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 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)

Transducin GTPase provides for rapid quenching of the cGMP cascade in rod outer segments

“…But the authors described only the linear part of the plot and could not calculate the Vmax value. At the same time our results contradict the data [9] that the GTPase reaction rate is not dependent on ROS concentration and may attain 20-30 turnovers per min at 37°C only when the GTP con-FEBS centration is about 10~-fold higher than in our experiments.…”

“…We could not determine this constant at physiological temperature, 37°C, because at high ROS concentrations the reaction was too fast to be monitored. But considering the fact that the temperature increase from 23°C to 37°C results in a 3-fold increase in the T GTPase rate [5,9], one can conclude that the rate of the reaction under physiological conditions is not less than 1 turnover per s.…”

“…Although it is known that PDE activation terminates after the hydrolysis of T-bound GTP [1], numerous measurements carried out in reconstituted systems indicate that the rate of GTPase reaction is too slow (tens of seconds) (review [5,6]) compared with the real cascade quenching time in an ROS suspension (seconds) [7]. To overcome the above contradiction two possibilities were postulated: (i) the rate of T GTPase under physiological conditions is higher than in reconstituted systems [6,8,9]; (ii) the rapid PDE quenching in ROS is caused by a mechanism, independent of T GTPase [10,11].…”

Mono ADP‐ribosylation of transducin catalyzed by rod outer segment extract

“…transducin is a heterotrimeric protein composed ore,/~ and :F subunits and the bindinB of GTP leads to the dissociation of the active T¢.GTP form from T,~. After hydrolysis of the GTP by its intrinsic l;uanosine triphosphatase (GTPase) activity [5]. T= returns to its T=.GDP-T/~y inactive form.…”

Molecular mechanism of visual transduction

“…Indirect measurements by light-scattering technique suggest that in very concentrated broken rods suspensions [80] or in resealed ROS from which T,GTP does not leak out [8], the GTPase rate is reduced to a few seconds. Very recently, direct measurements at high GTP concentration and 37°C suggcst also a fast GTPase rate [88].…”