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 exc hange assay. In exhaustive binding experiments, opsin activates transd ucin, with half-maximal exchange activity occurring at 0.8 mol of opsi n/mol of transducin. The opsin activity was light insensitive, hydroxy lamine-resistant, unaffected by stoichiometric concentrations of retin aloxime, and more heat labile than rhodopsin. The t1/2 of transducin a ctivation in the presence of excess opsin was 8.5 min, compared with 0 .7 min for metarhodopsin(II). The second-order rate constants were det ermined 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 pe r min/nM metarhodopsin(II). Opsin was able to activate more than one t ransducin, although there appeared to be a turnover-dependent inactiva tion of the apoprotein. Opsin showed a broad pH range (5.8-7.4) for op timal activity, with no activity in buffers of pH >9, whereas metarhod opsin(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 a ctivity is proposed.