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)

The kinetics and properties of the activation of adenylate cyclase by cholera enterotoxin have been examined primarily in toad erythrocytes, but also in avian erythrocytes, rat fat cells and cultured melanoma cells. When cholera toxin is incubated with intact cells it stimulates adenylate cyclase activity, as measured in the subsequently isolated plasma membranes, according to a triphasic time course. This consists of a true lag period of about 30 min, followed by a stage of exponentially increasing adenylate cyclase activity which continues for 110 to 130 min, and finally a period of slow activation which may extend as long as 30 hr in cultured melanoma cells. The progressive activation of adenylate cyclase activity by cholera toxin is interrupted by cell lysis; continued incubation of the isolated membranes under nearly identical conditions does not lead to further activation of the enzyme. The delay in the action of the toxin is not grossly dependent of the number of toxin-receptor (GM1 ganglioside) complexes, and is still seen upon adding a second dose of toxin to partially stimulated cells. Direct measurements indicate negligible intracellular levels of biologically active radioiodinated toxin in either a soluble or a nuclear-bound form. The effects are not prevented by Actinomycin D (20 mug/ml), uromycin (30 mug/ml), cycloheximide (30 mug/ml), sodium fluoride (10 mM) or sodium azide (1 mM); KCN, however, almost completely prevents the action of cholera toxin. The action of the toxin is temperature dependent, occurring at very slow or negligible rates below certain critical temperatures, the values of which depend on the specific animal species. Thetransition for toad erythrocytes occurs at 15 to 17 degrees C, while rat adipocytes and turkey erythrocytes demonstrate a discontinuity at 26 to 30 degrees C. The temperature effects are evident during the lag period as well as during the exponential phase of activation. The rate of decay of the stimulated adenylate cyclase activity of cultured melanoma cells indicates a half-time of about 36 hr. The rate of exponentially increasing activity and extent of enzyme activation are related to the number of bound toxin molecules according to a Langmuir adsorption isotherm and are half-maximal when about 2000 molecules of toxin are bound per cell. It is proposed that initially cholera toxin binds ineffectively, but that it is converted to an active form during the lag phase. This process may involve lateral motion of toxin-GM1 ganglioside complex within the plane of the membrane. The kinetics of adenylate cyclase activation are consistent with the possibility that during the exponential phase a bimolecular association is proceeding between the active form of the cholera toxin and some other membrane component. The possibility is considered that the cholera toxin molecule may bind directly to adenylate cyclase. These considerations may prove useful in understanding the possible interactions of active hormone-receptor complexes with adenylate cyclase in cell membranes.
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PMID:Mechanism of activation of adenylate cyclase by Vibrio cholerae enterotoxin. 80 48

The toxin of Vibrio cholerae dissociates into subunit A and an aggregate of subunit B (choleragenoid); the dissociation is rapid under denaturing conditions and slow at neutral pH. Subunit A has a molecular weight of 27,000 daltons (measured by sedimentation equilibrium or gel chromatography) and has two polypeptide chains (mol wt, approximately 22,000 and 5,000 daltons) joined by disulfide bonds. The molecular weight of subunit B in 6 M guanidine hydrochloride is 14,000 daltons when determined by sedimentation equilibrium or gel chromatography, although dodecylsulfate gel electrophoresis suggests a lower value. These results suggest a structure of AB4 for the toxin; studies of cross-linking with methyl-4-mercaptobutyrimidate confirm this structure. The properties of antibodies both to cholera toxin and to choleragenoid are compatible with this structure, but subunit A has very low immunogenicity. Subunit A by itself is active, and this activity is abolished by a large excess of antitoxin but not by choleragenoid, anticholeragenoid, or ganglioside GM1 (galactosyl-N-acetylgalactosaminyl [sialosyl] lactosyl ceramide; GGnSLC). It is suggested that the function of subunit B is not to interact directly with the adenylate cyclase system, but to bind to the cell membrane and facilitate the interaction of subunit A.
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PMID:The subunits of cholera toxin: structure, stoichiometry, and function. 81 50

Dynamic aspects of the binding of cholera toxin to lymphocyte membranes have been studied. We have shown that the receptor for this ligand--the GM1 ganglioside--can be laterally redistributed into aggregates and caps. Exogenous purified GM1 inserted into GM1-deficient human leukaemic cells can undergo a similar pattern of ligand-induced mobilisation. These observations may have important implications for both the general behaviour of cell surface glycolipids and the mode of action of cholera toxin on adenyl cyclase.
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PMID:Ligand-induced redistribution of lymphocyte membrane ganglioside GM1. 105 31

Cholera toxin (choleragen) can stimulate adenylate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)] activity in whole particulate fractions or purified plasma membranes of homogenates of isolated fat cells provided special precautions are taken to stabilize the enzyme during the required preincubation period. As observed with intact cells, the activation exhibits a protracted (about 25 min) lag phase, and it is blocked by ganglioside GM1 and choleragenoid ("binding" subunit of toxin). The 36,000 molecular weight subunit ("active" subunit), a hydrophobic polypeptide which does not block choleragen binding or action, can directly activate the enzyme in intact cells without a lag phase. Its effects are not blocked by ganglioside GM1 or choleragenoid, yet the stimulated activity exhibits reduced fluoride and enhanced isoproterenol sensitivity, properties characteristic of the choleragen-activated enzyme. Binding of the 125I-labeled 36,000 molecular weight subunit to cells is not saturable and is unaffected by gangliosides, choleragen, or choleragenoid, and the bound material behaves as an integral membrane protein; this protein may simply partition into the membrane matrix. With increasing time of incubation cell-bound choleragen may dissociate into its component subunits, but these remain in the membrane. Using a double antibody immunoprecipitin system, substantial precipitation of cyclase activity occurs with antisera against the 36,000 molecular weight subunit provided toxin activation has occurred. The normal process of activation may involve an initially inactive toxin--ganglioside complex which, as a result of lateral mobility and multivalent binding (lag phase), results in destabilization of the molecule with release of the "active" subunit into the membrane core where it can spontaneously associate with and perturb the cyclase complex.
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PMID:Mechanism of activation of adenylate cyclase by cholera toxin. 105 29

Fluorescein-labeled cholera toxin binds detectably to 40-60% of rat mesenteric lymph node cells and induces a temperature-dependent redistribution (patch and cap formation) of cell surface toxin receptors. The redistribution is inhibited by several "metabolic," "microtubule," and "microfilament" inhibitors, by concanavalin A, and by anticholera toxin IgG. Various studies indicate that cholera toxin is at least bivalent, and that this property may be related to both the induction of receptor redistribution and to the activation of adenylate cyclase. Membrane components which are probably identical to the sialo-glycolipid, GM1 ganglioside, appear to be mobile in the plane of the membrane. The possible role of toxin multivalency and receptor mobility in the mechanism of toxin action is considered.
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PMID:Mobility of cholera toxin receptors on rat lymphocyte membranes. 106 63

The ganglioside galactosy-N-acetylgalactosaminyl [sialosyl] lactosyl ceramide (GM1) is readily accumulated in pigeon red cell membranes soaked with [3H]GM1 (1-100 mug/ml) at 37 C for 30 min. This treatment enhances the activation of adenyl cyclase by the toxin of Vibrio cholerae. An attempt was made at correlation of the amount of incorporated GM1 with the increased binding of toxin and activation of adenyl cyclase. Cells with less than 2 mug of incorporated GM1 per 4 X 10(9) cells bind 5-10 mug more toxin than do untreated cells, which bind 0.25 mug per 4 X 10(9) cells. Cells with more than 2 mug of GM1 bound (per 4 X 10(9) CELls) which has been incorporated from micellar solutions of GM1 (greater than 20 mug/ml), do not bind any more extra toxin. In untreated cells, 0.1 mug of toxin is involved in the activation of adenyl cyclase. In GM1-treated cells 0.25-0.5 mug of toxin is involved, although at least 5 mug of toxin is bound. It is concluded that 90% of the extra toxin-binding sites on the GM1-treated cell are nonproductive.
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PMID:Aspects of the interaction of Vibrio cholerae toxin with the pigeon red cell membrane. 125 96

The ability of Fuc-GM1 ganglioside to mimic the receptor function of GM1 for cholera toxin (CT) has been investigated. For this purpose, rat glioma C6 cultured cells were enriched with Fuc-GM1 and the responsiveness to CT was compared with that of cells enriched with GM1 ganglioside. Fuc-GM1 was taken up by cells as rapidly and to the same extent as GM1. When comparable amounts of ganglioside were associated, the cells enriched with Fuc-GM1 bound the same amount of 125I-CT as did cells enriched with GM1. Under conditions in which GM1- and Fuc-GM1-enriched cells bound comparable amounts of CT, the Fuc-GM1-treated cells accumulated virtually the same amount of cyclic AMP as did GM1-treated cells, and activation of adenylate cyclase was also similar. The lag time preceding the CT-induced cAMP accumulation was the same in Fuc-GM1- and GM1-enriched cells. High-sensitivity isothermal titration calorimetry (ITC) experiments showed that the association constants of CT with Fuc-GM1 or GM1 ganglioside were comparable (4 x 10(7) M-1 and 1.9 x 10(7) M-1, respectively, at 25 degrees C). Also, the association constants of the B-subunit pentamer with Fuc-GM1 or GM1 ganglioside were comparable (about 3 x 10(7) M-1 and 7 x 10(7) M-1, respectively, at 25 degrees C).
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PMID:Fuc-GM1 ganglioside mimics the receptor function of GM1 for cholera toxin. 131 1

The massive secretion of salt and water in cholera-induced diarrhea involves binding of cholera toxin (CT) to ganglioside GM1 in the apical membrane of intestinal epithelial cells, translocation of the enzymatically active A1-peptide across the membrane, and subsequent activation of adenylate cyclase located on the cytoplasmic surface of the basolateral membrane. Studies on nonpolarized cells show that CT is internalized by receptor-mediated endocytosis, and that the A1-subunit may remain membrane associated. To test the hypothesis that toxin action in polarized cells may involve intracellular movement of toxin-containing membranes, monolayers of the polarized intestinal epithelial cell line T84 were mounted in modified Ussing chambers and the response to CT was examined. Apical CT at 37 degrees C elicited a short circuit current (Isc: 48 +/- 2.1 microA/cm2; half-maximal effective dose, ED50 integral of 0.5 nM) after a lag of 33 +/- 2 min which bidirectional 22Na+ and 36Cl- flux studies showed to be due to electrogenic Cl- secretion. The time course of the CT-induced Isc response paralleled the time course of cAMP generation. The dose response to basolateral toxin at 37 degrees C was identical to that of apical CT but lag times (24 +/- 2 min) and initial rates were significantly less. At 20 degrees C, the Isc response to apical CT was more strongly inhibited (30-50%) than the response to basolateral CT, even though translocation occurred in both cases as evidenced by the formation of A1-peptide. A functional rhodamine-labeled CT-analogue applied apically or basolaterally at 20 degrees C was visualized only within endocytic vesicles close to apical or basolateral membranes, whereas movement into deeper apical structures was detected at 37 degrees C. At 15 degrees C, in contrast, reduction to the A1-peptide was completely inhibited and both apical and basolateral CT failed to stimulate Isc although Isc responses to 1 nM vasoactive intestinal peptide, 10 microM forskolin, and 3 mM 8Br-cAMP were intact. Re-warming above 32 degrees C restored CT-induced Isc. Preincubating monolayers for 30 min at 37 degrees C before cooling to 15 degrees C overcame the temperature block of basolateral CT but the response to apical toxin remained completely inhibited. These results identify a temperature-sensitive step essential to apical toxin action on polarized epithelial cells. We suggest that this event involves vesicular transport of toxin-containing membranes beyond the apical endosomal compartment.
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PMID:Mechanism of cholera toxin action on a polarized human intestinal epithelial cell line: role of vesicular traffic. 131 83

This study addressed the question of whether the mucosal adjuvant property of cholera toxin (CT) and the structurally closely related Escherichia coli heat-labile toxin (LT) requires the enterotoxic and adenylate cyclase/cAMP activating property of these molecules. Therefore, we investigated the cytotoxic and adjuvant abilities of the enterotoxins and compared the results with those obtained with the non-toxic CT and LT derivatives; recombinant CTB (rCTB) and a mutated LT (mLT), which had a single amino acid substitution in position 112 (Glu----Lys) of the A subunit. Detailed functional studies revealed that, in contrast to the enterotoxins, both rCTB and mLT lacked ADP-ribosylating and cAMP-stimulating abilities. However, similar membrane ganglioside GM1-receptor binding ability of all the putative adjuvants was demonstrated. When the probe antigen, keyhole limpet hemocyanin (KLH), was given perorally together with CT or LT strong gut mucosal anti-KLH immune responses were stimulated, whereas no or very low anti-KLH responses were seen in the groups which received antigen admixed with rCTB or the mLT. Moreover, the specific serum antibody responses to the various immunization protocols closely paralleled the local anti-KLH response in the gut. From these results it appears that the adjuvant mechanism of LT, and probably also of CT, is linked to the ability to ADP-ribosylate and to stimulate cAMP formation. However, this study does not unequivocally rule out other possibilities such as interactions by the A1 fragment of CT or LT with other G-proteins than Gs alpha or events that parallel or precede the effects on the adenylate cyclase/cAMP system. Thus, the levels of ADP-ribosylation and cAMP-induction that are required and the key event or target cell that is responsible for the adjuvant effect of CT and LT remain to be elucidated. Studies are underway to address these issues.
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PMID:The adjuvant effect of Vibrio cholerae and Escherichia coli heat-labile enterotoxins is linked to their ADP-ribosyltransferase activity. 138 11

Low (nanomolar) concentrations of opioid agonists prolong the calcium-dependent component of the action potential duration (APD) of many dorsal root ganglion (DRG) neurons, whereas higher (micromolar) levels shorten the APD. Both effects are blocked by naloxone (1-10 nM). Opioid-induced APD prolongation appears to be mediated by excitatory opioid receptors that are positively coupled via a cholera toxin-A-sensitive Gs protein to adenylate cyclase/cyclic AMP-dependent ion conductances, whereas opioid-induced APD shortening is mediated by inhibitory receptors linked via pertussis toxin-sensitive Gi/Go proteins. Cholera toxin-B subunit, which binds to GM1 ganglioside, also selectively blocks opioid-induced APD prolongation. After brief treatment with GM1 ganglioside, the opioid agonists, dynorphin (1-13) or morphine, prolong the APD at femtomolar vs. the usual nanomolar concentrations, whereas no significant alterations were observed in the sensitivity of these GM1-treated cells to opioid inhibitory effects elicited by higher opioid concentrations. The present study shows that the opioid antagonists, naloxone or diprenorphine (1-30 nM), did not alter the APD of naive DRG neurons. In contrast, after GM1 treatment (1 microM, greater than 10 min), both opioid antagonists (but not (+)naloxone) unexpectedly prolonged the APD of most of the GM1-treated cells, but still continued to antagonize opioid-induced APD shortening. These results suggest that the supersensitivity of GM1-treated DRG neurons to the excitatory effects of opioid agonists and antagonists is due primarily to a remarkably increased efficacy of excitatory Gs-coupled opioid receptor functions, similar to the opioid excitatory supersensitivity that we have recently observed in chronic opioid-treated DRG neurons.
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PMID:After GM1 ganglioside treatment of sensory neurons naloxone paradoxically prolongs the action potential but still antagonizes opioid inhibition. 173 Oct 37


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