Isolation and characterization of methionine synthetase from human placenta.
The cobalamin-dependent enzyme, methionine synthetase, has been purified approximately 1000-fold to apparent homogeneity from human placenta with a 19% recovery. The final two steps of the purification utilized two different affinity columns. The first was a N5-methyltetrahydrofolate-cystamine-agarose column, and the second was a S-adenosylhomocysteine-agarose column. The enzyme was eluted from the first affinity column by buffer containing reducing agent which released the folate and the enzyme while elution from the second affinity column was accomplished with buffer containing 0.5 M sodium chloride. Criteria for purity were the observations that single peaks of enzyme activity, protein, and cobalamin with an apparent molecular weight of 160,000 were obtained by gel filtration and that holomethionine synthetase contained 1 mol of cobalamin/mol of protein. Furthermore, analysis by high performance liquid chromatography using a molecular weight sizing column demonstrated a single peak of protein with a corresponding cobalamin peak. This single peak of protein was progressively converted to a second protein peak that was enzymatically inactive, and this conversion was associated with a directly proportional loss of enzyme activity and cobalamin from the first peak. Methionine synthetase appeared to have a molecular weight of 160,000 on unreduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and subunits of Mr 90,000, 45,000, and 35,000 on reduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis.
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000032:analysis; D008780:Methyltransferases / Q000302:isolation & purification*; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D013053:Spectrophotometry; D014155:Transcobalamins / Q000032:analysis; D014805:Vitamin B 12 / Q000378:metabolism / Q000494:pharmacology
D000596:Amino Acids; D008670:Metals; D014155:Transcobalamins; D008780:Methyltransferases; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D014805:Vitamin B 12