Use of 8-azidoguanosine 5'-[gamma-32P]triphosphate as a probe of the guanosine 5'-triphosphate binding protein subunits in bovine rod outer segments.
In an in vitro incubation, 8-azidoguanosine 5'-[gamma-32P]triphosphate ( [gamma-32P]-8-azido-GTP) labeled bleached rhodopsin independent of ultraviolet light. Characterization of this labeling indicated that rhodopsin was phosphorylated with [gamma-32P]-8-azido-GTP as a phosphate donor. At low concentrations, ATP increased this labeling activity 5-fold. In the same incubation, [gamma-32P]-8-azido-GTP also labeled G alpha (Mr 40 000). This labeling was ultraviolet light dependent. G beta (Mr 35 000) was also labeled dependent for the most part upon ultraviolet light, but a smaller component of labeling appeared to result from phosphorylation. Differential labeling of G alpha and G beta was found to vary intricately with experimental conditions, especially prebleaching of rhodopsin, tonicity of the medium, and the presence or absence of 2-mercaptoethanol. Affinity labeling of G alpha and G beta by [gamma-32P]-8-azido-GTP in competition with ATP or GTP was kinetically complex, consistent with possible multiple binding sites for GTP on both subunits. Independent evidence for two or more binding sites on G alpha has been offered by other laboratories, and recently, at least one binding site on G beta and its analogues among the N proteins of adenylate cyclases has been identified.
D000818:Animals; D001386:Azides / Q000378:metabolism* / Q000528:radiation effects; D001665:Binding Sites; D002417:Cattle; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000528:radiation effects; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D010782:Photolysis; D010786:Photoreceptor Cells / Q000378:metabolism*; D012168:Retinal Pigments / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D014466:Ultraviolet Rays
C021961:8-azidoguanosine triphosphate; D001386:Azides; D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D012168:Retinal Pigments; D006160:Guanosine Triphosphate; D012243:Rhodopsin; D019204:GTP-Binding Proteins