Light-dependent phosphorylation of rhodopsin. Purification and properties of rhodopsin kinase.

Abstract

Rhodopsin kinase was extracted from bovine rod outer segments with 1 M ammonium chloride and purified by ammonium sulfate fractionation and chromatography on Sephacryl S-200 and Blue Sepharose CLGB.The purified kinase was essentially homogeneous in polyacrylamide gel electrophoresis.The molecular weight of kinase was estimated to be 50,000 to 53,000 from the electrophoretic mobility and by gel filtration.There is no evidence that the enzyme is composed of subunits.The enzyme was specific for rhodopsin; phosvitin, casein, histone, and protamine were not phosphorylated.ATP (Km = 8 PM, V,,,, = 40 nmol/ mg/min) was the preferred substrate to GTP (Km = 400 PM, vrmx = 2 nmol/mg/min).The activity was inhibited 90% by 1 mn Zn'+, 50% by 1 nm AMP, and 50% by 1 nm adenosine but not by cyclic AMP or cyclic GMP.Na+ (100 mn) was a potent inhibitor (90% inhibition) of the enzyme, while K+ (100 111~) was without effect.After phosphorylation and regeneration, phosphorylated pigment was separated from unphosphorylated pigment on epichlorohydrin triethanolamine cellulose.Phosphorylated pigment accounted for about 16% of the total pigment and contained about 5 mol of phosphate/mol of pigment.It was thus concluded that the substrate for the light-dependent phosphorylation of rod membranes is a small fraction of rhodopsin that possesses multiphosphorylation sites.To investigate the cellular location of the phosphorylation reaction, rod segments were prepared from living frogs in which newly formed discs had been labeled with [3HJleucine and phosphorylated.The 3H radioactivity per pigment was significantly higher in phosphorylated pigment than in unphosphorylated pigment.The result led us to conclude that newly formed discs (hence, the plasma membrane, as well, that is continuous with the disc infoldings) are preferentially phosphorylated.Light-dependent phosphorylation by ATP of a rod membrane protein, presumably rhodopsin, was discovered independently in three laboratories (l-3).Characteristics of the phosphorylation reaction elucidated with the whole retina, rod outer segments (ROS),' and crude aqueous extracts of rod outer segments include: (i) the reaction occurs only after rhodopsin is bleached by light, (ii) the action spectrum of the light effect coincides well with the absorption spectrum of rhodopsin (2), (iii) light does not activate protein kinase but * The costs of publication of this article were defrayed in part by the payment of page charges.