title: Purification and kinetic mechanism of S-adenosylmethionine: myelin basic protein methyltransferase from bovine brain.
abstract: The enzyme S-adenosylmethionine (AdoMet): myelin basic protein (MBP) methyltransferase was purified 250-fold from bovine brain with an overall yield of 130%, relative to crude supernatant. The purification involves acid-base and (NH4)2SO4 precipitation, chromatography over Sephadex G-100 and DEAE-cellulose, followed by preparative isoelectric focusing. The enzyme has a pI of 5.60 +/- 0.05, and the Mr is estimated to be between 71,000 (from SDS/polyacrylamide-gel electrophoresis) and 74,500 (from gel filtration). The enzyme is stable at 37 degrees C for over 2 h, is stable frozen and does not require metal ions or reductants. The enzyme shows a high specificity for MBP and does not accept polyarginine as a substrate; F1 histone is methylated at 37% of the rate of MBP. Methylation occurs on an arginine residue in a single h.p.l.c.-resolvable peptide from the tryptic cleavage of MBP. Simple saturation kinetics are observed with respect to both substrates, with Km values of 18 microM and 32 microM for MBP and AdoMet respectively. The simplest kinetic mechanism that is consistent with the data requires ordered rapid-equilibrium binding, with AdoMet as the first substrate. The enzyme isolated in this work is different, both physically and kinetically, from the histone-specific arginine methyltransferases described by other workers. A new, simple, assay system for the methylation of MBP is described.
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D007700:Kinetics; D004676:Myelin Basic Protein / Q000378:metabolism; D011496:Protein Methyltransferases / Q000378:metabolism*; D011484:Protein-Arginine N-Methyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004676:Myelin Basic Protein; D012436:S-Adenosylmethionine; D011496:Protein Methyltransferases; D011484:Protein-Arginine N-Methyltransferases