Purification and enzymatic properties of glyoxylate reductase II from baker's yeast.

Glyoxylate reductase II was purified about 2,400-fold from a cell extract of baker's yeast by protamine sulfate treatment, and column chromatographies on DEAE-cellulose, hydroxylapatite, Sephadex G-150, and phosphocellulose. The purified enzyme was electrophoretically homogeneous. The molecular weight was determined to be approximately 65,000 by gel filtration. The enzyme was greatly stabilized by the addition of 20% (v/v) glycerol. It catalyzed the reduction of glyoxylate and hydroxypyruvate and was specific for NADPH as an electron donor, but showed slight affinity towards NADH. The Michaelis constants for glyoxylate, hydroxypyruvate, NADPH, and NADH were found to be 16mM, 1.4 mM, 5.7 microM, and 0.43 mM, respectively. The enzyme was inhibited by p-chloromercuribenzoate (PCMB) and iodoacetate, but inhibition was prevented by dithiothreitol (DTT) or L-cysteine. The reduction of glyoxylate and hydroxypyruvate was not stimulated by anions.