Soluble ascorbate free radical reductase in the human lens.
A major and a minor ascorbate free radical (AFR) reductase were separated from the soluble fraction in the human lens cortex by DEAE-cellulose ion-exchange column chromatography. These AFR reductases also exhibited diaphorase activity using dichlorophenolindophenol and ferricyanide as electron acceptors. The major AFR reductase was partially purified by 5'AMP-Sepharose 4B affinity column chromatography. This partially purified AFR reductase showed a single band of diaphorase activity in native polyacrylamide disc gel electrophoresis. This activity band corresponded to the major protein observed in protein staining by Coomassie Brilliant Blue. However, the protein staining by Coomassie Brilliant Blue showed this activity band surrounded by diffused staining. Molecular weight of the partially purified AFR reductase was determined to be 32 kDa by gel filtration, and the apparent Km value for AFR was about 15 microM. This major lens AFR reductase could be distinguished from soluble Neurospora, Euglena and cucumber AFR reductases, and from two ubiquitous enzymes with reduction activity of AFR and/or foreign compounds, ie, NADH-cytochrome b5 reductase and DT-diaphorase, by their molecular weights, Km values and/or ion-exchange chromatographic behaviors.
Bando
M
M
Department of Ophthalmology, Tokai University School of Medicine, Kanagawa, Japan.
Obazawa
H
H
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001205:Ascorbic Acid / Q000378:metabolism*; D002386:Cataract / Q000201:enzymology*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005609:Free Radicals / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007904:Lens Cortex, Crystalline / Q000201:enzymology*; D008875:Middle Aged; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis / Q000302:isolation & purification*; D010088:Oxidoreductases; D012995:Solubility
D005609:Free Radicals; D010088:Oxidoreductases; D009247:NADH, NADPH Oxidoreductases; D008058:Dihydrolipoamide Dehydrogenase; D001205:Ascorbic Acid