Rhodopsin kinase prepared from bovine rod disk membranes quenches light activation of cGMP phosphodiesterase in a reconstituted system.
Rhodopsin kinase was extracted into a buffer containing 200 mM KCl and no MgCl2. The activity of the enzyme was stabilized with the use of a mixture of protease inhibitors, aprotinin, benzamidine, leupeptin, and pepstatin. The extract consisted of three major proteins of molecular weight (Mr) 65,000, 56,000, and 37,000, of which the Mr 65,000 protein was identified with the kinase activity since preparations containing the other proteins had no kinase activity and the Mr 65,000 protein was phosphorylated when the extract was incubated with ATP. A reconstituted cGMP phosphodiesterase (PDE) system consisting of peripheral protein-depleted rod disk membranes (RDM), GTP binding protein (G-protein), and PDE was used to test the effectiveness of the rhodopsin kinase preparation in mediating the ATP-dependent quench of light activation of PDE. In the absence of kinase, light-activated PDE activity lasted several minutes. In its presence, ATP and to a lesser extent GTP quenched the activation about as rapidly as in rod disk membranes. The influence of kinase was unaffected by increasing G-protein or PDE content of the reconstituted system but was slowed down by brighter flashes, showing that quench was caused by the inactivation of bleached rhodopsin and not of PDE or G-protein.
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism / Q000528:radiation effects*; D000818:Animals; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D004789:Enzyme Activation; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D019204:GTP-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D008027:Light; D010786:Photoreceptor Cells / Q000201:enzymology*; D011494:Protein Kinases / Q000378:metabolism*
D005136:Eye Proteins; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D019204:GTP-Binding Proteins