Ascorbate Free Radical Reductases and Diaphorases in Soluble Fractions of the Human Lens

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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 ionexchange 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 b_5 reductase. The major and minor AFR reductases were regarded as a major diaphorase group different from two ubiquitous diaphorases, i. e., NADH-cytochrome b_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.
東海大学の論文