Heterotrimeric G proteins are isoprenylated and methylated on their ga
mma subunits. Since methylation is the only reversible reaction in the
isoprenylation pathway, it could be a site of control of G protein ac
tivity. A method for selectively demethylating isoprenylated and methy
lated G proteins is reported here using retinal transducin (T) as a mo
del system. It was found that pig liver esterase is capable of complet
ely hydrolyzing T-beta gamma, but not T-alpha beta gamma, to its unmet
hylated form. This allows for the direct determination of the activiti
es of methylated and unmethylated T-beta gamma. The activities of the
T(beta gamma)s were determined by measuring their abilities to stimula
te GTP-gamma-S exchange in the presence of T-alpha and photoactivated
rhodopsin (R). It is reported here that, in detergent, unmethylated T
-beta gamma was at least as active as its methylated counterpart. Ther
efore, methylation does not affect the intrinsic ability of T-beta gam
ma to functionally interact with T-alpha and R. However, in disk memb
ranes an approximate 2-fold effect was observed, with the methylated T
-beta gamma being more efficient. Therefore, isoprenylated protein met
hylation may play a quantitative role in signal transduction, even tho
ugh the intrinsic activities of the methylated subunits in detergent m
ay be no different from their unmethylated counterparts. Finally, the
use of pig liver esterase to demethylate isoprenyIated proteins should
allow for a clarification of the physiological role(s) of isoprenylat
ed protein carboxymethylation in general.