64] Assay of phosphorylation of rhodopsinin vitro andin vivo

Abstract

This chapter discusses the assay of phosphorylation of rhodopsin in vitro and in vivo . Dark-adapted rhodopsin is unphosphorylated. Light induces a transient capacity into rhodopsin to be phosphorylated by a protein kinase and adenosine triphosphate (ATP). Guanosine triphosphate (GTP) may also serve as a substrate. The terminal phosphate group of ATP or GTP is transferred to opsin and bound covalently as a phosphate ester to serine and threonine residues, which are located close to the carboxyl terminus of opsin. The light-induced property of rhodopsin to be a substrate, however, decays slowly in the dark, allowing phosphorylation to take place only for a limited time after bleaching. Dephosphorylation may also be observed under certain conditions in rod outer segment (ROS) suspensions in vitro . However, it is easy to suppress the dephosphorylation activity completely in vitro , such that the activation and inactivation of the phosphorylation reaction can be studied in the absence of any dephosphorylation effects.