In Vitro Assay for Trans-Phosphorylation of Rhodopsin by Rhodopsin Kinase

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• 作者：Jeanne Rim ; Eva Faurobert ; James B. Hurley ; and Daniel D. Oprian
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：June 10, 1997
• 年：1997
• 卷：36
• 期：23
• 页码：7064 - 7070
• 全文大小：182K
• 年卷期：v.36,no.23(June 10, 1997)
• ISSN：1520-4995

文摘

Trans-phosphorylation of rhodopsin refers to a reaction in which arhodopsin kinase moleculethat has been activated by a light-activated rhodopsin moleculecollides with and phosphorylates a secondmolecule of rhodopsin that has not been activated by light. It hasbeen invoked as a mechanism forhigh-gain phosphorylation, a phenomenon that is observed at lowbleaching levels where up to severalhundred moles of phosphate are added to the rhodopsin pool per mole ofphotolyzed rhodopsin. Trans-phosphorylation is an appealing mechanism to propose for high-gainphosphorylation, but it has not beentested directly because of the difficulty inherent in unambiguousidentification of light-activated and darkforms of rhodopsin present in the same reaction mixture. We reporthere a direct assay fortrans-phosphorylation of rhodopsin. The assay is based on the useof a split receptor mutant of rhodopsin,SR(1-4/5-7), in which the fully functional protein isassembled from two separately expressed fragments.Because of different electrophoretic mobilities,SR(1-4/5-7) and wild-type rhodopsin can bemonitoredindependently for phosphorylation while in the same reaction mixture.Thus, if wild-type rhodopsin isexposed to light and then incubated in the dark withSR(1-4/5-7), ATP, and rhodopsin kinase,phosphorylation of SR(1-4/5-7) would be a clear demonstrationthat trans-phosphorylation has occurred.Despite numerous attempts using several different experimentalconfigurations, we have been unable todetect trans-phosphorylation of dark rhodopsin with thissystem.