title: Non-selenium glutathione peroxidase without glutathione S-transferase activity from bovine ciliary body.
abstract: A glutathione peroxidase was purified from bovine ciliary body by ammonium sulfate fractionation. Sephacryl S-300 gel filtration, diethylaminoethyl (DEAE)-cellulose chromatography and hydroxyapatite chromatography. The purified enzyme has an apparent mw of 112 kDa by gel filtration and 29 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme therefore is composed of four identical subunits. The ciliary enzyme is active with H2O2 (25), cumene hydroperoxide (170), t-butyl hydroperoxide (22), triphenylcarbinyl hydroperoxide (12), linoleic hydroperoxide (34) and 5-phenylpentenyl hydroperoxide (22): the numbers after substrates are K'm in microM. Glutathione is essential for the reaction; L-cysteine, dithiothreitol and 2-mercaptoethanol are inactive. Mercaptosuccinate (10 microM) inhibits the enzyme competitively (Ki = 7 microM) when cumene hydroperoxide is substrate, and uncompetitively (Ki = 10 microM) when H2O2 is substrate. No selenium was found in the enzyme by the fluorometric assay with 2.3-diaminonaphthalene. The enzyme demonstrates no glutathione S-transferase activity when tested with 1-chloro-2,4-dinitrobenzene, and several other compounds. A partial sequence of the enzyme shows some similarities both to Se-glutathione peroxidases and a glutathione S-transferase isozyme.
authors.affiliation: Kresge Eye Institute, Department of Ophthalmology, Wayne State University School of Medicine, Detroit, MI 48201.
authors.forename: H
authors.initials: H
authors.lastname: Shichi
authors.forename: J C
authors.initials: JC
authors.lastname: Demar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002924:Ciliary Body / Q000201:enzymology*; D005979:Glutathione Peroxidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012643:Selenium / Q000502:physiology*
chemical_list: D005979:Glutathione Peroxidase; D005982:Glutathione Transferase; D012643:Selenium