Activation of immobilized, biotinylated choleragen AI protein by a 19-kilodalton guanine nucleotide-binding protein.
Cholera toxin catalyzes the ADP-ribosylation that results in activation of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system, known as Gs. The toxin also ADP-ribosylates other proteins and simple guanidino compounds and auto-ADP-ribosylates its AI protein (CTA1). All of the ADP-ribosyltransferase activities of CTAI are enhanced by 19-21-kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors, or ARFs. CTAI contains a single cysteine located near the carboxy terminus. CTAI was immobilized through this cysteine by reaction with iodoacetyl-N-biotinyl-hexylenediamine and binding of the resulting biotinylated protein to avidin-agarose. Immobilized CTAI catalyzed the ARF-stimulated ADP-ribosylation of agmatine. The reaction was enhanced by detergents and phospholipid, but the fold stimulation by purified sARF-II from bovine brain was considerably less than that observed with free CTA. ADP-ribosylation of Gsa by immobilized CTAI, which was somewhat enhanced by sARF-II, was much less than predicted on the basis of the NAD:agmatine ADP-ribosyltransferase activity. Immobilized CTAI catalyzed its own auto-ADP-ribosylation as well as the ADP-ribosylation of the immobilized avidin and CTA2, with relatively little stimulation by sARF-II. ADP-ribosylation of CTA2 by free CTAI is minimal. These observations are consistent with the conclusion that the cysteine near the carboxy terminus of the toxin is not critical for ADP-ribosyltransferase activity or for its regulation by sARF-II. Biotinylation and immobilization of the toxin through this cysteine may, however, limit accessibility to Gsa or SARF-II, or perhaps otherwise reduce interaction with these proteins whether as substrates or activator.
Noda
M
M
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
Tsai
S C
SC
Adamik
R
R
Bobak
D A
DA
Moss
J
J
Vaughan
M
M
D001710:Biotin; D002772:Cholera Toxin; D003545:Cysteine; D004789:Enzyme Activation; D019204:GTP-Binding Proteins; D008970:Molecular Weight; D009243:NAD; D011065:Poly(ADP-ribose) Polymerases
D009243:NAD; D001710:Biotin; D002772:Cholera Toxin; D011065:Poly(ADP-ribose) Polymerases; D019204:GTP-Binding Proteins; D003545:Cysteine