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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1978 Jun;75(6):2669–2673. doi: 10.1073/pnas.75.6.2669

Mechanism of cholera toxin action: Covalent modification of the guanyl nucleotide-binding protein of the adenylate cyclase system

Dan Cassel 1,*, Thomas Pfeuffer 1,†
PMCID: PMC392624  PMID: 208069

Abstract

Treatment of pigeon erythrocyte membranes with cholera toxin and NAD+ enhanced the GTP stimulation and suppressed the F- activation of the adenylate cylase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. In the presence of NAD+ labeled with 32P in the AMP moiety the toxin catalyzed the covalent incorporation of radioactivity into membrane proteins with molecular weights (Mrs) of 200,000, 86,000, and 42,000. Extraction of toxin-treated membranes with Lubrol PX followed by affinity chromatography on a GTP-Sepharose column resulted in a 200-fold purification of the 42,000-Mr labeled protein and in its complete separation from the other labeled proteins. The fraction containing the purified GTP-binding component from toxin-treated membranes conferred an enhanced GTP-stimulated activity on adenylate cyclase solubilized from nontreated membranes. Likewise, the addition of GTP-binding fraction from nontreated membranes to an enzyme solubilized from toxin-treated membranes restored F- stimulation of the adenylate cyclase. The toxin-induced modification of adenylate cyclase and the incorporation of radioactivity into the 42,000-Mr protein were partially reversed upon incubation with toxin and nicotinamide at pH 6.1. The results indicate that cholera toxin affects the adenylate cyclase system by catalyzing an ADP-ribosylation of the 42,000-Mr component bearing the guanyl nucleotide regulatory site.

Keywords: pigeon erythrocyte membranes, ADP-ribosylation, fluoride activation

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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