Guanine nucleotide dependent formation of a complex between choleragen (cholera toxin) a subunit and bovine brain ADP-ribosylation factor.
Cholera toxin activates adenylyl cyclase by catalyzing the ADP-ribosylation of Gs alpha, the stimulatory guanine nucleotide binding protein of the cyclase system. This toxin-catalyzed reaction, as well as the ADP-ribosylation of guanidino compounds and auto-ADP-ribosylation of the toxin A1 protein (CTA1), is stimulated, in the presence of GTP (or GTP analogue), by 19-21-kDa proteins, termed ADP-ribosylation factors or ARFs. These proteins directly activate CTA1 in a reaction enhanced by sodium dodecyl sulfate (SDS) or dimyristoylphosphatidylcholine (DMPC)/cholate. To determine whether ARF stimulation of ADP-ribosylation is associated with formation of a toxin-ARF complex, these proteins were incubated with guanine nucleotides and/or detergents and then subjected to gel permeation chromatography. An active ARF-toxin complex was observed in the presence of SDS and GTP gamma S [guanosine 5'-O-(3-thiotriphosphate)] but not GDP beta S [guanosine 5'-O-(2-thiodiphosphate)]. Only a fraction of the ARF was capable of complex formation. The substrate specificities of complexed and noncomplexed CTA differed; complexed CTA exhibited markedly enhanced auto-ADP-ribosylation. In the presence of GTP gamma S and DMPC/cholate, an ARF-CTA complex was not detected. A GTP gamma S-dependent ARF aggregate was observed, however, exhibiting a different substrate specificity from monomeric ARF. These studies support the hypothesis that in the presence of guanine nucleotide and either SDS or DMPC/cholate, ARF and toxin exist as multiple species which exhibit different substrate specificities.
Tsai
S C
SC
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Adamik
R
R
Moss
J
J
Vaughan
M
M
D020727:ADP-Ribosylation Factors; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002417:Cattle; D002772:Cholera Toxin / Q000378:metabolism*; D019826:Cholic Acid; D002793:Cholic Acids / Q000494:pharmacology; D002850:Chromatography, Gel; D004134:Dimyristoylphosphatidylcholine / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
D002793:Cholic Acids; D008565:Membrane Proteins; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D012967:Sodium Dodecyl Sulfate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D019826:Cholic Acid; D004134:Dimyristoylphosphatidylcholine