IMMUNOCHEMICAL CHARACTERIZATION OF L-ISOASPARTYL-PROTEIN CARBOXYL METHYLTRANSFERASE FROM MAMMALIAN-TISSUES

Polyclonal antibodies were raised against a synthetic peptide correspo nding to a sequence of 14 amino acid residues found near the C-terminu s of L-isoaspartyl (D-aspartyl)-protein carboxyl methyltransferase (PC MT). The affinity-purified antibodies were used to detect the methyltr ansferase by Western-blot analysis in cytosolic and membrane fractions from several mammalian tissues. A protein of 27 kDa was detected in t he cytosol of most tissues; co-incubation with the peptide used for im munization 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 immu noprecipitated by the anti-PCMT antibodies and Protein A-agarose, indi cating that the native protein was recognized by the antibodies. PCMT was also immunodetected in crude membranes from brain, testes and hear t, and in purified membranes from kidney cortex. The expression of the methyltransferase was higher in bovine and human brain than in rat ti ssues. The bovine enzyme had a greater electrophoretic mobility, sugge sting small structural differences. The membrane-bound methyltransfera se could be extracted with detergents above their critical micellar co ncentration, 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 regardin g the expression of these enzymes in both soluble and membrane fractio ns of various cell types.