Studies on Menadione Reductase of Bakers' Yeast

Abstract

The crude extract was adsorbed on a Duolite CS-101 column, the yellow band eluted and added with ammonium sulfate (0.82 saturation). The precipitates were dissolved, fractionated with ammonium sulfate between 0.5 and 0.8 saturation, passed through an Amberlite IRC-50 column, the effluents chroma-tographed on a TEAE-cellulose column, eluted with 0.5 [image] phosphate buffer (pH 7.0), and, after desalting, crystallized. The preparation possessed menadione reductase, purified about 260-fold. The enzyme required DPNH2 as a sole H-donor for reducing a variety of substrates, including quinone derivatives, dyes, ferricyanide, and ferricytochrome c. Optimum pH was found at 5.8-5.9, but at 6. 5 with cytochrome c. The enzyme contained 1.12% flavinadenine dinucleotide(l mol./mol. enzyme of mol. wt. of 70,000), and gave the absorption bands at 272,370, 454 and 457 (shoulder) m[mu]. Dicumarol and DPN caused inhibition, which could be reversed by p-chloromercuribenzoate. Antimycin A and amytal had no effect on the enzyme''s activity.