Journal Article

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

Takahiro Ishikawa, Kazuya Yoshimura, Kosuke Sakai, Masahiro Tamoi, Toru Takeda and Shigeru Shigeoka

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 39, issue 1, pages 23-34
Published in print January 1998 | ISSN: 0032-0781
Published online January 1998 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/oxfordjournals.pcp.a029285
Molecular Characterization and Physiological Role of a Glyoxysome-Bound Ascorbate Peroxidase from Spinach

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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 substrate-velocity curves with the recombinant enzyme showed Michaelis-Menten type kinetics with AsA and hydrogen peroxide (H2O2); the apparent Km values for AsA and H2O2were 1.89±0.05 mM and 74±4.0μM, 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.

Keywords: Ascorbate peroxidase(EC 1.11.1.11); Glyoxysome; Hydrogen peroxide; Isozyme; Spinach (Spin-acia oleracea)

Journal Article.  0 words. 

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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