Figure 5.

Effect of 11-cis C19 retinylamine 1 on wild-type opsin. Wild-type opsin was purified by using 0.375 mg/ml asolectin and 0.25% CHAPS and then reconstituted into vesicles by removal of CHAPS on a Sephadex G-50 column. (A) Activation of transducin. Closed symbols, in the dark; open symbols, after exposure of the reaction mixture to light for 30 sec. Circles, opsin incubated in the absence of the analog for 30 min at room temperature then with 11-cis-retinal (231 μM) for 30 min before the assay. Squares, opsin incubated with 1 (100 μM) and 11-cis-retinal (231 μM) simultaneously for 30 min at room temperature before the assay. Triangles, opsin incubated with 1 (100 μM) for 30 min at room temperature then with 11-cis-retinal (231 μM) for 30 min at room temperature before the assay. All the assays were at pH 7.4. (B) Phosphorylation of wild-type opsin. Lane 1, wild-type opsin that was incubated in the absence of the analog for 30 min at room temperature then with 231 μM of 11-cis-retinal for 30 min; lane 2, incubation with 100 μM of 1 for 30 min at room temperature then with 231 μM of 11-cis-retinal for 30 min; and lane 3, incubation with 100 μM of 1 and 231 μM of 11-cis-retinal simultaneously for 30 min at room temperature.