Purification and cDNA cloning of chloroplastic monodehydroascorbate reductase from spinach.
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.
Sano
Satoshi
S
Graduate School of Agriculture, Kyoto Prefectural University, Kyoto 606-8522, Japan. satsano@kpu.ac.jp
Tao
Satoru
S
Endo
Yuko
Y
Inaba
Tomomi
T
Hossain
M Anwar
MA
Miyake
Chikahiro
C
Matsuo
Michinori
M
Aoki
Hideyuki
H
Asada
Kozi
K
Saito
Kazumi
K
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*
D018076:DNA, Complementary; D020033:Protein Isoforms; D009243:NAD; D009247:NADH, NADPH Oxidoreductases; C023927:monodehydroascorbate reductase (NADH)