Molecular characterization and physiological role of a glyoxysome-bound ascorbate peroxidase from spinach [Spinacia oleracea]

• Published in: Plant and cell physiology Vol. 39; no. 1; pp. 23 - 34
• Main Authors: Ishikawa, T. (Kinki Univ., Higashiosaka, Osaka (Japan). Faculty of Agriculture), Yoshimura, K, Sakai, K, Tamoi, M, Takeda, T, Shigeoka, S
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.01.1998
• Subjects: ACIDE ASCORBIQUE; ACIDO ASCORBICO; Agronomy. Soil science and plant productions; Amino Acid Sequence; Ascorbate peroxidase(EC 1.11.1.11); Ascorbate Peroxidases; ASCORBIC ACID; Base Sequence; Biological and medical sciences; Cloning, Molecular; CYTOPLASMIC ORGANELLES; DNA, Plant; Economic plant physiology; Enzymes; Fundamental and applied biological sciences. Psychology; Gene Expression; GENETICA MOLECULAR; GENETIQUE MOLECULAIRE; Glyoxysome; Hydrogen peroxide; Isozyme; Metabolism; Microbodies - enzymology; MOLECULAR GENETICS; Molecular Sequence Data; Molecular Weight; Nutrition. Photosynthesis. Respiration. Metabolism; ORGANITE CELLULAIRE; ORGANULOS CITOPLASMICOS; PEROXIDASAS; PEROXIDASES; Peroxidases - classification; Peroxidases - genetics; Peroxidases - isolation & purification; Peroxidases - physiology; PEROXYDASE; Phylogeny; PHYSIOLOGICAL REGULATION; Plant physiology and development; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; REGULACION FISIOLOGICA; REGULATION PHYSIOLOGIQUE; Sequence Homology, Amino Acid; Spinach (Spin-acia oleracea); SPINACIA OLERACEA; Spinacia oleracea - enzymology; Enzyme; Isozyme; Phylogenetic tree; L-Ascorbate peroxidase; Spinacia oleracea; Chenopodiaceae; Bound form; Characterization; Glyoxysome; Peroxidases; Vegetable crop; Dicotyledones; Angiospermae; Spermatophyta; Oxidoreductases; Molecular cloning; Localization; Catalyst activity; Aminoacid sequence
• ISSN: 0032-0781; 1471-9053
• DOI: 10.1093/oxfordjournals.pcp.a029285

cDNAs encoding two cytosolic and two chloroplastic ascorbate peroxidase (AsAP) isozymes from spinach have been cloned recently. We herein report the cloning of the fifth cDNA of an AsAP isozyme which localizes in spinach glyoxysomes (gAsAP). The open reading frame of the 858-base pair cDNA encoded 286 amino acid residues with a calculated molecular mass of 31,507 Da. By determination of the latency of AsAP activity in intact glyoxysomes, the enzyme, as well as monodehydroascorbate (MDAsA) reductase, was found to be located on the external side of the organelles. The cDNA was overexpressed in Escherichia coli. The enzymatic properties of the partially purified recombinant gAsAP were consistent with those of the native enzyme from intact glyoxysomes. The recombinant enzyme utilized ascorbate (AsA) as its most effective natural electron donor; glutathione (GSH) and NAD(P)H could not substitute for AsA. The substrate velocity curves with the recombinant enzyme showed Michaelis-Menten type kinetics with AsA and hydrogen peroxide (H2O2); the apparent K(m) values for AsA and H2O2 were 1.89 +- 4.0 mu-M, respectively. When the recombinant enzyme was diluted with AsA-depleted medium, the activity was stable over 180 min. We discuss the H2O2-scavenging system maintained by AsAP and the regeneration system of AsA in spinach glyoxysome