CLONING AND CHARACTERIZATION OF THE CYP2D1-BINDING PROTEIN, RETINOL DEHYDROGENASE

A CYP2D1-binding protein, 29 k-protein (p29), has been isolated and it s N-terminal amino acid sequence has been reported (Ohishi ct al. (199 3) Biochim. Biophys, Acta 1158, 227-236). In this study, p29 cDNA was isolated by PCR with oligonucleotide probes designed from the N-termin al amino acid sequence and p29 was found to be a microsomal retinol de hydrogenase, a member of the short-chain alcohol dehydrogenase family which metabolize hydroxysteroids and prostaglandins. CYP2D1 and p29 we re expressed in Saccharomyces cerevisiae to characterize these protein s. CYP2D1 had an absorption maximum at 448 nm in a GO-reduced form. Ex pressed p29 in yeast cells was detected with anti-p29 antibody. Solubi lized GYP2D1 and p29 from yeast microsomes were mixed and applied to a n anti-GYP2D1 antibody-binding column. Both proteins were retained in the column and eluted with glycine buffer (pH 2.8). However, when appl ied alone, p29 was not retained in the column. The findings indicated that GYP2D1 bound tightly with p29. Catalytic activities of p29 expres sed in yeast were investigated. p29 had retinal reductase activity in the presence of NADPH. Addition of GYP2D1 and NADPH-P450 reductase inc reased the retinal reductase activity of p29. These findings suggest t hat the complex of CYP2D1, p29, and NADPH-P450 reductase has an import ant role in the metabolism of retinoids. (C) 1998 Academic Press.