Stimulation of choleragen enzymatic activities by GTP and two soluble proteins purified from bovine brain.
Choleragen (cholera toxin) activates adenylate cyclase by catalyzing ADP-ribosylation of Gs alpha, the stimulatory guanine nucleotide-binding protein. It was recently found (Tsai, S.-C., Noda, M., Adamik, R., Moss, J., and Vaughan, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5139-5142) that a bovine brain membrane protein known as ADP-ribosylation factor or ARF, which enhances ADP-ribosylation of Gs alpha, also increases the GTP-dependent NAD:arginine and NAD:protein ADP-ribosyltransferase, NAD glycohydrolase, and auto-ADP-ribosylation activities of choleragen. We report here the purification and characterization of two soluble proteins from bovine brain that similarly enhance the Gs alpha-dependent and independent ADP-ribose transfer reactions catalyzed by toxin. Like membrane ARF, both soluble factors are 19-kDA proteins dependent on GTP or GTP analogues for activity. Maximal ARF effects were observed at a molar ratio of less than 2:1, ARF/toxin A subunit. Dimyristoyl phosphatidylcholine was necessary for optimal ADP-ribosylation of Gs alpha but inhibited auto-ADP-ribosylation of the choleragen A1 subunit and NAD:agmatine ADP-ribosyltransferase activity. It appears that the soluble factors directly activate choleragen in a GTP-dependent fashion. The relationships of the ARF proteins to the ras oncogene products and to the family of guanine nucleotide-binding regulatory proteins that includes Gs alpha remains to be determined.
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002772:Cholera Toxin / Q000378:metabolism*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000494:pharmacology*; D010430:Pentosyltransferases / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology
D009419:Nerve Tissue Proteins; D013873:Thionucleotides; D000246:Adenosine Diphosphate Ribose; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D036002:ADP Ribose Transferases; D010430:Pentosyltransferases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases