Purification and properties of methylenetetrahydrofolate reductase from pig liver.
Methylenetetrahydrofolate reductase from pig liver has been purified to homogeneity, as judged by several criteria: (i) a single band with a subunit molecular weight of 77,300 following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; (ii) a molecular weight determined by amino acid analysis of 74,500 per flavin, in agreement with the subunit molecular weight; and (iii) constant specific activities in the peak fractions during the final chromatography step. The purified enzyme exhibits a typical flavoprotein absorption spectrum. Methylenetetrahydrofolate reductase is a minor constituent of pig liver, and to obtain homogeneous enzyme, a 32,000-fold purification must be accomplished. The preparation described herein attains such purification in 5 steps and with a 14% yield. The enzyme isolated in this fashion is active and stable, and contains a stoichiometric complement of FAD. The enzyme is reducible under anaerobic conditions by 5-deazaflavin/EDTA/light or by NADPH. Reduction of 1 mol of enzyme-bound FAD requires 1.1 mol of NADPH. The reduced enzyme can be reoxidized by (6-R)-methylenetetrahydrofolate, again with nearly 1:1 stoichiometry. Steady state kinetic measurements of the NADPH-methylenetetrahydrofolate oxidoreductase activity give parallel line double reciprocal plots. The turnover number per mol of enzyme-bound flavin is 1600/min under Vmax conditions. The spectrum of the enzyme-bound flavin is significantly perturbed by the binding of S-adenosylmethionine, a metabolite known to be an allosteric modulator of the enzyme.
D000596:Amino Acids / Q000032:analysis; D000693:Anaerobiosis; D000818:Animals; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification*; D013053:Spectrophotometry; D013552:Swine
D000596:Amino Acids; D046911:Macromolecular Substances; D005182:Flavin-Adenine Dinucleotide; D010088:Oxidoreductases; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP)