Cloning and characterization of the CYP2D1-binding protein, retinol dehydrogenase.
A CYP2D1-binding protein, 29 k-protein (p29), has been isolated and its N-terminal amino acid sequence has been reported (Ohishi et al. (1993) Biochim. Biophys. Acta 1158, 227-236). In this study, p29 cDNA was isolated by PCR with oligonucleotide probes designed from the N-terminal amino acid sequence and p29 was found to be a microsomal retinol dehydrogenase, a member of the short-chain alcohol dehydrogenase family which metabolize hydroxysteroids and prostaglandins. CYP2D1 and p29 were expressed in Saccharomyces cerevisiae to characterize these proteins. CYP2D1 had an absorption maximum at 448 nm in a CO-reduced form. Expressed p29 in yeast cells was detected with anti-p29 antibody. Solubilized CYP2D1 and p29 from yeast microsomes were mixed and applied to an anti-CYP2D1 antibody-binding column. Both proteins were retained in the column and eluted with glycine buffer (pH 2.8). However, when applied alone, p29 was not retained in the column. The findings indicated that CYP2D1 bound tightly with p29. Catalytic activities of p29 expressed in yeast were investigated. p29 had retinal reductase activity in the presence of NADPH. Addition of CYP2D1 and NADPH-P450 reductase increased the retinal reductase activity of p29. These findings suggest that the complex of CYP2D1, p29, and NADPH-P450 reductase has an important role in the metabolism of retinoids.
Imaoka
S
S
Laboratory of Chemistry, Osaka City University Medical School, Japan.
Wan
J
J
Chow
T
T
Hiroi
T
T
Eyanagi
R
R
Shigematsu
H
H
Funae
Y
Y
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001921:Brain / Q000201:enzymology; D002384:Catalysis; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D018076:DNA, Complementary / Q000737:chemistry; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D012441:Saccharomyces cerevisiae
D018076:DNA, Complementary; D003577:Cytochrome P-450 Enzyme System; D000429:Alcohol Oxidoreductases; C022520:retinol dehydrogenase; D001189:Aryl Hydrocarbon Hydroxylases; C504568:Cyp2d1 protein, rat; D000072467:Cytochrome P450 Family 2; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase