Purification, Characterization And Full Length CDNA Cloning Of A Cytosolic NADPH-dependent Retinal Reductase From Rabbit Liver

Retinoids exert a variety of effects in diverse physiological functions in mammals. Disturbance of retinoids homeostasis could lead to diseases. The intracellular levels of retinoids are tightly controlled. The mechanisms underlying the regulation of retinoid metabolism by relevant enzymes, however, are not completely established.We focused on the cDNA that deposited in GenBank (accession number: AB045133) and annotated as peroxisomal NADPH-dependent retinol dehydrogenase/reductase. Huang (DY Huang and Y Ichikawa. Biochim Biophys Acta, Mar 1997; 1338(1): 47-59) reported the purification of a cytosolic retinol oxidoreductase, of which the subunit molecular weight is 34 kDa and the N-terminus are not identical to that of the AB045133, indicating that this enzyme is either the modified consequence of AB045133 or encoded by other gene. Additionally, the mouse and human orthologs of rabbit AB045133 possess two initiation sites and the native one remains controversial.In this study, we cloned the full-length cDNA of rabbit NRDR and demonstrated it harbored two initiation sites too. Furthermore, an enzyme capable of catalyzing the reduction of all-trans retinal to all-trans retinol was purified to homogeneity from New Zealand rabbit livers. In brief, subcellular fractions were prepared from rabbit liver homogenates by differential centrifugation and then the protein of interest was isolated from cytosolic fraction by successive chromatographies, including ion-exchange chromatography, hydrophobic interaction chromatography and size-exclusive chromatography. The subunit molecular weight of the purified protein, determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), was approximately 28kDa. The intact molecular weight was determined to be 27.368 kDa by MALDI-TOF mass spectrometry. Whereas analysis by size-exclusion chromatography showed the molecular weight was 63 kDa, indicating the native protein existed as a dimer. Using peptide mass fingerprint (PMF)...