Purification and characterization of chloroplast dehydroascorbate reductase from spinach leaves

Green leaves of plants require the high-level activity that can regenerate ascorbate during photosynthesis, One of such enzyme is dehydroascorbate red uctase (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 p urified both DHARs from spinach leaves. One form of DHAR originated in chlo roplasts; the other occurred in the subcellular compartment other than chlo roplasts, The chloroplast DHAR had K-m values of 70 muM and 1.1 mM for dehy droascorbate and reduced glutathione, respectively, The specific activity o f the purified enzyme corresponded to 360 mu mol of ascorbate formed per mi lligram of protein per minute. These properties were quite different from t hose 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 ma sses 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 ascorb ate is discussed.