Purification and cDNA Cloning of Chloroplastic Monodehydroascorbate Reductase from Spinach

Satoshi SANO; Satoru TAO; Yuko ENDO; Tomomi INABA; M. Anwar HOSSAIN; Chikahiro MIYAKE; Michinori MATSUO; Hideyuki AOKI; Kozi ASADA; Kazumi SAITO

doi:10.1271/bbb.69.762

Biochemistry & Molecular Biology Regular Papers

Purification and cDNA Cloning of Chloroplastic Monodehydroascorbate Reductase from Spinach

Satoshi SANO, Satoru TAO, Yuko ENDO, Tomomi INABA, M. Anwar HOSSAIN, Chikahiro MIYAKE, Michinori MATSUO, Hideyuki AOKI, Kozi ASADA, Kazumi SAITO

Author information

Satoshi SANO
Graduate School of Agriculture, Kyoto Prefectural University
Satoru TAO
Graduate School of Agriculture, Kyoto Prefectural University
Yuko ENDO
Graduate School of Agriculture, Kyoto Prefectural University
Tomomi INABA
Graduate School of Agriculture, Kyoto Prefectural University
M. Anwar HOSSAIN
Department of Biochemistry, University of Dhaka
Chikahiro MIYAKE
Plant Physiology Research Group, Research Institute of Innovative Technology for the Earth (RITE)
Michinori MATSUO
Graduate School of Agriculture, Kyoto University
Hideyuki AOKI
Hokuriku Research Center, National Agricultural Research Center (NARC)
Kozi ASADA
Faculty of Bioscience and Biotechnology, Fukuyama University
Kazumi SAITO
Graduate School of Agriculture, Kyoto Prefectural University

Keywords: active oxygen, ascorbate radical, chloroplastic isoform, monodehydroascorbate reductase (EC 1.6.4.7), spinach (Spinacea oleracea)

JOURNAL FREE ACCESS

2005 Volume 69 Issue 4 Pages 762-772

DOI https://doi.org/10.1271/bbb.69.762

Details

Abstract

The chloroplastic isoform of monodehydroascorbate (MDA) radical reductase was purified from spinach chloroplasts and leaves. The cDNA of chloroplastic MDA reductase was cloned, and its deduced amino acid sequence, consisting of 497 residues, showed high homology with those of putative organellar MDA reductases deduced from cDNAs of several plants. The amino acid sequence of the amino terminal of the purified enzyme suggested that the chloroplastic enzyme has a transit peptide consisting of 53 residues. A southern blot analysis suggested the occurrence of a gene encoding another isoform homologous to the chloroplastic isoform in spinach. The recombinant enzyme was highly expressed in Eschericia coli using the cDNA, and purified to a homogeneous state with high specific activity. The enzyme properties of the chloroplastic isoform are presented in comparison with those of the cytosolic form.

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