Transducin activation by the bovine opsin apoprotein.
The interaction of the bovine opsin apoprotein with transducin in rod outer segment membranes was investigated using a guanyl nucleotide exchange assay. In exhaustive binding experiments, opsin activates transducin, with half-maximal exchange activity occurring at 0.8 mol of opsin/mol of transducin. The opsin activity was light-insensitive, hydroxylamine-resistant, unaffected by stoichiometric concentrations of retinaloxime, and more heat-labile than rhodopsin. The t1/2 of transducin activation in the presence of excess opsin was 8.5 min, compared with 0.7 min for metarhodopsin (II). The second-order rate constants were determined to be 0.012 pmol of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) bound per min/nM opsin and 0.35 pmol of GTP gamma S bound per min/nM metarhodopsin (II). Opsin was able to activate more than one transducin, although there appeared to be a turnover-dependent inactivation of the apoprotein. Opsin showed a broad pH range (5.8-7.4) for optimal activity, with no activity in buffers of pH > 9, whereas metarhodopsin (II) exhibited activity at pH > 9. Regulation of opsin activity by stoichiometric amounts of retinal was observed, with inhibition by 11-cis-retinal and stimulation by all-trans-retinal. A model for opsin activity is proposed.
Surya
A
A
Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse 13210.
Foster
K W
KW
Knox
B E
BE
D000818:Animals; D001059:Apoproteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D011485:Protein Binding; D012172:Retinaldehyde / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism; D017299:Rod Opsins / Q000302:isolation & purification / Q000378:metabolism*; D015293:Transducin / Q000302:isolation & purification / Q000378:metabolism*
D001059:Apoproteins; D006898:Hydroxylamines; D017299:Rod Opsins; D019811:Hydroxylamine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015293:Transducin; D012172:Retinaldehyde