Purification, characterization and partial primary structure of morphine 6-dehydrogenase from rabbit liver cytosol.
Morphinone, a toxic metabolite, was formed from morphine by NAD(P)-dependent morphine 6-dehydrogenase(s) in both the cytosol and microsomal fractions of the rabbit liver at pH 7.4. The enzyme activity in the cytosol fraction was about twice that in the microsomal fraction and NAD served as the preferred cofactor in both fractions. The enzyme in the cytosol fraction was purified to a homogeneous protein by the use of various chromatographic techniques. The enzyme is a monomeric protein with a molecular weight of 36,000 and an isoelectric point of 6.4. The enzyme had a dual cofactor specificity but NAD was more efficiently utilized than NADP. With NAD, the enzyme showed an optimal pH of 9.4, and the Km and Vmax values toward morphine were 0.72 mM and 0.59 unit/mg protein, respectively. The enzyme also exhibited a significant activity for morphine analogs having an unsaturated bond at C-7,8 (codeine, ethylmorphine, and normorphine), alicyclic alcohols (3-hydroxyhexobarbital, 1-indanol, and cyclohexene-2-ol) and benzenedihydrodiol. In the reverse reaction, the enzyme exhibited highly restricted specificity for o-quinones. Sulfhydryl re-agents and quercetin inhibited the enzyme but pyrazole, barbital, and indomethacin had little effect on the enzyme activity. Androstanes, lithocholic acid, and estradiol potently inhibited the enzyme in a competitive manner toward morphine binding. The partial amino acid sequence of the random peptides obtained by the proteolytic digestion of the enzyme, which comprised about 40% of the whole protein, revealed a significant homology to the corresponding regions in the members of the aldo-keto reductase family. These results therefore indicate that the present enzyme is a new and unique member of the aldo-keto reductase family.
Yamano
S
S
Faculty of Pharmaceutical Sciences, Fukuoka University, Japan.
Ito
K
K
Ogata
S
S
Toki
S
S
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002847:Chromatography, Agarose; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004091:Hydromorphone / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D009020:Morphine / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D013256:Steroids / Q000494:pharmacology; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D013256:Steroids; C036083:morphinone; D009249:NADP; D009020:Morphine; D000429:Alcohol Oxidoreductases; C045221:morphine 6-dehydrogenase; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D004091:Hydromorphone