A. Clerc et N. Bennett, ACTIVATED CGMP PHOSPHODIESTERASE OF RETINAL RODS - A COMPLEX WITH TRANSDUCIN ALPHA SUBUNIT, The Journal of biological chemistry, 267(10), 1992, pp. 6620-6627
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.