title: Isolation of ascorbate free radical reductase from rabbit lens soluble fraction.
abstract: Ascorbate free radical (AFR) reductase with diaphorase activity was isolated from the rabbit lens soluble fraction to characterise some molecular properties of the enzyme. The isolation was accomplished using gel filtration (Sephadex G-75 superfine or Sephacryl S-200 HR), affinity chromatography (Affi-Gel Blue), native isoelectric focusing and two-dimensional gel electrophoresis. A major soluble AFR reductase was found at an isoelectric point of 8.4 and a molecular weight of 31 kDa, and a few minor enzymes were also detected in the range of pI 7.0-8.6. An unknown N-terminal partial amino acid sequence was determined in one peptide fragment prepared from the major enzyme fraction. From the sequence analysis, it is discussed that the lens soluble AFR reductase may differ from NADH-cytochrome b5 reductase reported to be involved in the membrane-bound AFR reductase activity of mitochondria, microsomes and plasma membrane.
authors.affiliation: Department of Ophthalmology, Tokai University School of Medicine, Isehara, Japan.
authors.forename: Masayasu
authors.initials: M
authors.lastname: Bando
authors.forename: Takashi
authors.initials: T
authors.lastname: Inoue
authors.forename: Mikako
authors.initials: M
authors.lastname: Oka
authors.forename: Kayako
authors.initials: K
authors.lastname: Nakamura
authors.forename: Kenji
authors.initials: K
authors.lastname: Kawai
authors.forename: Hajime
authors.initials: H
authors.lastname: Obazawa
authors.forename: Shizuko
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Makoto
authors.initials: M
authors.lastname: Takehana
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional; D007525:Isoelectric Focusing; D007908:Lens, Crystalline / Q000201:enzymology*; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011817:Rabbits; D012995:Solubility
chemical_list: D009247:NADH, NADPH Oxidoreductases; C023927:monodehydroascorbate reductase (NADH)