GTP HYDROLYSIS BY PURIFIED ALPHA-SUBUNIT OF TRANSDUCIN AND ITS COMPLEX WITH THE CYCLIC-GMP PHOSPHODIESTERASE INHIBITOR

The single-turn GTP hydrolysis by isolated and soluble transducin has been time-resolved using a rapid flow filtration technique which takes advantage of the GTP-requiring detachment of transducin alpha-subunit s (Talpha) from photoactivated rhodopsin (R). Illuminated rod outer s egment (ROS) fragments to which holo-transducin is tightly bound are r etained on a syringe filter that is washed continuously with a buffer containing no GTP. When the flow is switched to a buffer with GTP, Tal phaGTP is specifically eluted and injected into a cuvette where GTP hy drolysis is monitored via the associated change in the Talpha intrinsi c tryptophan fluorescence. Low concentrations of GTP elute the complet e pool of Talpha from the filter-retained ROS fragments in less than 1 s. This directly demonstrates that, upon GTP loading, Talpha becomes instantly soluble in physiological buffers (120 mM KCl and 2 mM MgCl2) . When all alone, Talpha hydrolyzes its bound GTP in 21 +/- 1 s (1/e t ime at 25-degrees-C). Replacing chloride by other anions increases the GTPase rate by 2-fold. The K50 for chloride inhibition of GTPase is a pproximately 2 mM. Slower GTP hydrolysis is observed for cholera-toxin -modified transducin or when GTPalphaS (Sp) replaces GTP in the elutin g buffer. No signal is observed when GTPgammaS is used. The GTPase rat e is unaffected when TalphaGTP binds to the inhibitory subunit (PDEgam ma) of the cGMP phosphodiesterase (PDE), although this binding is fast and of high affinity. This suggests that in a more complete system, w here transducin and PDE deactivations take less than 1 s, additional f actor(s) must speed up the GTPase rate of Talpha.