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.
Ishikawa
T
T
Department of Biochemistry, Wakayama Medical College, Wakayama 641-0012, Japan.
Casini
A F
AF
Nishikimi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D012333:RNA, Messenger; D010088:Oxidoreductases; C020666:glutathione dehydrogenase (ascorbate); D001205:Ascorbic Acid