Ascorbate Synthesizing System in Rat Liver Microsomes

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Abstract
Gulonolactone oxidase [EC 1.1.3.8] was effectively solubilized from rat liver micro-somes by the action of detergents but not with proteases. We established a new method of purification of the enzyme. After digestion with trypsin [EC 3.4.4.4] which removed cytochrome b5 from the microsomes, the oxidase was solubilized by the treatment with Emasol 1130, a non-ionic detergent. The solubilized enzyme was further purified by DEAE-Sephadex A-50 and Sepharose 6B column chromatog-raphy. Judging from the gel filtration, an apparent molecular weight of the enzyme was estimated to be about 100, 000. The presence of a gulonolactone-reducible pigment associated with enzyme activity was detected with partially purified preparation, which was almost free from cytochrome b5. The gulonolactone reduced minus oxidized difference spectrum of the enzyme had only one trough at 450–470 μ. L-Gulono-γHactone and L-galactono-γ-lactone were only specific substrates for the reduction of the pigment. The specific content of the pigment increased in roughly parallel to the specific activity of the enzyme during the purification procedures. Based on above lines of evidence, we concluded that the pigment, which exhibits a difference spectrum by the reduction with gulonolactone, is a specific prosthetic group of gulonolactone oxidase. The proposed possibility that the enzyme has two active sites is discussed with the results of the inhibition studies using PCMS and Na2S.

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