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.

T Nishino,K Tsushima

doi:10.1016/s0021-9258(18)67374-0

Abstract

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.

Highlights

INHIBITION OF MILK XANTHINE OXIDASE BY FOLIC ACID AND SEPARATION OF THE ENZYME INTO TWO FRACTIONS ON SEPHAROSE 4B/FOLATEGEL*
Resolution amined after complete removal of the contaminant of active and inactive forms was reported by Edmondson et which was responsible for time-dependentinactivation al. [9] who used an allopurinol analoguaes an affinity ligand
The highly active enzyme sample was prepared by affinity chromatography onthe folate column, the contentof the inactive form in the enzyme preparation has not been analyzed indetail previously

Summary

Takeshi Nishino and Keizo Tsushima

Inhibition of xanthine oxidase by folic acid was reex- in enzyme samples prepared nboyrmal procedures. The highly active enzyme sample was prepared by affinity chromatography onthe folate column, the contentof the inactive form in the enzyme preparation has not been analyzed indetail previously. Using a Sepharose 4B/folate column, folic acid was In this paper we re-examined whether folic acid was an confirmed to be a competitive inhibitor of xanthine inhibitor and analyzed in detail the content of theinac - oxidase. Folic acid was purified by DEAE-cellulosecolumn chromatography according to themethod of Inhibition of xanthine oxidase by folic acid was first re- Shiota et al [14]. This article must be hereby marked “aduer- activity defined as theabsorbance change/min at 295 nm, monitoring tisernent” in accordance with 18U.S.C. Section 1734 solelyto indicate conversion of xanthine to uric acid, dividedby the enzyme absorbance this fact.

RESULTS

Fraction h m t m

ESR slow signal