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DJ Kelleher and GL Johnson
Department of Biochemistry, University of Massachusetts Medical School, Worcester 01655.
Rhodopsin kinase was purified from bovine retina rod outer segments as a 62-64-kDa protein that phosphorylated purified rhodopsin reconstituted into egg phosphatidylcholine/phosphatidylethanolamine liposomes. A competition binding assay in which transducin competes with rhodopsin kinase for binding sites on rhodopsin was used to assess the interaction of purified transducin subunits with rhodopsin. Preincubation of purified holotransducin with rhodopsin, in the absence of guanosine triphosphate, blocked the ability of the kinase to phosphorylate rhodopsin. Transducin-dependent inhibition of phosphorylation was relieved when guanosine 5'-(3-O-thio)triphosphate was present during the preincubation. Resolved alpha and beta gamma transducin subunits, in the absence of guanosine triphosphate, were each capable of specifically blocking phosphorylation of rhodopsin. A maximally effective concentration of T alpha or T beta gamma (1 microM) subunits inhibited phosphorylation of rhodopsin (0.23 microM) 45-65%. A similar concentration of reconstituted transductin (T alpha and T beta gamma) or native holotransducin (T alpha beta gamma) inhibited phosphorylation greater than 98%. The results indicate that rhodopsin must have a binding site for T beta gamma as well as a binding site for T alpha, and each subunit influences the recognition of bleached rhodopsin by rhodopsin kinase.
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