Short communications. Subunit A from cholera toxin is an activator of adenylate cyclase in pigeon erythrocytes

Heyningen, Simon van; King, C. A.

doi:10.1042/bj1460269

Cited by 80 publications

(26 citation statements)

References 16 publications

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“…The initial step in the activation process is presumed to be the binding of the B protomer of choleragen to cell surface receptors, presumably the monosialoganglioside GM1 (2-7). The A protomer is then thought to penetrate the membrane and activate the cyclase (8)(9)(10)(11). In cell homogenates, where the binding step can apparently be bypassed, the A protomer alone can activate adenylate cyclase in an NAD-dependent reaction (12)(13)(14).…”

mentioning

confidence: 99%

Hydrolysis of nicotinamide adenine dinucleotide by choleragen and its A protomer: possible role in the activation of adenylate cyclase.

Moss¹,

Manganiello²,

Vaughan³

1976

Proc. Natl. Acad. Sci. U.S.A.

207

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Choleragen and the isolated A protomer catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide.The protein with NADase activity (NAD nucleosidase; NAD glycohydrolase, EC 3.2.2.5) migrated on polyacrylamide gels with choleragen, and chromatographed on Bio-Gel P-60 columns with the A protomer. The NADase activity of choleragen and of the A protomer was increased markedly in acetate and phosphate buffers, and enhanced over 10-fold by dithiothreitol in high concentration. NAD hydrolysis was proportional to choleragen concentration; the Michaelis constant for NAD was about 4 mM with both choleragen and the A protomer. The demonstration that the A protomer of choleragen catalyzes an enzymatic reaction involving activation of the ribosyl-nicotinamide bond of NAD, a reaction analogous to those catalyzed by diphtheria toxin, supports the hypothesis that activation of adenylate cyclase by choleragen involves the ADP-ribosylation of an appropriate acceptor protein.

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mentioning

confidence: 99%

Hydrolysis of nicotinamide adenine dinucleotide by choleragen and its A protomer: possible role in the activation of adenylate cyclase.

Moss¹,

Manganiello²,

Vaughan³

1976

Proc. Natl. Acad. Sci. U.S.A.

207

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“…Van Heyningen and King have shown recently with partially lysed erythrocytes that, provided the toxin subunit of protein I1 can enter the cells, it will stimulate the intracellular adenylate cyclase system [16].…”

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confidence: 99%

Studies of the Subunit Structure of Choleragen

Sattler

Wiegandt

Staerk³

et al. 1975

Eur J Biochem

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The two choleragen protein constituents were isolated and characterized. Protein I has a molecular weight of approximately 54 000. It consists of subunits of approximate molecular weight 10 000. Protein I1 with molecular weight of approximately 32000 is cleaved by 2-mercaptoethanol into two fragments, protein 11, (N-terminal Asx, M , = 25000) and protein 11, (N-terminal Ser, M , = 7000).Proteins 111 and 112 could be recombined by oxidation to yield protein 11. Upon treatment of choleragen with 2-mercaptoethanol protein 11, precipitates quantitatively. The remaining protein consisting of proteins I and II,, was quantitatively precipitated by ganglioside GGtetl. Of the separated choleragen subunit proteins, only protein I and not protein I1 complexed specifically with ganglioside GGtetl. The isolated proteins I and I1 were considerably less toxic in the skin test but almost full toxicity was recovered after mixing the two proteins I and 11. Antisera against protein I and protein 11 revealed no immuno-cross reactivity between the two proteins. Both antisera inhibited the biological effects of choleragen in the skin and ileal loop tests. A molecular model for the constitution of choleragen is proposed.The pathogenic effects of cholera bacteria in vivo are caused by the action of a secreted toxin upon the mucosal cells. This cholera exo-entero-toxin, choleragen, induces major irreversible membrane permeability changes including the massive outflow of fluid into the intestine. Choleragen also acts on numerous other cell types by way of stimulation of adenylate cyclase (for review see [3]).The work of van Heyningen and collaborators first identified ganglioside to be the cell receptor for choleragen [l 1. These authors discovered that choleragen complexes very specifically with the major monosialo-ganglioside of brain, ganglioside GGtetl .In an earlier investigation it was shown that both the lipophilic and the hydrophilic moiety of the glycolipid contribute to the strength of the binding [Z]. It was also reported that the precipitation of choleragen with ganglioside GGtetl leads to the formation of an apparently high-molecular-weight aggregate of one of the two protein subunits of the toxin. Theother protein subunit, however, showed a less tight binding to the toxin-ganglioside complex and could be readily dissociated by detergents.The ganglioside complexing ability does not appear to be directly related to the toxic activity of the choleragen in vivo. This is shown by the fact that ganglioside is also bound to choleragenoid, which itself is nontoxic and consists only of one type protein of the original choleragen. Furthermore, gangliosidoides i.e. analogues of the ganglioside GGtetl, display comparable binding properties to choleragen, but in contrast to the natural ganglioside still do not inhibit its toxicity in vivo.In order to study the role of the subunits of choleragen in the binding to ganglioside GGtJ as well as choleragenicity, the two proteins were separated and isolated in pure form.The two choleragen prot...

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“…However, if choleragen does dissociate appreciably under these conditions, for ~hy~~rn, the A subunit may be sufficient for adenyl cyclase activation, as reported for two vertebrate cells [6,34]. Proteolytic activities in the growth medium may also be important.…”

Section: Discussionmentioning

confidence: 99%

“…While the mechanism of the rapid activation in Physarum is presently unknown, the simplest explanation may reside in an accelerated activation sequence following the membrane binding of choleragen. Activation of adenyl cyclase in membrane preparations from vertebrate cells by the A or A, protomer occursessentially without alag [8, 13,34]. In Physa~m, reactivity with the whole plasmodium may be accelerated by, e.g., ready accessibility of the A protomer to the regulatory unit of the cyclase.…”

Section: Discussionmentioning

confidence: 99%

Choleragen‐induced cyclic nucleotide response in a lower eukaryote

1980

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Short communications. Subunit A from cholera toxin is an activator of adenylate cyclase in pigeon erythrocytes

Abstract: Intact cholera toxin and its purified subunit A both activate the adenylate cyclase of pigeon erythrocyte membranes, but subunit B does not. The activation by subunit A is unaffected by treatments that inhibit whole toxin by interfering with the binding of subunit B to cell membranes.

Cited by 80 publications

References 16 publications

Hydrolysis of nicotinamide adenine dinucleotide by choleragen and its A protomer: possible role in the activation of adenylate cyclase.

Hydrolysis of nicotinamide adenine dinucleotide by choleragen and its A protomer: possible role in the activation of adenylate cyclase.

Studies of the Subunit Structure of Choleragen

Choleragen‐induced cyclic nucleotide response in a lower eukaryote

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