Purification and characterization of rat liver naloxone reductase that is identical to 3alpha-hydroxysteroid dehydrogenase.
1. Rat liver cytosol produced exclusively 6beta-naloxol from naloxone in the presence of either NADPH or NADH at pH 7.4. The amount of 6beta-naloxol formed with NADPH was about four times that with NADH. The enzyme responsible for this reaction, termed naloxone reductase, was purified to a homogeneous protein by various chromatographic techniques. 2. The purified enzyme is a monomeric protein with a molecular weight of 34000 and an isoelectric point of 5.9, and it has a dual co-factor specificity for NADPH and NADH. The enzyme catalysed the reduction of various carbonyl compounds as well as naloxone analogues, and the dehydrogenation of 3alpha-hydroxysteroids and alicyclic alcohols. Indomethacin, quercetin and sulphhydryl reagents potently inhibited the enzyme, but pyrazole and barbital had no effect on the enzyme activity. 3. Identity of naloxone reductase and 3alpha-hydroxysteroid dehydrogenase in rat liver was demonstrated by comparing the elution profiles of the two enzyme activities during purification, the ratios of the two enzyme activities at each purification steps, and thermal stability and susceptibility to inhibitors for the two enzyme activities. 4. Amino acid sequences of five peptides obtained by proteolytic digestion of the purified enzyme were completely identical to the corresponding regions of previously reported 3alpha-hydroxysteroid dehydrogenase.
Yamano
S
S
Faculty of Pharmaceutical Sciences, Fukuoka University, Japan. pp031903@Psat.fukuoka-u.ac.jp
Nakamoto
N
N
Toki
S
S
D015096:3-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D007213:Indomethacin / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009270:Naloxone / Q000378:metabolism; D011794:Quercetin / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D020539:Sequence Analysis, Protein; D020245:p-Chloromercuribenzoic Acid / Q000494:pharmacology
D004791:Enzyme Inhibitors; D009243:NAD; D009270:Naloxone; D009249:NADP; D020245:p-Chloromercuribenzoic Acid; D011794:Quercetin; D015096:3-Hydroxysteroid Dehydrogenases; D000429:Alcohol Oxidoreductases; C045221:morphine 6-dehydrogenase; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D005033:Ethylmaleimide; D007213:Indomethacin