Molecular Cloning and Functional Expression of Rat Liver Glutathione-dependent Dehydroascorbate Reductase

We have isolated a cDNA clone for a novel glutathione-dependent dehydroascorbate reductase from a rat liver cDNA library in lambdagt11 by immunoscreening. The authenticity of the clone was confirmed as follows: first, the antibody that had been purified through affinity for the protein expressed by the cloned lambdagt11 phage recognized only the enzyme in a crude extract from rat liver; and second, two internal amino acid sequences of purified enzyme were identified in the protein sequence predicted from the cDNA. The predicted protein consists of 213 amino acids with a molecular weight of 24,929, which is smaller by approximately 3,000 than the value obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This discrepancy of the molecular weight was explained by post-translational modification because the recombinant protein expressed by a mammalian system (Chinese hamster ovary cells) was of the same size as rat liver enzyme but larger than the protein expressed by a bacterial system (Escherichia coli). Chinese hamster ovary cells, originally devoid of glutathione-dependent dehydroascorbate reductase activity, was made to elicit the enzyme activity (1.5 nmol/min/mg of cytosolic protein) by expression of the recombinant protein. Additionally, the cells expressing the enzyme were found to accumulate 1.7 times as much ascorbate as the parental cells after incubation with dehydroascorbate. This result points to the importance of the dehydroascorbic acid reductase in maintaining a high concentration of ascorbate in the cell

The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen

The reduction of nitro blue tetrazolium (NitroBT) with NADH mediated by phenazine methosulfate (PMS) under aerobic conditions was inhibited upon addition of superoxide dismutase. This observation indicated the involvement of superoxide aninon radical (O2-) in the reduction of NitroBT, the radical being generated in the reoxidation of reduced PMS. Similarly, the reduction of NitroBT coupled to D-amino acid oxidase-PMS system under aerobic conditions was also inhibited by superoxide dismutase. A simple method for detecting superoxide dismutase is described

Reactivity of D-amino acid oxidase with artificial electron acceptors

1. 1.|The fully reduced form of D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3] reacted with a number of electron acceptors and the order of reactivity was: benzoquinone > phenazine methosulfate (PMS) > oxygen > 2,6-dichlorophenolindophenol (DCIP) > methylene blue > ferricyanide. The stoichiometric reaction between the fully reduced enzyme and PMS was established. 2. 2.|A mechanism for the catalytic oxidation of D-arginine by the enzyme using PMS as electron acceptor involved the fully reduced enzyme which was reoxidized by PMS. 3. 3.|When D-alanine was used as substrate, PMS, ferricyanide, DCIP and methylene blue served as electron acceptor. Analyzing the kinetic data of the catalytic oxidation of D-alanine as well as D-α-aminobutyric acid when PMS was used as acceptor, it was revealed that the purple intermediate formed through the reaction of the enzyme with the substrate was involved in the mechanism of the oxidation. The rate of reaction of PMS with the intermediate was fairly small compared with that of PMS with the fully reduced enzyme. 4. 4.|PMS reacted stoichiometrically with the semiquinoid form of the enzyme at a faster rate than with the fully reduced enzyme

Increased cellular resistance to oxidative stress by expression of cyanobacterium catalase-peroxidase in animal cells

AbstractTo exploit prokaryotic antioxidant enzymes for protection of animal cells from oxidative damage, we expressed catalase-peroxidase of cyanobacterium Synechococcus PCC 7942 in 104C1 cells. The gene for this enzyme was inserted into the mammalian expression vector pRc/CMV. The stable transfectants obtained had higher specific activities of catalase and as a result became more resistant to H2O2 or paraquat than the parental cells. Subcellular fractionation and immunoblot analysis revealed that the expressed catalase-peroxidase was confined to the cytosol; this localization may be the basis for the effective protection of the transfectants from the oxidative cell damage