Contribution of the guanosinetriphosphatase activity of G-protein to termination of light-activated guanosine cyclic 3',5'-phosphate hydrolysis in retinal rod outer segments

Abstract

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 .mu.M at 22.degree. C and < 1 and 10-25 .mu.M at 37.degree. 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.degree. 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 measurablke 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 also resides in the G-protein. In reconstituted RDM containing excess amounts of rhodopsin kinase, a condition under which rapid inactivation of bleached rhodopsin leaves PDE activity entirely dependent upon activated G-protein, PDE activated by dim light flash turned off rapidly, with a time constant of 4.7 s. These observations show that the GTPase activity of G-protein is close to being adequate to inactivate light-activated G-protein.