Purification and characteristics of photoreceptor light-activated guanosinetriphosphatase.
We describe a reconstitution of light-activated vertebrate photoreceptor GTPase and a purification of the GTP-binding protein (G protein), which is a component of the GTPase and modulates the light-activated phosphodiesterase (PDE) enzyme system. Rod outer segments (ROS) of bull frogs were treated with ethylenediaminetetraacetic acid (EDTA), and the GTPase and PDE fractions were solubilized (EDTA supernatant). When the EDTA supernatant and EDTA-treated membrane fraction (EDTA-washed membranes) were recombined, light-dependent GTPase activity appeared. In the reconstituted system, the Km for GTP as substrate was 0.5 microM; the optimum pH was 7.5-8.0. The isoelectric point of GTPase in EDTA supernatant was 4.8. G protein was purified 400-fold from ROS, and the molecular weight of G protein was determined to be 40 000 by polyacrylamide gel electrophoresis. The amount of G protein in ROS was calculated as at least 1 molecule per 400 rhodopsin molecules. By recombining (in the presence or absence of GTP) purified G protein, PDE, H fraction (an additional component of GTPase), and illuminated or unilluminated EDTA-washed membranes (as a source of rhodopsin), we showed that illuminated rhodopsin, G protein, PDE, and GTP are the minimum requirements for light-dependent PDE activity. We discuss the significance of these findings in the regulation of the light-activated GTPase and PDE activities, especially with regard to the mechanism of activation.
D000818:Animals; D002417:Cattle; D003624:Darkness; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D008081:Liposomes; D008970:Molecular Weight; D010713:Phosphatidylcholines; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010786:Photoreceptor Cells / Q000201:enzymology*; D011892:Rana catesbeiana; D012243:Rhodopsin / Q000302:isolation & purification; D012374:Rod Cell Outer Segment / Q000201:enzymology*
D008081:Liposomes; D010713:Phosphatidylcholines; D012243:Rhodopsin; D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases