Purification and molecular properties of ascorbate peroxidase from bovine eye.
Ascorbate peroxidase (APX) is a hydrogen peroxide-scavenging peroxidase which uses ascorbate (AsA) as the specific electron donor. APX has not been isolated in mammals. Ocular tissue contains AsA at high concentrations, and we detected APX activity in bovine retinal pigment epithelium (RPE) and choroid. We purified APX from bovine RPE and choroid by four chromatographic steps. The purified APX was a monomeric hemoprotein with a molecular mass of 43 kDa. The amino acid sequence of the amino-terminal region of the purified APX showed a high degree of homology to that of plants. The primary product of the APX reaction was identified as the monodehydroascorbate radical. The APX showed high specificity for AsA as an electron donor. This is the first isolation and characterization of APX from mammals, and its role in the protection against active species of oxygen in ocular tissue is discussed.
Wada
N
N
Research Institute for Food Science, Kyoto University, Japan.
Kinoshita
S
S
Matsuo
M
M
Amako
K
K
Miyake
C
C
Asada
K
K
D000595:Amino Acid Sequence; D000818:Animals; D060387:Ascorbate Peroxidases; D001205:Ascorbic Acid / Q000378:metabolism; D002417:Cattle; D002829:Choroid / Q000737:chemistry / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D003683:Dehydroascorbic Acid / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005123:Eye / Q000201:enzymology; D016166:Free Radical Scavengers / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012160:Retina / Q000737:chemistry / Q000196:embryology; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013379:Substrate Specificity
D016166:Free Radical Scavengers; D006861:Hydrogen Peroxide; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid