The characterization of a membrane-bound protein carboxylmethylation system in brain

Abstract

The membrane-bound component of the cerebral protein carboxylmethylation system, consisting of the membrane-bound enzyme protein carboxylmethyltransferase II (PCMT) and of selected membrane-bound methyl accepting proteins (MAP), is described. The cellular localization of this membrane-bound protein carboxylmethylation system is shown to include, in addition to nerve cell bodies and purified synaptosomes, astrocytes and oligodendroglia. The membrane-bound nature of the protein carboxylmethylation system was investigated and these studies revealed a tight association which exposure to several detergents could only partially solubilize. The membrane-bound PCMT could be shown to undergo activation after treatment with Na-deoxycholate and CHAPs, while after its detergent-induced solubilization PCMT activation was observed after Na-deoxycholate, Nonidet P-40 and Lubrol-P(X). Solubilization of the carboxylmethylation system in CHAPS appeared to be more effective at 0 degrees C than at 25 degrees C or 37 degrees C. Detergent treatment was shown to be deleterious to the MAPs as PCMT substrates, particularly when the exposure was extended to more than 1 h. These observations prompted exposure of the brain membranes and of their Lubrol-P(X) and Nonidet P-40 extracts to NH(4)OH, treatment which promotes the conversion of protein asparagine residues to atypical l-isoaspartate residues, recently shown (in synthetic peptides) to be the single most effective residue recognized for carboxylmethylation by PCMT. We found up to a 400% enhancement of the carboxylmethylation of solubilized membrane MAPs by the equally solubilized PCMT (which resisted the alkaline treatment virtually unscathed) after 90 min at 37 degrees C in 0.05 M NH(4)OH. However, when brain membrane Lubrol-P(x) extracts were first subjected to bis(I,I-trifluoroacetoxy)-iodobenzene, a reagent which converts the carboxyamide group of protein-bound asparagine to the corresponding primary amine, the amount of MAPs susceptible to be acted upon by 0.05 M NH(4)OH became greatly reduced. Finally, acidic slab gel electrophoresis of membrane-bound MAPs, carboxyl-[(3)H]-methylated by the membrane-bound PCMT, revealed the presence of about 12 radioactive protein bands, ranging in MW from under 20 KDa to about 90 KDa.

The carboxylmethylation of cerebral membrane-bound proteins increases with age

“…Recently we have characterized in some detail a membrane-bound (mb) component of brain PCMT [20,21] which effectively carboxylmethylates mb methyl-accepting proteins (MAPs). We have also shown that exposing mb-MAPs or their Lubrol-P x extracts to mild alkali leads to a marked increase in their recognition by PCMT [22]. Since, in vitro, one of the likely consequences of the mild alkaline treatment appears to be the deamidation of selected protein-bound asparagines [23], and/or aspartates [24], followed by the formation, in their place, of unnatural isoaspartate(s) [25,26], it is reasonable to assume that mb-MAPs constitute unique targets for the mb-PCMT, because they contain unnatural aspartate residues.…”

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

“…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.…”

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

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

“…After their extraction from the membranes with a detergent, these enzymes appear very similar to the cytosolic enzymes with regard to their catalytic and structural properties (Brown;1984;Iqbal and Steenson, 1976;Boivin et al, 1993), suggesting a similar physiological role. In this context, it is noteworthy that increased deamidation of membrane-associated substrates by alkaline treatment results in an increased activity of the brain membrane-associated methyltransferase (Sellinger et al, 1987). The role of the membrane-associated PCMTs may thus be similar to that of the cytosolic one, and could involve recognition of L-isoaspartylcontaining proteins not accessible to the cytosolic enzyme in vivo.…”

“…A number of reports describing membrane-associated PCMTs have appeared over the years (Iqbal and Steenson, 1976;Brown, 1984;Sellinger et al, 1987;Saido et al, 1987;Gingras et al, 1991). However, these enzymes have not been characterized in great detail.…”