Diphosphopyridine Nucleotide L-Gulonic Acid Dehydrogenase from Guinea Pig Liver

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Abstract
The enzymatic mechanism of the formation of L-xylulose from d-glucuronic acid in mammalian tissues has been studied by many investigators and the participation of l-gulonic acid dehydrogenases specific to triphos-phopyridine* and diphosphopyridine nucleotide has been established in the liver and kidney of guinea pigs (1), or other species of animals (2-4). The reduction of D-glucuronic acid with TPNH in the presence of TPN-L-gulonic dehydrogenase has been confirmed as the first step of L-xylulose formation (1, 3). D-glucuronic acid+TPNH+H+ ⇄ L-gulonic acid+TPN+ In the previous paper from this laboratory, L-gulonic acid, the product of the above reaction, was shown to undergo second reaction by the DPN-l-gulonic dehydrogenase (1). When a crude preparation of the liver or kidney of guinea pigs was used, l-gulonic acid was converted to l-xylulose with simultaneous release of carbon dioxide in the presence of DPN. L-gulonic acid+DPN+→ L-xylulose+CO2 + DPNH+H+ However, ketohexonic acid, probably 3-keto-l-gulonic acid, was proposed as the reaction product of DPN-specific gulonic acid dehydrogenase by Lehninger and his coworkers (2, 3), and this compound was suspected as the direct precursor of l-ascorbic acid or l-xylulose, in the following manner: L-gulonic acid+DPN→ 3-keto-L-gulonic acid+DPNH+H+ 3-keto-L-gulonic acid→ L-ascorbic acid+H2O 3-keto-L-gulonic acid→ L-xylulose+C02 Thus, the DPN-specific enzyme was considered by these workers as an enzyme similar to malic enzyme, but the enzyme has not been purified as yet, and the nature of DPN-L-gulonic dehydrogenase has not been clarified completely. The present report deals with the purification and properties of DPN-L-gulonic dehydrogenase from guinea pig liver which is unable to synthesize L-ascorbic acid in vivo or in vitro.