Molecular Characterization and Physiological Role of a Glyoxysome-Bound Ascorbate Peroxidase from Spinach

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概要

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• cDNAs encoding two cytosolic and two chloroplastic ascorbate peroxidase(AsAP) isozymes from spinach have been cloned recently[Ishikawa et al.(1995)FEBS Lett.367:28, (1996)FEBS Lett.384:289]. 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(E.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 substratevelocity curves with the recombinant enzyme showed Michaelis-Menten type kinetics with AsA and hydrogen peroxide(H_2O_2); the apparent K_m values for AsA and H_2O_2 were 1.89±0.05 mM and 74±4.0 μM, respectively. When the recombinant enzyme was diluted with AsA-depleted medium, the activity was stable over 180 min. We discuss the H_2O_2-scavenging system maintained by AsAP and the regeneration system of AsA in spinach glyoxysome.

• 日本植物生理学会の論文

著者

• Takeda Toru

  Department of Radiology, Institute of Clinical Medicine, University of Tsukuba

• Ishikawa Tomohiro

  Department Of Agricultural Chemistry School Of Agriculture Nagoya University

• Tada T

  Dep. Of Advanced Bioscience Fac. Of Agriculture Kinki Univ.

• ISHIKAWA Takahiro

  Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane Uni

• SHIGEOKA Shigeru

  Department of Advanced Bioscience, Faculty of Agriculture, Kinki University

• Takeda Toru

  Dep. Of Advanced Bioscience Fac. Of Agriculture Kinki Univ.

• YOSHIMURA Kazuya

  Department of Food and Nutrition, Faculty of Agriculture, Kinki University

• Tamoi Masahiro

  Department of Food and Nutrition, Faculty of Agriculture, Kinki University

• Sakai Kosuke

  Department of Food and Nutrition, Faculty of Agriculture, Kinki University

• Takeda Takumi

  Wood Research Institute Kyoto University

• Ishikawa T

  Fac. Of Life And Environmental Sci. Shimane Univ.

• Sakai Kosuke

  Department Of Food And Nutrition Faculty Of Agriculture Kinki University

• Maruta Takanori

  Dep. Of Advanced Bioscience Fac. Of Agriculture Kinki Univ.

• Tamoi Masahiro

  Department Of Advanced Bioscience Faculty Of Agriculture Kinki University

• Shigeoka Shigeru

  Department Of Advanced Bioscience Faculty Of Agriculture Kinki University

• Yoshimura Kazuya

  Department Of Food And Nutritional Science College Of Bioscience And Biotechnology Chubu University

• Takeda T

  Dep. Of Advanced Bioscience Fac. Of Agriculture Kinki Univ.

• Kojima Yusuke

  Department Of Food And Nutritional Science College Of Bioscience And Biotechnology Chubu University

• Shigeoka Shigeru

  Dept. Adv. Biosci. Fac. Agr. Kinki Univ

• Maruta Takanori

  Department Of Food And Nutrition Faculty Of Agriculture Kinki University

• Ishikawa Takahiro

  Department Of Clinical Cell Biology And Medicine Chiba University Graduate School Of Medicine

• Takeda Toru

  Department Of Agricultural Chemistry Faculty Of Agriculture Hokkaido Universty:(present Office)kansa

• Ishikawa Takahiro

  Department of Applied Bioscience and Biotechnology, Faculty of Life and Environmental Science, Shimane University

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