ACTIVATED CGMP PHOSPHODIESTERASE OF RETINAL RODS - A COMPLEX WITH TRANSDUCIN ALPHA SUBUNIT

Purified G-protein (transducin) activated with the nonhydrolyzable ana log guanosine 5'-O-(thiotriphosphate) (GTP(gamma)S) and cGMP phosphodi esterase (PDE) from retinal rods are added to protein-stripped disc me mbranes. Specific binding of the mainly soluble alpha subunit of G-pro tein with GTP(gamma)S bound (G-alpha-GTP(gamma)S, activator of the PDE ) to the disc membrane in the presence of PDE is measured from gel sca ns or experiments with labeled G-protein alpha subunit (G-alpha). Its variation as a function of G concentration matches the theoretical var iation of G-alpha involved in the activation of PDE calculated with pr eviously estimated dissociation constants (Bennett, N., and Clerc, A. (1989) Biochemistry 28, 7418-7424), and the G-alpha bound/PDE ratio at saturation is close to 2. No increase of G-alpha binding to the membr ane is observed when purified inhibitory subunit of PDE (PDE(gamma)) i s added together with or instead of total PDE, and excess PDE(gamma) r emains soluble. These results suggest that activated PDE is a complex with the activator G-alpha-GTP rather than PDE from which the inhibito ry subunits have been removed. A method for purifying PDE(gamma) with a high yield of recovery and activity is described.