MODULATION OF THE GTPASE ACTIVITY OF TRANSDUCIN - KINETIC-STUDIES OF RECONSTITUTED SYSTEMS

We seek to define the influence of retinal cGMP phosphodiesterase (PDE ) on the GTPase activity of transducin (T). A novel stopped-flow/fast filtration apparatus [Antonny, B., et al. (1993) Biochemistry 32, 8646 -8653] is used to deliver T alpha GTP free of rod outer segment (ROS) membranes to a suspension of phospholipid vesicles bearing holoPDE. As measured by a pH electrode, the decay of cGMP hydrolysis from these s amples, which contain no other proteins but T alpha and holoPDE, requi res GTP hydrolysis and occurs in 40 s. The addition of T beta gamma to the vesicles does not accelerate this deactivation. When ROS membrane s are urea-stripped, reconstituted with transducin + holoPDE, and illu minated, the injection of an amount of GTP that is substoichiometric t o holoPDE gives a cGMP hydrolysis pulse that lasts far 30 s. However, the same reconstitution performed with ROS stripped by extensive dilut ion in isotonic buffer results in a deactivation time of only 8 s, whi ch resembles the 7 s observed with native ROSs. With these isotonicall y stripped ROSs, when GTP injection comes after a first injection with GTP gamma S, the cGMP hydrolysis pulse is lengthened and lasts for 17 s; with urea-washed ROS, no such lengthening is observed. These resul ts clearly demonstrate that holoPDE by itself cannot enhance the GTPas e activity of transducin, even when the two proteins are localized on a membrane surface. Instead, they point to the existence of a membrane -bound, urea-sensitive protein factor that activates the GTPase of Ta in the transducin-holoPDE complex.