1] Inhibition of rhodopsin phosphorylation by S-modulins: purification, reconstitution, and assays

Abstract

Publisher Summary S-Modulin is a calcium-binding protein found in frog retinal rods. S-modulin activity was first detected while performing an electrophysiological measurement of cGMP phosphodiesterase (PDE) activity, using a truncated preparation of frog rod outer segment (tROS) that can be internally perfused with a bathing solution. In a later study, it was shown that S-modulin regulates PDE activation by inhibiting rhodopsin phosphorylation at high Ca 2+ concentrations. At almost the same time that S-modulin was reported, another Ca 2+ -binding protein was reported. This protein, named recoverin, was found in bovine retina. Later study showed that recoverin is the bovine homolog of S-modulin. This chapter describes: (1) an electrophysiological method by which to detect S-modulin activity, (2) purification of S-modulin and its cone homolog s26 (frog visinin) from frog retina and purification of recoverin from bovine retina, (3) purification of S-modulin, s26, and recoverin that are expressed in Escherichia coli, and (4) reconstitution and assay of S-modulin activity.