Contribution of the guanosinetriphosphatase activity of G-protein to termination of light-activated guanosine cyclic 3',5'-phosphate hydrolysis in retinal rod outer segments.
Light activation of GTP binding to G-protein and its eventual hydrolysis are hypothesized to lead to activation and inactivation of cGMP phosphodiesterase (PDE) in vertebrate rod disk membranes (RDM). However, the reported GTPase rate of 3 per minute is too slow to account for the observed rapid inactivation of PDE. Our investigations on GTPase activity showed that RDM isolated in the dark have considerable dark GTPase activity, which is enhanced by light. In dark and light, the enzyme exhibits biphasic substrate dependence with two Km's for GTP of 2-3 and 40-80 microM at 22 degrees C and less than 1 and 10-25 microM at 37 degrees C. The Km's were not influenced by light. On the basis of G-protein content of the RDM, the Vmax's for the two activities at 37 degrees C in light are 4-5 and 20-30 GTPs hydrolyzed per minute per G-protein. RDM washed free of soluble and peripheral proteins do not have measurable GTPase activity in the dark or light. Purified G-protein alone also did not turn over GTP, apparently because bleached rhodopsin is required for it to bind GTP. Reconstitution of washed membranes with purified G-protein restores both the low- and high-Km GTPase activities. Inactivation of G-protein as measured by PDE turnoff and dissociation signal recovery is found to be faster at higher than lower [GTP], consistent with the observation that the higher GTPase activity associated with the higher Km alos resides in the G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Sitaramayya
A
A
Pennsylvania College of Optometry, Philadelphia 19141.
Casadevall
C
C
Bennett
N
N
Hakki
S I
SI
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D006152:Cyclic GMP / Q000378:metabolism*; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010786:Photoreceptor Cells / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism* / Q000528:radiation effects
D005136:Eye Proteins; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D010744:Phosphoric Monoester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D006152:Cyclic GMP