cDNA cloning, overproduction and characterization of rat adrenodoxin reductase.
We isolated a full-length cDNA clone for rat adrenodoxin reductase (AdR). The precursor of rat AdR was predicted to consist of 34 amino-terminal residues of extrapeptide for transport into mitochondria and the following 460 residues of the mature peptide region. The deduced amino acid sequence was 70.8 and 61.8% homologous to those of bovine and human AdRs in the extrapeptide region, respectively, and 88.5% homologous to both the sequences of bovine and human AdRs in the mature peptide region. The predicted mature form of rat AdR was directly expressed in Escherichia coli, using cDNA, and was purified with a yield of 32 mg/l of culture. The purified recombinant rat AdR showed an absorption spectrum characteristic of a flavoprotein with peaks at 270, 378 and 450 nm and shoulders at 280, 425 and 474 nm. The extinction coefficient was estimated to be 10.9 mM(-1) cm(-1) at 450 nm. The absorbance ratio at 270 nm/450 nm was 7.1. From the &theta;(208) value in the circular dichroism spectrum, the alpha-helix content in the rat AdR was calculated to be 30%. In NADPH-cytochrome c reductase activity reconstituted with adrenodoxin (Ad), the apparent K(m) value of rat AdR for NADPH was 0.32 microM, a value significantly lower than that of bovine AdR (1.4 microM). The rat AdR showed a higher affinity to the heterologous redox partner (bovine Ad, K(m)=9.3 nM) than to the native partner (rat Ad, K(m)=16.7 nM), whereas the affinity of bovine AdR was slightly higher to the native partner (bovine Ad, K(m)=37.1 nM) than to the heterologous partner (rat Ad, K(m)=46.8 nM). The K(m) values showed a reverse correlation to the difference of pI values between the redox partners. These results indicate that AdR binds to Ad mainly by ionic interaction.
Sagara
Y
Y
Department of Medical Biology, Kochi Medical School, Oko-cho, Nankoku, Kochi, Japan.
Watanabe
Y
Y
Kodama
H
H
Aramaki
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D051381:Rats; D016415:Sequence Alignment
D018076:DNA, Complementary; D005287:Ferredoxin-NADP Reductase