title: Molecular characterization of Euglena ascorbate peroxidase using monoclonal antibody.
abstract: Ascorbate peroxidase (EC 1.11.1.11) has been purified to electrophoretic homogeneity from Euglena gracilis Z. The enzyme showed a molecular mass of 58 kDa on SDS-PAGE and gel filtration, indicating that Euglena ascorbate peroxidase exists as a monomeric form. The substrate specificity for an electron donor and the stability of the purified enzyme were similar to those of cytosolic isozymes from higher plants. One of the characteristic properties was that Euglena ascorbate peroxidase reduces organic hydroperoxides as well as hydrogen peroxide. The N-terminal amino-acid sequence showed no significant similarity to any other ascorbate peroxidase from higher plants. However, the sequence of the peptides from the purified enzyme exhibited a high degree of homology to sequences of cytosolic and chloroplastic ascorbate peroxidases. Monoclonal antibodies against the purified Euglena ascorbate peroxidase were prepared. Two monoclonal antibodies (EAP1 and EAP2) showed high homology to cytosolic ascorbate peroxidases of higher plants, as judged by Western blot analysis. The EAP1 was also specific for chloroplastic ascorbate peroxidase from spinach. These findings indicate that Euglena ascorbate peroxidase exists in highly homologous regions with the ascorbate peroxidases of higher plants.
authors.affiliation: Department of Food and Nutrition, Faculty of Agriculture, Kinki University, Nara, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Ishikawa
authors.forename: T
authors.initials: T
authors.lastname: Takeda
authors.forename: H
authors.initials: H
authors.lastname: Kohno
authors.forename: S
authors.initials: S
authors.lastname: Shigeoka
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D060387:Ascorbate Peroxidases; D003429:Cross Reactions; D005056:Euglena gracilis / Q000201:enzymology*; D007527:Isoenzymes / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000276:immunology / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D010544:Peroxidases; D060387:Ascorbate Peroxidases