Fumarate reductase was highly purified from the cytosol fraction of the cells of baker's yeast, which were anaerobically cultured, with a yield of 6% by.7 steps. The purified enzyme is homogeneous in disc-gel electrophoresis, and has a molecular weight of 58,800. Its isoelectric point is 5.3. It was reported that fumarate reductase in brewer's yeast was separated into two groups, which were named types I and II, on hydroxyapatite chromatography.4) Type I of the enzyme was partially purified and the molecular properties were described by Tisdale et al.5) Hauber et al^showed that the yeast when cultivated anaerobically had a high fumarate reductase activity. In this paper we report the purification and characterization of the enzyme prepared from the cytoplasm of the baker's yeast. MATERIALS AND METHODSOrganism and cultivation.

Fumarate reductase apoenzyme having the ability to reconstitute active enzyme was obtained by dialyzing the holoenzyme against 1 M KBr. The dissociation constant of the FAD-apoenzyme complex was 2.3 X 10(-8) M. The denatured holoenzyme and apoenzyme possessed seven sulfhydryl (SH) groups as determined with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). In the native apoenzyme, five SH-groups reacted with DTNB, and four of them were completely protected by the addition of FAD, while in the native holoenzyme, one was modified without inactivation. These results indicate that one SH-group is located on the surface of the enzyme molecule, four at or near the FAD-binding site, and two deeply embedded in the molecule. The modification of the apoenzyme caused inhibition of binding of FAD, resulting in loss of the ability to reconstitute enzymatic activity. Analyses of the data by statistical and kinetic methods suggested that a reactive SH-group is involved among the four SH-groups in the binding of FAD to the apoenzyme.

The rapid and effective purification of soluble fumarate reductase from baker's yeast achieved by Blue Sepharose CL-6B chromatography. Cibacron Blue F3GA, the chromophore of Blue Sepharose, inhibited the activity of fumarate reductase. The enzyme bound to the column was selectively eluted by flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) or riboflavin. The purified enzyme was essentially homogeneous as indicated by polyacrylamide gel electrophoresis under nondenaturing conditions and under denaturing conditions in sodium dodecylsulfate. By this procedure, the enzyme could be rapidly purified with high yield from yeast cells.

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