Regulation by GTPgammaS of protein carboxylmethyltransferase activity in kidney brush border membranes.

The increase in carboxyl methylation induced by guanosine 5',3-O-(thio)triphosphate (GTPgammaS) in brush border membranes from rat kidney cortex was studied, and the methyltransferase activities affected by this nucleotide analog were identified. Addition of GTPgammaS to brush border membranes stimulated the carboxyl methylation in a time-dependent manner while adenosine and guanine nucleotides were ineffective. The GTPgammaS-dependent carboxyl methylation was inhibited by the chelating agents EDTA (63%) and 1,10-phenanthroline (68%), suggesting that this activity required divalent cations. The methyl ester groups induced by the addition of GTPgammaS to brush border membranes were unstable, with about 80% of them hydrolyzed following 1 h incubation at 37 degrees C. The GTPgammaS stimulation of the carboxyl methylation in brush border membranes was unaffected by the detergent 3-[(3cholamido)-dimethylammonio]-1-propanesulfonic acid up to a concentration of 0.4% (w/v). At this latter detergent concentration, the activity of prenylated protein methyltransferase (PPMT) was strongly inhibited and that of l-isoaspartyl/d-aspartylmethyltransferase (PIMT) was increased twofold, as measured with their respective exogenous substrates, N-acetyl-S-farnesyl cysteine and ovalbumin. GTPgammaS increased the methylation of several substrates in brush border membranes. The induced methylation in substrates migrating between 20 and 36 kDa was strongly decreased by the competitive inhibitor farnesylthioacetic acid, a synthetic farnesylated substrate for PPMT, while a delta-sleep-inducing peptide containing an L-isoaspartyl residue inhibited that of substrates with molecular weights above 36 kDa, suggesting that PIMT activity was also involved. This interpretation was strengthened by the observation that the increased methylation induced by GTPgammaS in these membrane substrates was completely lost following their analysis by gel electrophoresis under alkaline conditions. Taken together, these results indicate that both PPMT and PIMT activities are regulated by guanine nucleotides in brush border membranes of rat kidney.