ASYMMETRICAL DISTRIBUTION OF L-ISOASPARTYL PROTEIN CARBOXYL METHYLTRANSFERASES IN THE PLASMA-MEMBRANES OF RAT-KIDNEY CORTEX

We have studied the distribution of membrane-associated L-isoaspartyl protein carboxyl methyltransferases (PCMTs) in plasma membranes purifi ed from rat kidney cortex. Addition of CHAPS to brush-border membranes (BBM) and basolateral membranes (BLM) was required to measure optimal membrane-dependent methylation of ovalbumin and TS-isoD-YSKY, substra tes of L-isoaspartyl PCMTs. Extraction of both membrane-associated enz ymes was achieved with detergents, but not with high-salt solutions, s uggesting a strong membrane attachment. However, upon phase partitioni ng using Triton X-114, both enzymes were predominantly associated with the detergent-poor phase, suggesting a relatively hydrophilic nature. The enzymes showed similar catalytic properties such as substrate rec ognition and affinity towards the methyl donor, S-adenosyl-L-methionin e. The activity of the BBM enzyme, however, was about 2-fold higher th an that of the BLM enzyme. Identification of the endogenous substrates located in the two plasma membranes by acidic gel electrophoresis in the presence of a cationic detergent revealed significant differences in the methyl-accepting proteins of both membranes. The BBM-methylated proteins had sizes of 35, 50 and 54 kDa, whereas the major BLM-methyl ated substrates were of 97 and 100 kDa. The enzymes showed distinct be haviour on Mono Q anion-exchange chromatography. The PPM-associated PC MT did not bind to the column, being eluted in the flow-through, where as the BLM enzyme bound to the column and was eluted at 0.15 M NaC1. M oreover, the two enzymes had different molecular masses under both den aturing and nondenaturing conditions, the BLM PCMT migrating at an app arent molecular mass of 29 kDa, compared with 27 kDa for the BBM enzym e. Taken together, these results show the presence of two distinct L-i soaspartyl PCMTs in the plasma membranes of the kidney cortex.