title: Ascorbate free radical reductases and diaphorases in soluble fractions of the human lens.
abstract: 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.
authors.affiliation: Department of Ophthalmology, School of Medicine, Tokai University, Kanagawa, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Bando
authors.forename: H
authors.initials: H
authors.lastname: Obazawa
mesh_terms: 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
chemical_list: D009247:NADH, NADPH Oxidoreductases; C023927:monodehydroascorbate reductase (NADH); D008058:Dihydrolipoamide Dehydrogenase