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

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摘要：

Treatment of pigeon erythrocyte membranes with cholera toxin and NAD+enhanced the GTP stimulation and suppressed the F-activation of the adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. In the presence of NAD+labeled with32P 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-Mrlabeled 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-Mrprotein 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-Mrcomponent bearing the guanyl nucleotide regulatory site.

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关键词：

Biochemistry Pigeon erythrocyte membranes ADP-ribosylation Fluoride activation

DOI：

10.1073/pnas.75.6.2669

被引量：

1755

年份：

1978