Immunochemical characterization of L-isoaspartyl-protein carboxyl methyltransferase from mammalian tissues.
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.
Boivin
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D
GRTM, Département de Chimie-biochimie, Université du Québec à Montréal, Canada.
Bilodeau
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Béliveau
R
R
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002417:Cattle; D006801:Humans; D007120:Immunochemistry; D007668:Kidney / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D013737:Testis / Q000201:enzymology
D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase