Abstract
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.
Highlights
Visual transduction in the rod cell is mediated by the photoreceptor, rhodopsin
Accompanying the conformational changes are the deprotonation of the Schiff base, followed by the hydrolysis and release of the chromophore, yielding the apoprotein and all-trans-retinal (for a recent review, see Birge (1990)
There are a number of conformational states of light-activated rhodopsin, several lines of evidence suggest that metarhodopsin(II) is a key catalyst of transducin activation in the rod outer segment
Summary
Protein and Lipid Preparations-Rod outer segments were prepared in dim red light from frozen bovine retinae The purified transducin bound between 0.91.0 mol ofGTPyS'/mol of protein, as determined by exhaustive reaction with [35S]GTPyS in the presence of excess light-activated rhodopsin (see below). Initial Rate Measurements-Transducin (150 pmol) was added in the dark to 750 J.LI containing 10 mM Tris acetate, pH 7.4, 100 mM NaCI, 5 mM MgCI 2, 5 mM 2-mercaptoethanol, 2 J.LM [35S]GTPyS and either opsin or rhodopsin. Data from the exhaustive binding experiments (Fig. 2) was fit to the Hill equation, which yielded estimates for the concentration of opsin or metarhodopsint ll) required to activate. The initial rate data for both opsin and metarhodopsin(Il) were fitted to single parameter exponential decay equations These equations yielded values for the tY" for the reaction. The second-order rate constant was determined from the slope of the linear portion of the fit for the opsin or metarhodopsin(II) concentration in the linear range for transducin activation
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