A 27.368 kDa retinal reductase in New Zealand white rabbit liver cytosol encoded by the peroxisomal retinol dehydrogenase-reductase cDNA: purification and characterization of the enzyme.
We obtained a full-length cDNA based on a sequence deposited in GenBank (accession No. AB045133), annotated as rabbit peroxisomal NADP(H)-dependent retinol dehydrogenase-reductase (NDRD). The rabbit NDRD gene, like its mouse and human homologs, harbors 2 initiation sites, one of which theoretically encodes a 29.6 kDa protein with 279 amino acids, and the other encodes a 27.4 kDa protein with 260 amino acids. The purification of a rabbit cytosolic retinol oxidoreductase with a subunit molecular mass of 34 kDa and an N terminus that is not completely identical to that of NDRD, has been reported. An enzyme responsible for the all-trans retinal reductase activity in the liver cytosol of New Zealand white rabbit was purified to homogeneity using differential centrifugation and successive chromatographic analyses. The subunit molecular mass of the purified enzyme, revealed by SDS-PAGE, was approximately 27 kDa. The intact molecular mass, measured by MALDI-TOF mass spectrometry, was 27.368 kDa. The 60 kDa relative mobility observed in size-exclusion chromatography indicates that the native protein probably exists as a dimer. The purified enzyme was positively confirmed to be the product of NDRD by peptide mass fingerprinting, tandem mass spectrometry, and N-terminal sequencing. Taken together, the results suggested that the native protein is truncated at the N terminus.
Du
Kun
K
Center for Molecular Biology, Shantou University Medical College, Shantou, Guangdong 515041, China.
Liu
Ge-Fei
GF
Xie
Jian-Ping
JP
Song
Xu-Hong
XH
Li
Rui
R
Liang
Bin
B
Huang
Dong-Yang
DY
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D020675:Peroxisomes / Q000201:enzymology* / Q000235:genetics; D011817:Rabbits
D018076:DNA, Complementary; D000429:Alcohol Oxidoreductases; C084155:alcohol dehydrogenase (NAD(P)+)