title: Purification and cDNA cloning of chloroplastic monodehydroascorbate reductase from spinach.
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.
authors.affiliation: Graduate School of Agriculture, Kyoto Prefectural University, Kyoto 606-8522, Japan. satsano@kpu.ac.jp
authors.forename: Satoshi
authors.initials: S
authors.lastname: Sano
authors.forename: Satoru
authors.initials: S
authors.lastname: Tao
authors.forename: Yuko
authors.initials: Y
authors.lastname: Endo
authors.forename: Tomomi
authors.initials: T
authors.lastname: Inaba
authors.forename: M Anwar
authors.initials: MA
authors.lastname: Hossain
authors.forename: Chikahiro
authors.initials: C
authors.lastname: Miyake
authors.forename: Michinori
authors.initials: M
authors.lastname: Matsuo
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Aoki
authors.forename: Kozi
authors.initials: K
authors.lastname: Asada
authors.forename: Kazumi
authors.initials: K
authors.lastname: Saito
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics*; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004579:Electron Transport; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D010802:Phylogeny; D020033:Protein Isoforms / Q000378:metabolism; D016415:Sequence Alignment; D018724:Spinacia oleracea / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D020033:Protein Isoforms; D009243:NAD; D009247:NADH, NADPH Oxidoreductases; C023927:monodehydroascorbate reductase (NADH)