Asymmetrical distribution of l-isoaspartyl protein carboxyl methyltransferases in the plasma membranes of rat kidney cortex

Abstract

We have studied the distribution of membrane-associated L-isoaspartyl protein carboxyl methyltransferases (PCMTs) in plasma membranes purified 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, substrates of L-isoaspartyl PCMTs. Extraction of both membrane-associated enzymes was achieved with detergents, but not with high-salt solutions, suggesting a strong membrane attachment. However, upon phase partitioning 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 recognition and affinity towards the methyl donor, S-adenosyl-L-methionine. The activity of the BBM enzyme, however, was about 2-fold higher than 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-methylated substrates were of 97 and 100 kDa. The enzymes showed distinct behaviour on Mono Q anion-exchange chromatography. The BBM-associated PCMT did not bind to the column, being eluted in the flow-through, whereas the BLM enzyme bound to the column and was eluted at 0.15 M NaCl. Moreover, the two enzymes had different molecular masses under both denaturing and nondenaturing conditions, the BLM PCMT migrating at an apparent molecular mass of 29 kDa, compared with 27 kDa for the BBM enzyme. Taken together, these results show the presence of two distinct L-isoaspartyl PCMTs in the plasma membranes of the kidney cortex.

Immunochemical characterization of l-isoaspartyl-protein carboxyl methyltransferase from mammalian tissues

“…Surprisingly, a protein with a slightly higher molecular mass was also detected in BBM, BLM, and mitochondria, but not in cytosol, IM and nuclei from rat kidney ( Figure 6). This extra band appears to be due to non-specific binding, since (i) only the 27 kDa protein displays enzymic activity after renaturation from polyacrylamide gels [24,25] and (ii) this additional band is not present in cytosol and membranes from other tissues (see Figure 4). Figure 7 shows the expression of PCMT in cytosol and membrane fractions from brains of rats of various ages.…”

“…Although PCMT activity is found predominantly in the cytosolic fraction of cells, there has been reports of membraneassociated PCMT activities from brain [22, 23,52], erythrocytes [26] and kidney [24,25,53]. In rat kidney, immunoreactive PCMT is detected in different membrane fractions as well as in the cytosol.…”

“…However, equal amounts of isoenzymes I and II were found in the microsomal fraction from rat brain [46]. Furthermore, more than 95 % of the PCMT associated with the kidney BBM is the more basic isoenzyme I [24], whereas most of the PCMT activity associated with the kidney basolateral membrane is catalysed by isoenzyme II [25]. Thus the membrane localization of PCMTs does not appear to be isoform-specific.…”

“…in both compartments, at least in bovine brain [22,23], rat kidney [24,25] and chicken erythrocytes [26].…”

Isolation of Renal Brush Borders

“…Density gradient centrifugation (Boffeli et al, 1997;Yoshioka et al, 1997), density gradient centrifugation phase partitioning (Hammond et al, 1993;Gingras et al, 1994), differential centrifugation (Biber et al, 1981;Yusufi et al, 1994), flow cytometry (Hammond, 1990), and free-flow electrophoresis (Mircheff et al, 1984) all have been employed to separate brush border membranes and basolateral membranes, which exhibit subtle differences in density and net surface charge.…”