REGULATION BY GTP-GAMMA-S OF PROTEIN CARBOXYLMETHYLTRANSFERASE ACTIVITY IN KIDNEY BRUSH-BORDER MEMBRANES

The increase in carboxyl methylation induced by guanosine 5',3-O-(thio )triphosphate (GTP gamma S) in brush border membranes from rat kidney cortex was studied, and the methyltransferase activities affected by t his nucleotide analog were identified, Addition of GTP gamma S to brus h border membranes stimulated the carboxyl methylation in a time-depen dent manner while adenosine and guanine nucleotides were ineffective, The GTP gamma S-dependent carboxyl methylation was inhibited by the ch elating agents EDTA (63%) and 1,10-phenanthroline (68%), suggesting th at this activity required divalent cations, The methyl ester groups in duced by the addition of GTP gamma S to brush border membranes were un stable, with about 80% of them hydrolyzed following 1 h incubation at 37 degrees C, The GTP gamma S stimulation of the carboxyl methylation in blush border membranes was unaffected by the detergent -[(3-cholami do)-dimethylammonio]-1-propanesulfonic acid up to a concentration of 0 .4% (w/v), At this latter detergent concentration, the activity of pre nylated protein methyltransferase (PPMT) was strongly inhibited and th at of L-isoasparty]/D-aspartylmethyltransferase (PIMT) was increased t wofold, as measured with their respective exogenous substrates, N-acet yl-S-farnesyl cysteine and ovalbumin, GTP gamma S increased the methyl ation of several substrates in brush border membranes, The induced met hylation in substrates migrating between 20 and 36 kDa was strongly de creased by the competitive inhibitor farnesylthioacetic acid, a synthe tic farnesylated substrate for PPMT, while a delta-sleep-inducing pept ide containing an L-isoaspartyl residue inhibited that of substrates w ith 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 GTP gamma S in these membra ne substrates was completely lost following their analysis by gel elec trophoresis under alkaline conditions, Taken together, these results i ndicate that both PPMT and PIMT activities are regulated by guanine nu cleotides in brush border membranes of rat kidney. (C) 1998 Academic P ress.