Interaction of milk xanthine oxidase with folic acid. Inhibition of milk xanthine oxidase by folic acid and separation of the enzyme into two fractions on Sepharose 4B/folate gel.
Inhibition of xanthine oxidase by folic acid was reexamined after complete removal of the contaminant which was responsible for time-dependent inactivation (Lewis, A. S., Murphy, L., Mcalla, C., Fleary, M., and Purcell, S. (1984) J. Biol. Chem. 259, 12-15; Spector, T., and Ferone, R. (1984) J. Biol. Chem. 259, 10784-10786). From turnover experiments using stopped flow equipment with a limited amount of xanthine and excess oxygen, and from kinetic analyses with an oxygen electrode, folic acid was found to be an inhibitor of xanthine oxidase. The inhibition was competitive with xanthine with a Ki value of 4.2 X 10(-5) M. From the behavior of the enzyme in affinity chromatography using a Sepharose 4B/folate column, folic acid was also confirmed to be a competitive inhibitor of xanthine oxidase. When enzyme which had been pretreated with oxipurinol was applied to the affinity column, two fractions of xanthine oxidase were separated. The first fraction was found to contain the fully active form (double-active dimers) from the analyses of spectral changes on addition of xanthine, oxipurinol titration, and ESR slow signal, whereas the second fraction was assumed to contain mixed dimers and double-inactive dimers. The ratio of the content of the first fraction to that of the second fraction supports the hypothesis that there are three enzyme species and that there is no interaction either in catalytic activity or in sulfuration or desulfuration reactions between the two subunits.
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D004578:Electron Spin Resonance Spectroscopy; D005492:Folic Acid / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008892:Milk / Q000201:enzymology*; D010117:Oxypurinol / Q000494:pharmacology; D019820:Xanthine; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014970:Xanthines / Q000494:pharmacology
D046911:Macromolecular Substances; D014970:Xanthines; D019820:Xanthine; D005492:Folic Acid; D014969:Xanthine Oxidase; D010117:Oxypurinol