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 phosphodiesterase (PDE). The purified proteins were recombined with isolated rod disk membranes. Reactions were triggered by flash photolysis of the receptor rhodopsin, and monitored by changes of near-infrared light scattering (LS signals). PDE-related LS signals from membrane preparations have been described by others [Caretta, A., & Stein, P.J. (1986) Biochemistry 25,2335-2341; Bennett, N., & Clerc, A. (1992) Biochemistry 31, 1858-1866]. In the present study, the previous overall light-scattering (turbidity) approach was improved by a specific optical setup and millisecond time resolution. This allowed isolation of a fast ''PDE signal'' which monitors G(t)-PDE interaction on the membrane and in real time. The assignment to the reaction is made by the following observations: (i) preactivation of PDE by purified G(alpha)GTP(gamma)S gradually suppresses the signal; (ii) the peak amplitude of the signal is stoichiometric with respect to both G(t) and PDE; and (iii) the rise and delay time fit into a kinetic model for a reaction of activated G(t)(G*). A comparative investigation, relating the PDE signal with G(t) activation and PDE enzymatic activity, led to the following results: (i) the apparent dissociation constant for the interaction of G* with the first 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 form, slows the release of G* from the membrane. The results suggest a kinetic 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 takes G* to couple to PDE in vitro fits well into the time frame for this reaction, as determined in situ by electrophysiology.