38] Rhodopsin kinase

Abstract

Publisher Summary This chapter focuses on rhodopsin kinase. Rhodopsin kinase—the enzyme involved in the reaction—catalyzes the transfer of the terminal (γ) phosphate group of adenosine triphosphate (ATP) to the opsin protein. In the assay method of rhodopsin kinase, bovine rod outer segments are treated with urea to prepare the substrate. This treatment denatures membrane-associated rhodopsin kinase but has little effect on rhodopsin. The substrate is then incubated in the light with rhodopsin kinase, [y -32p ]ATP, and [adenine- 3 H]ATP. The inclusion of [ 3 H]ATP is to estimate the extent of binding of ATP itself. In an alternative method, rod membranes are incubated with [γ -32P ]ATP in dark and then in light. The kinase activity is determined by subtracting the 32 P incorporated in dark from the 32 P incorporated in light. The phosphorylated protein is identified as rhodopsin after the separation of the labeled protein by chromatographic and electrophoretic methods.