A protocol for the separation of phosphorhodopsin from phospho-opsin has been developed. The method takes advantage of the finding that, while 0.5% N,N-dimethyldodecylamine-N-oxide completely solubilises membrane-embedded phosphorhodopsin, at this concentration of detergent, phospho-opsin is only sparingly soluble. Phosphorhodopsin solubilised in this manner may be freed from contaminant phospho-opsin by chromatography on hydroxyapatite. Using this method, the rhodopsin-kinase-catalysed phosphorylation of photoexcited rhodopsin and native rhodopsin was studied in rod outer-segment membranes at different levels of bleaching. Prior to analysis of the phosphorylation mixture, the phosphorylated form of photoexcited rhodopsin was converted into phospho-opsin by treatment with NH2OH. It was found that, while at a 5% bleach level the amount of phosphorhodopsin produced was 15% that of phospho-opsin, at 60% bleaching the phosphorhodopsin was less than 1% of phospho-opsin. The phosphorylation reaction under different bleaching conditions was also studied in a completely soluble system (using 2% dodecyl maltoside) and the pattern of phosphate incorporation into rhodopsin versus opsin was identical to that in the membrane system. We have previously proposed that rhodopsin kinase normally exists in an inactive form and is only activated following interaction with photoexcited rhodopsin. The present work strengthens this conclusion and also shows that, following activation, the kinase preferentially phosphorylates photoexcited rhodopsin but can also act upon unbleached rhodopsin. Two possible mechanisms for the activation of the kinase are considered. From the distribution of phosphorhodopsin and phosphoopsin at different bleaching levels, the relative rates of the phosphorylation of photoexcited rhodopsin (k(R)*) and rhodopsin (k(R)) were calculated. k(R)*/k(R) values for the membrane system of 71 +/- 20 and, for the solubilised system, of 80 +/- 19 were obtained. The algebraic equation used to obtain these values highlights the fact that the ratio of the concentrations of the two substrates, photoexcited rhodopsin and rhodopsin, in a sample, determines the final distribution of phosphate between bleached and unbleached rhodopsin. This conclusion may contribute to the understanding of 'high-gain' phosphorylation observed previously.
