Mechanism of cholera toxin action: covalent modification of the guanyl nucleotide-binding protein of the adenylate cyclase system.
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 (32)P in the AMP moiety the toxin catalyzed the covalent incorporation of radioactivity into membrane proteins with molecular weights (M(r)s) 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-M(r) 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-M(r) 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-M(r) component bearing the guanyl nucleotide regulatory site.
D000247:Adenosine Diphosphate Sugars / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000495:Allosteric Site; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002772:Cholera Toxin / Q000494:pharmacology*; D010856:Columbidae; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000187:drug effects; D005459:Fluorides / Q000494:pharmacology; D006160:Guanosine Triphosphate; D006801:Humans; D009243:NAD / Q000378:metabolism*; D012266:Ribose / Q000378:metabolism
D000247:Adenosine Diphosphate Sugars; D002352:Carrier Proteins; D009243:NAD; D012266:Ribose; D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D000262:Adenylyl Cyclases; D005459:Fluorides