Use of 8-azidoguanosine 5'-(gamma-/sup 32/P)triphosphate as a probe of the guanosine 5'-triphosphate binding protein subunits in bovine rod outer segments

Journal Article · Tue Jul 02 00:00:00 EDT 1985 · Biochemistry; (United States)

OSTI ID:6171205

Kohnken, R E; Mc Connell, D G

In an in vitro incubation, 8-azidoguanosine 5'-(gamma-/sup 32/P)triphosphate ( (gamma-/sup 32/P)-8-azido-GTP) labeled bleached rhodopsin independent of ultraviolet light. Characterization of this labeling indicated that rhodopsin was phosphorylated with (gamma-/sup 32/P)-8-azido-GTP as a phosphate donor. At low concentrations, ATP increased this labeling activity 5-fold. In the same incubation, (gamma-/sup 32/P)-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-/sup 32/P)-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.

• Research Organization: Michigan State Univ., East Lansing
• OSTI ID: 6171205
• Journal Information: Biochemistry; (United States), Vol. 14
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
GUANYLIC ACID
RECEPTORS
RHODOPSIN
LABELLING
PHOSPHORYLATION
AFFINITY
ATP
AZIDES
BIOCHEMICAL REACTION KINETICS
CATTLE
MOLECULAR WEIGHT
PHOTOLYSIS
ULTRAVIOLET RADIATION
ANIMALS
CHEMICAL REACTIONS
DECOMPOSITION
DOMESTIC ANIMALS
ELECTROMAGNETIC RADIATION
KINETICS
MAMMALS
NITROGEN COMPOUNDS
NUCLEOTIDES
ORGANIC COMPOUNDS
PHOTOCHEMICAL REACTIONS
PIGMENTS
PROTEINS
RADIATIONS
REACTION KINETICS
RUMINANTS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques