NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells. Purification and properties.
NAD-dependent methylenetetrahydrofolate dehydrogenase is expressed in transformed or established mammalian cell lines in vitro but only in the developmental tissues of normal adult animals (Mejia, N. R. and MacKenzie, R. E. (1985) J. Biol. Chem. 260, 14616-14620). The enzyme, which contains methenyltetrahydrofolate cyclohydrolase activity as well, has been purified 6000-fold from Ehrlich ascites tumor cells. The preparation is homogeneous by sodium dodecyl sulfate gel electrophoresis (Mr = 34,000), and results from cross-linking with bis(sulfosuccinimidyl)suberate are consistent with a dimeric structure (Mr = 68,000) for the native bifunctional enzyme. The dehydrogenase is specific for NAD and requires both a divalent cation, Mg2+ or Mn2+, for activity and as well is stimulated by inorganic phosphate. When compared to the usual NADP-dependent methylenetetrahydrofolate dehydrogenase from mouse liver, the NAD-dependent dehydrogenase activity of the murine tumor enzyme shows a greater affinity for the polyglutamate forms of folate.
D000619:Aminohydrolases / Q000378:metabolism*; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D045064:Methenyltetrahydrofolate Cyclohydrolase; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology*; D009249:NADP / Q000494:pharmacology; D010088:Oxidoreductases / Q000378:metabolism*; D010710:Phosphates / Q000494:pharmacology; D013388:Succinimides
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D010710:Phosphates; D013388:Succinimides; D009243:NAD; D008345:Manganese; D009249:NADP; C035760:bis(sulfosuccinimidyl)suberate; D010088:Oxidoreductases; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D000619:Aminohydrolases; D045064:Methenyltetrahydrofolate Cyclohydrolase; D008274:Magnesium