Light-regulated binding of rhodopsin kinase and other proteins to cattle photoreceptor membranes
Biochemistry 17(21): 4389-4395
ISSN/ISBN: 0006-2960 PMID: 718845 DOI: 10.1021/bi00614a006
Rhodopsin kinase, the enzymatic activity which catalyzes the light-induced phosphorylation of rhodopsin by ATP, could be readily extracted into aqueous buffers from dark-adapted, but not from illuminated, rod outer segments. Kinase activity was up to 17 times higher in dark extracts than in corresponding light extracts, regardless of ionic strength and composition of the extracting buffer. The kinase bound to freshly bleached rod outer segment membranes but was released in the dark with a half-time of about 20-30 min at 20.degree. C. The light-induced binding was not influenced by MgCl2, (ethylenedinitrilo)tetraacetic acid or KF. This reversible binding was used to partially purify the enzyme. The kinase was bound to bleached photoreceptor disk membranes, other soluble proteins were washed out and the kinase was then extracted in the dark. The most prominent protein in such purified extracts had a MW of 48,000 and no kinase activity; its function is as yet unknown. Rhodopsin kinase, the next most prominent protein reversibly bound, had a MW of 67,000-69,000, as determined by molecular-sieve chromatography of the active enzyme and by sodium dodecyl sulfate gel electrophoresis. Gel electrophoresis also showed significantly higher amounts of these 2 proteins in crude dark extracts as compared with crude light extracts. Under some conditions, a 3rd protein (about 37,000 daltons) was present in dark extracts and absent in light extracts. The decay in the dark of the capacity of bleached membranes to bind kinase had a time course which corresponded approximately to the decay of phosphorylation activity after bleaching. Apparently rhodopsin or the membrane, after bleaching, transiently exhibits binding sites for the kinase and for the other proteins, and the activity of the phosphate transfer, and perhaps of other reactions, is regulated by this light- and time-dependent binding.