Characterization of the isozymes of bovine adrenal medullary phenylethanolamine N-methyltransferase.
Bovine adrenal medullary phenylethanolamine N-methyltransferase (EC 2.1.1.28) has been purified to apparent homogeneity. The enzymatically active monomer has a relative molecular weight of 30,000 and can be separated into at least four active charged isozymes. These isozymes, designated PNMT-1, PNMT-2, PNMT-3 and PNMT-4, have isoelectric points of 5.1, 5.2, 5.3 and 5.4, respectively. Kinetic parameters have been determined for each isozyme. The Kms for phenylethanolamine range from 11.9 to 45.9 microM; the Kms for S-adenosylmethionine range from 1.13 to 1.47 microM; and the Kis for the competitive inhibitor, S-adenosylhomocysteine, range from 0.12 to 0.22 microM. For isozymes PNMT-1 and PNMT-4, and Kms for S-adenosylhomocysteine are not significantly different. Vmax values for all of the isozymes do not change significantly in the presence of S-adenosylhomocysteine. Treatment of the purified isozymes with various endo- and exoglycosidases does not alter electrophoretic mobility. Hence, carbohydrate substitution must be minimal. No high mannan, complex sugars or terminal N-acetylglucosamine residues are present. The absence of carbohydrate is further supported by the inability of Schiff-periodic acid to stain the protein. Limited thermolysin digests of each isozyme show distinct peptide cleavage products. In conjunction with the kinetic and glycosylation data, this suggests that the isozymes of phenylethanolamine N-methyltransferase may be primary structural variants.
D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D002384:Catalysis; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification*; D007700:Kinetics; D010625:Phenylethanolamine N-Methyltransferase / Q000302:isolation & purification*
D007527:Isoenzymes; D010625:Phenylethanolamine N-Methyltransferase