Mechanism of cholera toxin activation by a guanine nucleotide-dependent 19 kDa protein.
Cholera toxin causes the devastating diarrheal syndrome characteristic of cholera by catalyzing the ADP-ribosylation of Gs alpha, a GTP-binding regulatory protein, resulting in activation of adenylyl cyclase. ADP-ribosylation of Gs alpha is enhanced by 19 kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors or ARFs. We investigated the effects of agents known to alter toxin-catalyzed activation of adenylyl cyclase on the stimulation of toxin- and toxin subunit-catalyzed ADP-ribosylation of Gs alpha and other substrates by an ADP-ribosylation factor purified from a soluble fraction of bovine brain (sARF II). In the presence of GTP, sARF II enhanced activity of both the toxin catalytic unit and a reduced and alkylated fragment ('A1'), as a result of an increase in substrate affinity with no significant effects on Vmax. Activation of toxin was independent of Gs alpha and was stimulated 4-fold by sodium dodecyl sulfate, but abolished by Triton X-100. sARF II therefore serves as a direct allosteric activator of the A1 protein and may thus amplify the pathological effects of cholera toxin.
Noda
M
M
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Tsai
S C
SC
Adamik
R
R
Moss
J
J
Vaughan
M
M
D036002:ADP Ribose Transferases / Q000378:metabolism; D020727:ADP-Ribosylation Factors; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002772:Cholera Toxin / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology*; D007700:Kinetics; D008565:Membrane Proteins / Q000494:pharmacology*; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D008565:Membrane Proteins; D000246:Adenosine Diphosphate Ribose; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D006160:Guanosine Triphosphate; D017830:Octoxynol; D002772:Cholera Toxin; D036002:ADP Ribose Transferases; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D000262:Adenylyl Cyclases