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

Abstract

Polyclonal antibodies were raised against a synthetic peptide corresponding to a sequence of 14 amino acid residues found near the C-terminus of L-isoaspartyl (D-aspartyl)-protein carboxyl methyltransferase (PCMT). The affinity-purified antibodies were used to detect the methyltransferase by Western-blot analysis in cytosolic and membrane fractions from several mammalian tissues. A protein of 27 kDa was detected in the cytosol of most tissues; co-incubation with the peptide used for immunization abolished the detection. The identity of the 27 kDa protein as a PCMT was demonstrated by renaturation of PCMT activity from SDS/polyacrylamide gels. The methyltransferase from brain cytosol was immunoprecipitated by the anti-PCMT antibodies and Protein A-agarose, indicating that the native protein was recognized by the antibodies. PCMT was also immunodetected in crude membranes from brain, testes and heart, and in purified membranes from kidney cortex. The expression of the methyltransferase was higher in bovine and human brain than in rat tissues. The bovine enzyme had a greater electrophoretic mobility, suggesting small structural differences. The membrane-bound methyltransferase could be extracted with detergents above their critical micellar concentration, but not with salt, alkaline or urea solutions suggesting that the binding of the enzyme to membranes is hydrophobic by nature. Anti-PCMT antibodies provide an interesting tool for studies regarding the expression of these enzymes in both soluble and membrane fractions of various cell types.

A conserved deamidation site at asn 2 in the catalytic subunit of mammalian cAMP‐dependent protein kinase detected by capillary LC‐MS and tandem mass spectrometry

“…is specific for L-isoAsp-and D-Asp-containing substrates (Aswad, 1984;Murray & Clarke, 1984), and catalyzes their partial conversion to aspartate in vivo (for review of the mechanism, see Clarke 1985Clarke , 1988Aswad & Johnson, 1987;Wright, 1991). It is interesting to note the reciprocal correlation between the abundance of this "repair" enzyme and the observed ratios of isoAsp:Asp in enzyme from the corresponding tissues: the reported cellular levels of PCMT are much higher in heart muscle than in skeletal muscle, at both the level of mRNA and protein (Boivin et al, 1995). The action of PCMT thus may provide an explanation for the observed isoAsp:Asp isomer ratios in the selected tissues, although this possibility requires further investigation.…”

“…A 1 : 10 and 1 :5 isoAsp:Asp ratio as observed for amino-terminal peptides from heart tissues and skeletal muscle tissues, respectively, could indicate either (1) an additional mechanism of deamidation that does not result in isoaspartate formation, (2) a selective degradation of isoaspartatecontaining isoforms, or (3) the action of PCMT. PCMT, ubiquitous in all mammalian tissues (Boivin et al, 1995). is specific for L-isoAsp-and D-Asp-containing substrates (Aswad, 1984;Murray & Clarke, 1984), and catalyzes their partial conversion to aspartate in vivo (for review of the mechanism, see Clarke 1985Clarke , 1988Aswad & Johnson, 1987;Wright, 1991).…”

Down‐regulation of protein l‐isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulin

“…Membranes were blocked overnight at 4°C in TBS containing 0.1% Tween 20 (TBS-T) and 5% non-fat dry milk. Membranes were incubated for 1 h at room temperature with a polyclonal antibody against PIMT previously raised by our laboratory (Boivin et al 1995), a monoclonal antibody against b-actin (Sigma, Oakville, Canada), a monoclonal antibody recognizing neuron-specific enolase (NSE) (Calbiochem, La Jolla, CA, USA), a monoclonal antibody against glial fibrillary acidic protein (GFAP) (Sigma, Oakville, Canada) or a monoclonal antibody against endothelial nitric oxide synthase (eNOS) (Transduction Laboratories, Lexington, KY, USA) diluted in TBS-T containing 3% bovine serum albumin (BSA), and then incubated for 1 h at room temperature with horseradish peroxidase-coupled goat anti-mouse or donkey anti-rabbit IgG (Amersham Pharmacia Biotech, Baie d'Urfé, Canada) diluted 1 : 2500 in TBS-T containing 5% dry milk. Immunoreactive proteins were revealed with an enhanced chemiluminescence (ECL) western blotting kit (Amersham Pharmacia Biotech).…”

Accelerated protein damage in brains of PIMT+/− mice; a possible model for the variability of cognitive decline in human aging

“…PIMT is the product of single “housekeeping” gene (Pcmt1) that is expressed in all mammalian tissues and cell types studied to date, with highest levels reported for brain, retina, and testis (Boivin, et al, 1995; DeVry, et al, 1996; Diliberto and Axelrod, 1976; Mizobuchi, et al, 1994; Qin, et al, 2014). Mice that are heterozygous (HZ, −/+) for Pcmt1 exhibit 50-52% of the enzyme activity found in wild-type (WT, +/+) mice, regardless of the tissue (Yamamoto, et al, 1998).…”