Use of [.gamma.-32P]-8-azidoguanosine 5'-triphosphate as a probe of the guanosine 5'-triphosphate binding protein subunits in bovine rod outer segments

Abstract

In an in vitro incubation, 8-azidoguanosine 5''-[.gamma.-32P]triphosphate([.gamma.-32P]-8-azido-GTP) labeled bleached rhodopsin independent of UV. 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. (MW 40,000). This labeling was UV light dependent. G.beta. (MW 35,000) was also labeled dependent for the most part upon UV, but a smaller component of labeling appeared to result from phosphorylation. Differential labeling of G.alpha. and G.beta. ws 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 2 or more binding sites on G.alpha. was offered by other laboratories, and recently, at least 1 binding site on G.beta. and its analogs among the N proteins of adenylate cyclases was identified.