THE STOICHIOMETRY OF G-ALPHA(S) PALMITOYLATION IN ITS BASAL AND ACTIVATED STATES

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.