Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.6.1.1 (adenylate cyclase)
 19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Choleragen and its A protomer catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. NADase activity was inhibited by gangliosides GM1 (galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide), GM2 (N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide), GM3 (N-acetylneuraminyl-galactosylglucosylceramide), and GD1a (N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-E1N-acetylneuraminyl]-galactosylglucosylceramide). These gangliosides also increased the intensity of the tryptophanyl fluorescence of the isolated A protomer (lambda max = 328 nm). GM1 but not GM2, GM3, and GD1a caused a "blue shift" in the fluorescence spectrum of the B protomer. These results are consistent with other evidence that the specificity of GM1 as the choleragen receptor resides in its carbohydrate moiety. The NADase activity of choleragen was similar to that of diphtheria toxin previously described [J. Kandel, R. J. Collier & D. W. Chung (1974) J. Biol. Chem. 249, 2088-2097]. As with diphtheria toxin, analogues of NAD were inhibitory, adenine being the most effective. Significant inhibition was also noted with adenosine, AMP, ADP-ribose, nicotinamide, nicotinamide mononucleotide, and NADP. NADP was hydrolyzed only slowly by choleragen. In the NADase reaction catalyzed by diphtheria toxin, water serves as an acceptor for the ADP-ribose moiety of NAD in lieu of the natural acceptor molecule, which is elongation factor II (Kandel et al., 1974). It seems probable that the natural protein acceptor for ADP-ribose in the reaction catalyzed by choleragen is adenylate cyclase or a protein component of a cyclase complex that regulates enzymatic activity.
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PMID:Effect of gangliosides and substrate analogues on the hydrolysis of nicotinamide adenine dinucleotide by choleragen. 1 71

Rat liver membrane adenylate cyclase (EC 4.6.1.1) that has been stimulated more than 10-fold by cholera toxin (choleragen) has a 3-fold greater sensitivity to stimulation by glucagon. Choleragen similarly increases the sensitivity of cyclase to other peptide (ACTH, vasoactive intestinal polypeptide) and nonpeptide (catecholamines) hormones in this and other tissues. The rate of 125I-labeled glucagon-membrane dissociation is decreased about 2-fold in toxin-treated liver membranes. Toxin-activated cyclase activity of fat cell membranes is retained upon solubilization with Lubrol PX. Provided 125I-labeled choleragen is first incubated with cells under conditions resulting in enzyme activation, the solubilized cyclase activity migrates with a component of 125I-labeled choleragen on gel filtration chromatography. Agarose derivatives containing the "active" subunit (molecular weight 36,000) of the toxin can specifically adsorb solubilized adenylate cyclase. Toxin-stimulated cyclase can be immunoprecipitated with antitoxin or anti-"active" subunit antibodies. There is a large excess of membrane receptors (ganglioside GM1) which, with the use of choleragenoid, can be shown to be functionally equivalent with respect to cyclase activation. Choleragenoid, an inactive competitive antagonist of toxin binding, can occupy and block a large proportion of toxin receptors without affecting toxin activity. A scheme of toxin action is proposed that involves lateral membrane diffusion of the initially inactive toxin-receptor complex with subsequent direct interaction with and modulation of adenylate cyclase. The basic features of this scheme may be pertinent to the mechanisms by which hormone receptors normally modulate adenylate cyclase.
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PMID:Mechanism of action of cholera toxin and the mobile receptor theory of hormone receptor-adenylate cyclase interactions. 16 20

The adenylate cyclase activity of intact pigeon erythrocytes begins to rise after about 20 min of exposure to cholera toxin. The maximum rate at which the cyclase activity increases appears to be limited by the number of toxin molecules which can reach an intracellular target. If the erythrocytes are made permeable to the toxin by a bacterial hemolysin, no such limit exists, and adenylate cyclase activity starts to rise immediately upon the addition of toxin, and continues to rise to a maximum at an initially constant rate which is dependent upon the concentration of toxin. On lysed erythrocytes, the addition of cholera antitoxin immediately prevents any further rise in adenylate cyclase activity, but does not reverse any activation already achieved. Erythrocyte lysates may also be activated by isolated peptide A1 of cholera toxin, although activation of adenylate cyclase of intact erythrocytes requires the complete toxin molecule. In the intact cells, toxin first attaches by its Component B to surface receptors of which there are about 30 per erythrocyte. Subsequently, peptide A1 but not Component B is inserted into the erythrocyte. It takes only about 1 min at 37 degrees for peptide A1 to be sufficiently deep within the cell membrane to be inaccessible to extracellular antitoxin, but its complete transit through the membrane appears to take longer. The surface receptors are used only once, for they remain blocked by Component B. The number of receptors available on the surface may be increased by soaking cells in ganglioside GM1. Cholera toxin also decreases the rate of apparently spontaneous loss of adenylate cyclase activity and increases the response to epinephrine. Theophylline inhibits the action of cholera toxin.
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PMID:The mechanism of action of cholera toxin in pigeon erythrocyte lysates. 16 43

Studies with chemically modified cholera toxin derivatives showed that all treatments that decreased the ability of toxin to bind to mouse thymus cells or to polystyrene-coupled GM1 ganglioside caused a concomitant reduction in the toxin's ability to increase adenosine 3':5'-cyclic phosphate (cyclic AMP) in thymus cells and skin vascular permeability in rabbits. Dissociation of the H (heavy) and L (light) subunits abolished the biologic activity without inhibiting receptor binding, as did treatment with arginyl-specific reagents (which did not change the aggregation state of the toxin). When thymus cells were incubated with 125I-labelled toxin at 37C,only about 1% of the total cell-bound radioactivity was recovered in the cytosol supernate. Similar values were found for cells incubated with toxin at 0C, and with 125I-labelled choleragenoid at 37C or 0C. Thymus cells rapidly bound less than or equal to equal to 5 X 10(4) cholera toxin molecules per cell at both 0C and 37C. Much less, however, of the radioactive toxin bound at 37C than of that bound at 0C was displaced by addition of unlabelled toxin or choleragenoid. Similar temperature-related irreversible binding was noted with 125I-labelled choleragenoid. The relative amounts of H and L subunits in the irreversibly cell-bound and in the displaced 125I-labelled toxin were indistinguishable. Treatment of thymus cells at 37C, but not at 0C, with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide caused a 10-fold reduction of adenylate cyclase stimulation by cholera toxin without inhibiting activation by epinephrine or prostaglandin E1, or appreciably altering the basal, unstimulated enzyme activity. The carbodiimide inhibited the cyclic AMP response to cholera toxin when added shortly after the toxin had bound to the cells (early in the lag phase).
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PMID:Cholera toxin and the adenylate cyclase-activating signal. 17 82

The enterotoxin from Vibrio cholerae is a protein of 100,000 mol wt which stimulates adenylate cyclase activity ubiquitously. The binding of biologically active 125I-labeled choleragen to cell membranes is of extraordinary affinity and specificity. The binding may be restricted to membrane-bound ganglioside GM1. This ganglioside can be inserted into membranes from exogenous sources, and the increased toxin binding in such cells can be reflected by an increased sensitivity to the biological effects of the toxin. Features of the toxin-activated adenylate cyclase, including conversion of the enzyne to a GTP-sensitive state, and the increased sensitivity of activation by hormones, suggest analogies between the basic mechanism of action of choleragen and the events following binding of hormones to their receptors. The action of the toxin is probably not mediated through intermediary cytoplasmic events, suggesting that its effects are entirely due to processes involving the plasma membrane. The kinetics of activation of adenylate cyclase in erythrocytes from various species as well as in rat adipocytes suggest a direct interaction between toxin and the cyclase enzyme which is difficult to reconcile with catalytic mechanisms of adenylate cyclase activation. Direct evidence for this can be obtained from the comigration of toxin radioactivity with adenylate cyclase activity when toxin-activated membranes are dissolved in detergents and chromatographed on gel filtration columns. Agarose derivatives containing the "active" subunit of the toxin can specifically absorb adenylate cyclase activity, and specific antibodies against the choleragen can be used for selective immunoprecipitation of adenylate cyclase activity from detergent-solubilized preparations of activated membranes. It is proposed that toxin action involves the initial formation of an inactive toxin-ganglioside complex which subsequently migrates and is somehow transformed into an active species which involves relocation within the two-dimensional structure of the membrane with direct perturbation of adenylate cyclase molecules (virtually irreversibly). These studies suggest new insights into the normal mechanisms by which hormone receptors modify membrane functions.
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PMID:Structure and function of cholera toxin and hormone receptors. 17 37

NCTC 2071 cells are unable to synthesize the monosialoganglioside GM1. When grown in chemically defined medium these cells contained no detectable GM1 and did not accumulate 3': 5'-cyclic AMP in response to choleragen. Incubation of the cells with [3H]GM1 permitted quantification of ganglioside uptake which was dependent on time and concentration of [3H]GM1 in the medium. Responsiveness to choleragen was demonstrated with binding of as few as 17,000 molecules of [3H]GM1 per cell; a maximal response was observed with 10(5) molecules per cell. With increasing cellular content of GM1, the rate of rise in intracellular cyclic AMP in response to choleragen was increased. With greater than 1 X 10(5) molecules of GM1 per cell, the delay between addition of choleragen and the cyclic AMP response was inversely proportional to choleragen concentration; less than 250 molecules of choleragen per cell caused a significant increase in cyclic AMP after 8 hr of incubation. Although the responsiveness of intact cells to choleragen was dependent on GM1, choleragen activation of adenylate cyclase in homogenates with 0.6 mM NAD was independent of added ganglioside. These observations are consistent with the view that exogenous ganglioside GM1 can be functionally integrated into the surface membrane of intact cells and serve as the choleragen receptor. Furthermore, although exogenous GM1 is required for choleragen responsiveness in intact cells, the ganglioside does not play an obligatory role in cell homogenates, where the surface receptor can presumably be bypassed.
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PMID:Functional incorporation of ganglioside into intact cells: induction of choleragen responsiveness. 17 69

The adenyl cyclase-activating enterotoxin of Vibrio cholerae was shown to contain two types of subunit: six smaller units (L) that are responsible for the binding to cell membrane receptors and a larger unit (H) that mediates the toxic action. The receptor was identified as the ganglioside GM1 (galactosyl-N-acetylgalactosaminyl [sialosyl] lactosyl ceramide), and the results suggested that penetration of the toxin molecule into the membrane follows the rapid binding to GM1. The relationship of these findings to the mechanism of protective immunity, which is mediated by antibodies to the enterotoxin as well as those to the cell wall lipopolysaccharide of V. cholerae, was investigated. The antitoxic antibodies were directed mainly against the L subunit and protected by preventing binding of toxin; the antibacterial antibodies probably interfered with adhesion of V. cholerae to the intestine. The finding that the immune responses to toxin and bacteria act synergistically in protection against experimental cholera indicates that an improved cholera vaccine should contain both toxoid and lipopolysaccharide as antigens. In the rabbit, either subcutaneous or enteral immunization gave rise to intestinal synthesis of specific antibodies to V. cholerae.
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PMID:Mechanisms of disease and immunity in cholera: a review. 19 73

Choleragen exerts its effect on cells through activation of adenylate cyclase. Choleragen initially interacts with cells through binding of the B subunit of the toxin to the ganglioside GM1 on the cell surface. Subsequent events are less clear. Patching or capping of toxin on the cell surface may be an obligatory step in choleragen action. Studies in cell-free systems have demonstrated that activation of adenylate cyclase by choleragen requires NAD. In addition to NAD, requirements have been observed for ATP, GTP, and calcium-dependent regulatory protein. GTP also is required for the expression of choleragen-activated adenylate cyclase. In preparations from turkey erythrocytes, choleragen appears to inhibit an isoproterenol-stimulated GTPase. It has been postulated that by decreasing the activity of a specific GTPase, choleragen would stabilize a GTP-adenylate cyclase complex and maintain the cyclase in an activated state. Although the holotoxin is most effective in intact cells, with the A subunit having 1/20th of its activity and the B subunit (choleragenoid) being inactive, in cell-free systems the A subunit, specifically the A1 fragment, is required for adenylate cyclase activation. The B protomer is inactive. Choleragen, the A subunit, or A1 fragment under suitable conditions hydrolyzes NAD to ADP-ribose and nicotinamide (NAD glycohydrolase activity) and catalyzes the transfer of the ADP-ribose moiety of NAD to the guandino group of arginine (ADP-ribosyltransferase activity). The NAD glycohydrolase activity is similar to that exhibited by other NAD-dependent bacterial toxins (diphtheria toxin, Pseudomonas exotoxin A), which act by catalyzing the ADP-ribosylation of a specific acceptor protein. If the ADP-ribosylation of arginine is a model for the reaction catalyzed by choleragen in vivo, then arginine is presumably an analog of the amino acid which is ADP-ribosylated in the acceptor protein. It is postulated that choleragen exerts its effects on cells through the NAD-dependent ADP-ribosylation of an arginine or similar amino acid in either the cyclase itself or a regulatory protein of the cyclase system.
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PMID:Mechanism of action of choleragen. 21 41

Reaction of cholera toxin with NN'-bis(carboximidomethyl)tartaramide dimethyl ester produced several cross-linked species that had subunit B (which binds to the cell surface) and peptides A1 (which activates adenylate cyclase) and A2 all covalently joined together. This cross-linded material had activity with pigeon erythrocytes that was comparable in all respects with that of native toxin. It activated the adenylate cyclase of whole cells, showing a characteristic lag phase, and this activation was increased if the cells had been preincubated with ganglioside GM1, but abolished if the protein had been preincubated with the ganglioside. It activated the enzyme in lysed cells more strongly and without the lag phase. These results show that the toxin is active even when peptide A1 cannot be released from the rest of the molecule.
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PMID:Activity of covalently cross-linked cholera toxin with the adenylate cyclase of intact and lysed pigeon erythrocytes. 60 47

Cholera diarrhoea is due to the action of a toxin that acts on all animal cells by stimulating the enzyme adenylate cyclase, which catalyses the production oc cyclic AMP from ATP. In intestinal brush border cells raised cyclic AMP levels result in increased secretion of chloride ions, leading to fluid accumulation in the gut. Escherichia coli produces a similar toxin. The receptor for cholera toxin on the cell membrane appears to be a complex containing the ganglioside GGnSLC (or GM1). Cholera toxin is a protein composed of two different kinds of subunits linked non-covalently. Each toxin molecule has one subunit A and four or more subunits B. Subunit B is inactive but binds to the ganglioside GGnSLC on the cell surface. Subunit A does not bind to cell membranes or gangioside and is slightly toxic to intact cells but strongly and instantly active in lysed cells. The binding of whole toxin through the B subunit to the cell is followed by a lag before subunit A penetrates the cell membrane (leaving subunit B on the surface) and stimulates the adenylate cyclase. The stimulation of adenylate cyclase depends on the presence of NAD and other co-factors present in the cell sap.
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PMID:The nature and action of cholera toxin. 79


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