Labeling of a GTP-binding regulatory protein of rat brain adenylate cyclase system by cholera toxin-catalyzed ADP-ribosylation.
Cholera toxin ADP-ribosylated several proteins in synaptic plasma membranes of rat brains such as peptides of 66,000, 48,000, 38,000, 25,000, and 18,000 daltons. A 42,000-dalton protein reported to be the GTP-binding regulatory protein in several tissues was faintly labeled and its content varied from preparation to preparation. Adenylate cyclase activity was concomitantly enhanced by more than twice in proportion to the ADP-ribosylation. Accumulated evidence indicated that a protein of 48,000 daltons (48K protein) was a component which constituted the GTP-binding regulatory protein of adenylate cyclase system in rat brain. Among ADP-ribosylated proteins, the 48K protein was the major substrate for the ADP-ribosylation, and the content of this protein was estimated to be about 15 pmol/mg membrane protein. When synaptic membranes were solubilized and applied on a Ultrogel AcA34 gel filtration column to separate the regulatory protein from other components of adenylate cyclase, the 48K protein--but not other ADP-ribosylated proteins--co-eluted with the regulatory protein activity assayed by the reconstitution with catalytic unit of adenylate cyclase. The 48K protein was also detected in myelin fraction which lack adenylate cyclase activity. When solubilized proteins from myelin were subjected to the gel filtration in the AcA34 column, the regulatory protein activity eluted in the 48K protein fractions where the regulatory protein from synaptic membranes appeared. Myelin contained basic protein-like peptides of 20,000 and 18,000 daltons ADP-ribosylated by cholera toxin. These peptides were solubilized from the membranes with acid. Soluble proteins in brain cytosol were ADP-ribosylated by cholera toxin, which was dependent on the presence of nucleotides.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002772:Cholera Toxin / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism; D019204:GTP-Binding Proteins; D008565:Membrane Proteins / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*
D008565:Membrane Proteins; D009702:Nucleoside Diphosphate Sugars; D011956:Receptors, Cell Surface; D000246:Adenosine Diphosphate Ribose; D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases