Ascorbate free radical reductases and diaphorases in soluble fractions of the human lens.
Major and minor ascorbate free radical (AFR) reductases, with diaphorase activity, and three other diaphorases were separated from the human lens soluble fraction by DEAE-cellulose ion-exchange column chromatography. They were characterized for adsorptivity to ion-exchange and 5'AMP-Sepharose 4B affinity columns, kinetic properties, and substrate specificity. The latter diaphorases were closely correlated with NADH-cytochrome beta 5 reductase. The major and minor AFR reductases were regarded as a major diaphorase group different from two ubiquitous diaphorases, i.e., NADH-cytochrome beta 5 reductase and DT-diaphorase. A major AFR reductase was partially purified approximately 50 fold over the lens soluble fraction by ion-exchange, affinity, and gel filtration (Sephacryl S-200 HR) column chromatography. From the partially purified enzyme, 2 bands, one sharp and one diffuse, were obtained by native polyacrylamide gel electrophoresis. Two proteins, of 20 and 24 kDa, were identified in the active enzyme bands by SDS-polyacrylamide gel electrophoresis. This suggests that the 20 and/or 24 kDa proteins may be components of the major AFR reductase.
Bando
M
M
Department of Ophthalmology, School of Medicine, Tokai University, Kanagawa, Japan.
Obazawa
H
H
D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D008058:Dihydrolipoamide Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007908:Lens, Crystalline / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity
D009247:NADH, NADPH Oxidoreductases; C023927:monodehydroascorbate reductase (NADH); D008058:Dihydrolipoamide Dehydrogenase