Journal Article

Purification and Characterization of Chloroplast Dehydroascorbate Reductase from Spinach Leaves

Taise Shimaoka, Akiho Yokota and Chikahiro Miyake

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 41, issue 10, pages 1110-1118
Published in print October 2000 | ISSN: 0032-0781
Published online October 2000 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcd035
Purification and Characterization of Chloroplast Dehydroascorbate Reductase from Spinach Leaves

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Green leaves of plants require the high-level activity that can regenerate ascorbate during photosynthesis. One of such enzyme is dehydroascorbate reductase (DHAR), but the molecular and enzymological properties of the enzyme remain to be fully characterized. In this study, we showed that two major DHAR existed in spinach leaves. The two DHARs occupied at least over 90% of total DHAR activity. The amount of the two DHARs was almost the same. We purified both DHARs from spinach leaves. One form of DHAR originated in chloroplasts; the other occurred in the subcellular compartment other than chloroplasts. The chloroplast DHAR had Km values of 70 µM and 1.1 mM for dehydroascorbate and reduced glutathione, respectively. The specific activity of the purified enzyme corresponded to 360 µmol of ascorbate formed per milligram of protein per minute. These properties were quite different from those of trypsin inhibitor, which has been reported to be the plastid DHAR. The other DHAR had the very similar properties to those of chloroplast DHAR. Chloroplast and the other DHARs functioned as a monomer with molecular masses of 26 kDa and 25 kDa, respectively. cDNA for the chloroplast DHAR was cloned with the determined amino-terminal amino acid sequence. The primary sequence predicted from the cDNA included the plastid-targeting sequence. Finally, the significance of chloroplast DHAR in the regeneration of ascorbate is discussed.

Keywords: Key words: Ascorbate — Chloroplast — Dehydroascorbate reductase — Oxidative stress — Spinach.; Abbreviations: APX, ascorbate peroxidase; Chl, chlorophyll; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; MDA, monodehydroascorbate; MDAR, monodehydroascorbate reductase; SOD, superoxide dismutase; GR, glutathione reductase; IAA, iodoacetamide.; The nucleotide sequences reported in this paper have been recorded in the GenBank under accession numbers AF195783 (spinach chloroplast DHAR) and AF195887 (Arabidopsis chloroplast DHAR).

Journal Article.  5560 words.  Illustrated.

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

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