Guanosine 5 '-(3-O-thio)triphosphate stimulates protein carboxyl methylation in cell membranes

Using guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), we previously repo rted that protein carboxyl methyltransferase activities in kidney brush bor der membranes were increased by the GTP analog (Arch, Biochem. Biophys. 351 , 149-158, 1998), Here, we investigated the distribution and characterized the effect of GTP gamma S on protein carboxyl methylation activity. The ana lysis of species distribution of carboxyl methylation in kidney brush borde r membranes showed that the GTP gamma S strongly stimulated this activity i n rat (15.9-fold), mouse (14.7-fold), human (2.9-fold), and rabbit (2.7-fol d). Analysis of GTP gamma S-dependent carboxyl methylation in rat tissues a nd cell fractions indicated that the activity was mainly localized in membr anes of intestine, lung, and kidney, with the highest activity found in liv er. To characterize the methyltransferase activity modulated by GTP gamma S in liver membranes, their sensitivity to the detergent 3-[(3-cholamido)dim ethylammonio]-1-propanesulfonic acid (Chaps) was used. Methylation of N-ace tyl-S-farnesyl cysteine, a prenylated protein methyltransferase (PPMT) subs trate was strongly inhibited (86%) in the presence of Chaps, while the meth ylation of bovine calmodulin and ovalbumin, both of which are substrates fo r the protein L-isoaspartyl/D-aspartyl methyltransferase (PIMT), was slight ly reduced by the detergent (0-12%). The GTP gamma S-dependent carboxyl met hylation of endogenous substrates in liver membranes was decreased by 35% i n the presence of Chaps, suggesting that PPMT was not the predominant methy ltransferase involved in the methylation stimulated by GTP gamma S in liver membranes. Electrophoretic analysis showed that radioactive methylation of several substrates induced by GTP gamma S in liver membranes was reduced b y adding calmodulin, Interestingly, addition of GTP gamma S partially inhib ited the methylation of two PIMT substrates, ovalbumin (24%) and bovine cal modulin (19%), when incubated with liver membranes. Immunoprecipitation of PIMT from liver and lung membranes strongly inhibited (88-94%) the methylat ion stimulated by GTP gamma S, Altogether, these data support the hypothesi s that GTP gamma S could regulate PIMT activity and may provide new insight s into the function of the methyltransferase, (C) 1999 Academic Press.