The light-activated GTP-dependent cyclic GMP phosphodiesterase complex of bovine retinal rod outer segments. Dark resolution of the catalytic and regulatory proteins.
A cyclic GMP phosphodiesterase associated with retinal rod outer segment (ROS) membranes is fully activated only in the presence of light and GTP. Activity can be easily depleted from the membranes by hypotonic washing, which in darkness removes two major soluble proteins. One of these has cGMP phosphodiesterase activity which is no longer activated by light or GTP. The other lacks phosphodiesterase activity but copurifies with the catalytic protein unless special measures are taken. In the present report, these 2 proteins removed from the ROS in darkness were resolved in a manner which was qualitatively and quantitatively dependent upon concentrations of 2-mercaptoethanol and EDTA. We designated the catalytic protein as P and the other protein G, because it has been reported to exhibit GTP-related activities. The unresolved P and G proteins behaved as a single complex on native gels, analytical ultracentrifugation, sucrose gradient sedimentation, and gel filtration. The P:G complex had the same Mr as purified P and more stable phosphodiesterase activity. Removal of G from P destabilized the catalytic activity and allowed aggregation of P. With loss of activity by purified P, multiple slow migrating protein bands appeared upon native gel electrophoresis. Destabilization of P could be partially prevented by addition of Mg2+ before physical separation of P from G. If 2-mercaptoethanol was removed simultaneously, dissociation of the P:G complex and destabilization of P were prevented. These findings imply that the G protein is essential to the catalytic stability of P when both are removed in darkness from the ROS membrane.
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000302:isolation & purification / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D003624:Darkness; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000494:pharmacology*; D008027:Light; D008055:Lipids / Q000032:analysis; D046911:Macromolecular Substances; D008970:Molecular Weight; D010786:Photoreceptor Cells / Q000201:enzymology*; D012374:Rod Cell Outer Segment / Q000201:enzymology*
D000596:Amino Acids; D002241:Carbohydrates; D008055:Lipids; D046911:Macromolecular Substances; D006160:Guanosine Triphosphate; D015106:3',5'-Cyclic-GMP Phosphodiesterases