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 t 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`-(-thio)triphosphate (GTPS) bound per min/nM opsin and 0.35 pmol of GTPS 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.

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