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

Autor:	R. E. Kohnken, D. G. Mc Connell
Rok vydání:	1985
Předmět:	Affinity labeling GTP' biology Chemistry Binding protein Adenylate kinase Guanosine Biochemistry chemistry.chemical_compound Rhodopsin Ultraviolet light biology.protein Binding site
Zdroj:	Biochemistry. 24:3803-3809
ISSN:	1520-4995 0006-2960
DOI:	10.1021/bi00335a057
Popis:	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.
Databáze:	OpenAIRE
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