Using guanosine 5'-(3-O-thio)triphosphate (GTPγS), we previously reported that protein carboxyl methyltransferase activities in kidney brush border membranes were increased by the GTP analog (Arch. Biochem. Biophys. 351, 149-158, 1998). Here, we investigated the distribution and characterized the effect of GTPγS on protein carboxyl methylation activity. The analysis of species distribution of carboxyl methylation in kidney brush border membranes showed that the GTPγS strongly stimulated this activity in rat (15.9-fold), mouse (14.7-fold), human (2.9-fold), and rabbit (2.7-fold). Analysis of GTPγS-dependent carboxyl methylation in rat tissues and cell fractions indicated that the activity was mainly localized in membranes of intestine, lung, and kidney, with the highest activity found in liver. To characterize the methyltransferase activity modulated by GTPγS in liver membranes, their sensitivity to the detergent 3-[(3- cholamido)dimethylammonio]-1-propanesulfonic acid (Chaps) was used. Methylation of N-acetyl-S-farnesyl cysteine, a prenylated protein methyltransferase (PPMT) substrate was strongly inhibited (86%) in the presence of Chaps, while the methylation of bovine calmodulin and ovalbumin, both of which are substrates for the protein L-isoaspartyl/D-aspartyl methyltransferase (PIMT), was slightly reduced by the detergent (0-12%). The GTPγS-dependent carboxyl methylation of endogenous substrates in liver membranes was decreased by 35% in the presence of Chaps, suggesting that PPMT was not the predominant methyltransferase involved in the methylation stimulated by GTPγS in liver membranes. Electrophoretic analysis showed that radioactive methylation of several substrates induced by GTPγS in liver membranes was reduced by adding calmodulin. Interestingly, addition of GTPγS partially inhibited the methylation of two PIMT substrates, ovalbumin (24%) and bovine calmodulin (19%), when incubated with liver membranes. Immunoprecipitation of PIMT from liver and lung membranes strongly inhibited (88-94%) the methylation stimulated by GTPγS. Altogether, these data support the hypothesis that GTPγS could regulate PIMT activity and may provide new insights into the function of the methyltransferase.
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