Rod outer segment phosphodiesterase binding and activation in reconstituted membranes.
Light exposure of rhodopsin in rod outer segment (ROS) membranes activates several cyclic GMP phosphodiesterase (PDE) molecules via a GTP-binding protein (G protein). Both PDE and G protein are surface-associated (peripheral) enzymes, which may be extracted from ROS by hypotonic media, individually purified, and recombined in isotonic media with purified rhodopsin-phospholipid vesicles to yield membranes of low dark and high light phosphodiesterase activity. In isotonic media, the PDE strongly associates with phospholipid membranes as well as with ROS and rhodopsin-phospholipid membranes. Because only membrane-associated PDE is readily light activated, the PDE activity saturates when the available binding sites are occupied. At a constant G-protein concentration, the PDE activity observed at saturation is 4 times greater for unilamellar rhodopsin-phospholipid vesicles with a lipid to rhodopsin ratio of 460 than for those with a ratio of 120. Thus, PDE association with membrane in isotonic media is dependent on the phospholipid content rather than the rhodopsin content. Several G proteins per PDE are necessary to maximize the PDE activity of reconstituted membranes; therefore, a weak association between activated G protein and PDE is indicated. Both peripheral enzymes readily transfer between membrane surfaces. Rhodopsin-phospholipid vesicles devoid of enzyme activity were exposed to a light flash and then mixed in the dark in isotonic media with unilluminated ROS membranes which contained PDE and G protein. PDE activity was observed within 2 s after mixing. Subsequent separation and evaluation of the denser ROS membranes and the less dense vesicles demonstrated that both PDE and G protein were associated with the vesicles as well as the ROS membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D004789:Enzyme Activation / Q000528:radiation effects; D019204:GTP-Binding Proteins / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D008027:Light; D008081:Liposomes / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010786:Photoreceptor Cells / Q000201:enzymology*; D012243:Rhodopsin / Q000502:physiology; D012374:Rod Cell Outer Segment / Q000201:enzymology*
D008081:Liposomes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D012243:Rhodopsin; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D019204:GTP-Binding Proteins