Palmitoylation is the dynamic modification of proteins by the addition
of palmitate to cysteine residues. The alpha subunits of heterotrimer
ic G proteins undergo palmitoylation on their amino terminus, and acti
vation of alpha(s) accelerates its palmitate turnover. In previous stu
dies, palmitoylation was assessed by incorporation or turnover of [H-3
]palmitate. These studies did not determine the fraction of alpha s( )
that is palmitoylated because the specific activity of [H-3]palmitoyl-
CoA within cells is indeterminate. We developed an HPLC method to dete
rmine the fraction of alpha(s) that was palmitoylated in the basal and
activated states. COS and S49 cells were radiolabeled with [S-35]meth
ionine, and alpha(s) was immunoprecipitated from the particulate fract
ion. The immunoprecipitated proteins were separated by reverse phase H
PLC into two peaks that were determined to contain the modified and un
modified forms of alpha(s). Approximately 77% of the endogenous alpha(
s) in COS cells and 70% in S49 lymphoma cells were palmitoylated. The
fraction of alpha(s) that was modified did not change after treatment
with isoproterenol, a beta-adrenergic receptor agonist that causes tur
nover of palmitate on alpha(s). These results suggest that receptor ac
tivation of alpha(s) caused a rapid turnover of palmitate to maintain
most of alpha(s) in its palmitoylated form.