G-PROTEIN EFFECTOR COUPLING - A REAL-TIME LIGHT-SCATTERING ASSAY FOR TRANSDUCIN PHOSPHODIESTERASE INTERACTION

We describe a real-time assay for the interaction of the G-protein of retinal rods, transducin (G(t)), with its effector, cGMP phosphodieste rase (PDE). The purified proteins were recombined with isolated rod di sk membranes. Reactions were triggered by flash photolysis of the rece ptor rhodopsin, and monitored by changes of near-infrared light scatte ring (LS signals). PDE-related LS signals from membrane preparations h ave been described by others [Caretta, A., & Stein, P.J. (1986) Bioche mistry 25,2335-2341; Bennett, N., & Clerc, A. (1992) Biochemistry 31, 1858-1866]. In the present study, the previous overall light-scatterin g (turbidity) approach was improved by a specific optical setup and mi llisecond time resolution. This allowed isolation of a fast ''PDE sign al'' which monitors G(t)-PDE interaction on the membrane and in real t ime. The assignment to the reaction is made by the following observati ons: (i) preactivation of PDE by purified G(alpha)GTP(gamma)S graduall y suppresses the signal; (ii) the peak amplitude of the signal is stoi chiometric with respect to both G(t) and PDE; and (iii) the rise and d elay time fit into a kinetic model for a reaction of activated G(t)(G ). A comparative investigation, relating the PDE signal with G(t) acti vation and PDE enzymatic activity, led to the following results: (i) t he apparent dissociation constant for the interaction of G with the f irst binding site on membrane-bound PDE is less than 2.5 nM; (ii) the time interval between formation of G and its interaction with PDE is less than 5 ms; (iii) membrane-bound PDE, even in its preactivated for m, slows the release of G from the membrane. The results suggest a ki netic competition mechanism in which activated G(t) stays long enough on the membrane to favor its capture by the PDE. The 5-ms time it take s G to couple to PDE in vitro fits well into the time frame for this reaction, as determined in situ by electrophysiology.