RHODOPSIN-STIMULATED ACTIVATION DEACTIVATION CYCLE OF TRANSDUCIN - KINETICS OF THE INTRINSIC FLUORESCENCE RESPONSE OF THE ALPHA-SUBUNIT

The intrinsic tryptophan fluorescence of the α subunit of transducin (αT) has been shown to be sensitive to the binding of guanine nucleotides, with the fluorescence being enhanced by as much as 2-fold upon the binding of GTP or nonhydrolyzable GTP analogues [cf. Phillips and Cerione (1988) J. Biol. Chem. 263, 15498-15505]. In this work, we have used these fluorescence changes to analyze the kinetics for the activation (GTP binding)-deactivation (GTPase) cycle of transducin in a well-defined reconstituted phospholipid vesicle system containing purified rhodopsin and the αT and βγT subunits of the retinal GTP-binding protein. Both the rate and the extent of the GTP-induced fluorescence enhancement are dependent on [rhodopsin], while only the rate (and not the extent) of the GTPγS-induced enhancement is dependent on the levels of rhodopsin. Comparisons of the fluorescence enhancements elicited by GTPγS and GTP indicate that the GTPγS-induced enhancements directly reflect the GTPγS-binding event while the GTP-induced enhancements represent a composite of the GTP-binding and GTP hydrolysis events. At high [rhodopsin], the rates for GTP binding and GTPase are sufficiently different such that the GTP-induced enhancement essentially reflects GTP binding. A fluorescence decay, which always follows the GTP-induced enhancement, directly reflects the GTP hydrolytic event. The rate of the fluorescence decay matches the rate of [32P]Pi production due to [γ−32P]GTP hydrolysis, and the decay is immediately reversed by rechallenging with GTP. The GTP-induced fluorescence changes (i.e., the enhancement and ensuing decay) could be fit to a simple model describing the activation-deactivation cycle of transducin. The results of this modeling suggest the following points: (1) the dependency of the activation-deactivation cycle on [rhodopsin] can be described by a simple dose response profile; (2) the rate of the rhodopsin-stimulated activation of multiple αT(GDP) molecules is dependent on [rhodopsin] and when [αT]> [rhodopsin], the activation of the total αT pool may be limited by the rate of dissociation of rhodopsin from the activated αT(GTP) species; and (3) under conditions of optimal rhodopsin-αT coupling (i.e., high [rhodopsin]), the cycle is limited by GTP hydrolysis with the rate of Pi release, or any ensuing conformational change, being at least as fast as the hydrolytic event. © 1990, American Chemical Society. All rights reserved.