Molecular Characterization and Physiological Role of a Glyoxysome-Bound Ascorbate Peroxidase from Spinach |
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Authors: | Ishikawa Takahiro; Yoshimura Kazuya; Sakai Kosuke; Tamoi Masahiro; Takeda Toru; Shigeoka Shigeru |
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Institution: | Department of Food and Nutrition, Faculty of Agriculture, Kinki University Nakamachi, Nara, 631 Japan |
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Abstract: | cDNAs encoding two cytosolic and two chloroplastic ascorbateperoxidase (AsAP) isozymes from spinach have been cloned recentlyIshikawa et al. (1995) FEBS Lett. 367: 28, (1996) FEBS Lett.384: 289]. We herein report the cloning of the fifth cDNA ofan AsAP isozyme which localizes in spinach glyoxysomes (gAsAP).The open reading frame of the 858-base pair cDNA encoded 286amino acid residues with a calculated molecular mass of 31,507Da. By determination of the latency of AsAP activity in intactglyoxysomes, the enzyme, as well as monodehydroascorbate (MDAsA)reductase, was found to be located on the external side of theorganelles. The cDNA was overexpressed in Escherichia coli (E.coli). The enzymatic properties of the partially purified recombinantgAsAP were consistent with those of the native enzyme from intactglyoxysomes. 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-velocitycurves with the recombinant enzyme showed Michaelis-Menten typekinetics with AsA and hydrogen peroxide (H2O2); the apparentKm values for AsA and H2O2were 1.89±0.05 mM and 74±4.0µM,respectively. When the recombinant enzyme was diluted with AsA-depletedmedium, the activity was stable over 180 min. We discuss theH2O2-scavenging system maintained by AsAP and the regenerationsystem of AsA in spinach glyoxysome.
1Present address: Department of Biochemistry, Wakayama MedicalCollege, 27 Kyubancho, Wakayama, 640 Japan |
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