pertussis toxin. 298

Hypothyroidism is associated with an enhanced sensitivity of rat fat cells to the inhibitory action of adenosine and adenosine agonists. The sensitivity of the forskolin-stimulated cyclic AMP response of rat fat cells to the adenosine agonist N6-phenylisopropyladenosine is amplified 3-fold by hypothyroidism. Forskolin-stimulated adenylate cyclase activity is more sensitive to inhibition by this adenosine agonist in membranes of fat cells isolated from hypothyroid as compared to euthyroid rats. Hypothyroidism does not significantly alter the number of affinity of binding sites for N6-cyclohexyl[3H]adenosine or N6-phenylisopropyladenosine in membranes of rat fat cells. GTP-induced inhibition of forskolin-stimulated adenylate cyclase was markedly enhanced in the hypothyroid state, suggesting an alteration in the inhibitory regulatory component (Ni)-mediated control of adenylate cyclase. Incubating membranes with [alpha-32P]NAD+ and preactivated pertussis toxin results in the radiolabeling of two peptides with Mr = 40,000 and 41,000 as visualized in autoradiograms of polyacrylamide gels run in sodium dodecyl sulfate. The amount of label incorporated by pertussis toxin into these two peptides (putative subunits of Ni) per mg of protein of membrane is increased 2-3-fold in the hypothyroid state. The amount of the stimulatory regulatory component, Ns, in fat cell membranes is not altered by hypothyroidism (Malbon, C. C., Graziano, M. P., and Johnson, G. L. (1984) J. Biol. Chem. 259, 3254-3260). The amplified response of hypothyroid rat fat cells to the inhibitory action of adenosine appears to reflect a specific increase in the activity and abundance of Ni.
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PMID:Fat cell adenylate cyclase system. Enhanced inhibition by adenosine and GTP in the hypothyroid rat. 298 25

In rat pancreatic islet membranes exposed to [alpha-32P]NAD, cholera toxin stimulated the labelling of three peptides with Mr close to 22 000, 42 000 and 48 000, respectively. In the islets, the toxin-stimulated ADP-ribosylation of the heavy form of the Ns alpha-subunit predominated over that of the light form, in mirror image of the situation found in the exocrine pancreas. When intact islets were preincubated with cholera toxin, the adenylate cyclase activity of a subcellular particulate fraction was increased. The responsiveness of adenylate cyclase to GTP was also augmented, but that to NaF was decreased. In intact islets, the production of cyclic AMP and the glucose-stimulated release of insulin were also enhanced after pretreatment with cholera toxin. These findings reveal the presence in pancreatic islets of the guanyl nucleotide regulatory protein of adenylate cyclase, with an unusual predominance of the heavy form of the Ns alpha-subunit.
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PMID:Stimulation by cholera toxin of ADP-ribosylation of membrane proteins, adenylate cyclase and insulin release in pancreatic islets. 298 11

As noted previously, in N1E-115 neuroblastoma cells, carbamylcholine, a muscarinic cholinergic agonist, increased cGMP over 15-fold and decreased basal and prostaglandin E1 (PGE1)-stimulated cAMP content. In contrast to the stimulatory effects of PGE1 on cAMP, which were immediate, the carbamylcholine-induced decrease in basal and PGE1-stimulated cAMP exhibited a delay. The delay in carbamylcholine inhibition was independent of the extent of adenylate cyclase activation. Although basal cAMP content was suppressed within 30 sec after addition of carbamylcholine, inhibition was not maximal for at least 2 min following agonist addition; the delay was similar in cells exposed to PGE1 for 10 min prior to carbamylcholine but could be eliminated by incubation of the cells with muscarinic cholinergic agonist for 5 min prior to addition of prostaglandin. N1E-115 neuroblastoma cells possess a 41,000-Da membrane protein believed to be a component of the inhibitory GTP-binding protein of adenylate cyclase that is ADP ribosylated by pertussis toxin. Incubation of the cells with pertussis toxin prior to the addition of carbamylcholine reduced the maximal extent of inhibition of cAMP content and prevented the [32P]ADP-ribosylation of a 41,000-Da protein by toxin and [32P]NAD in membrane preparations from these cells. Incubation of cells with pertussis toxin, however, did not significantly alter the dose-response curve for carbamylcholine effects on cGMP. Even high concentrations of carbamylcholine, effective in stimulating cGMP, had minimal effects on cAMP content in toxin-treated cells; thus, ADP-ribosylation of Gi converts the adenylate cyclase but not the guanylate cyclase system to an agonist-insensitive state.
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PMID:Effects of pertussis toxin on cAMP and cGMP responses to carbamylcholine in N1E-115 neuroblastoma cells. 299 40


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