Purification and Characterization of Two Major Forms of Naloxone Reductase from Rabbit Liver Cytosol, New Members of Aldo-Keto Reductase Superfamily

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概要

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• Rabbit liver cytosol produced approximately equal amounts of 6α-naloxol and 6β-naloxol from naloxone in the presence of NADPH at pH 7.4,and contained at least four forms of naloxone reductase. The two major forms, NR1 and NR2,which catalyze the stereospecific reduction of naloxone to 6α-naloxol and 6β-naloxol, respectively, were purified to apparent homogeneity by various chromatographic techniques. Both enzymes are monomeric proteins with similar molecular weights of 35000-36000,but NR1 is a basic protein with an isoelectric point (pI) of 9.3,while NR2 is an acidic protein (pI of 5.9). NR1 and NR2 gave the maximal activities at pH 8.0 and 6.1,respectively. NR1 exhibited considerable activity with NADH as well as with NADPH, whereas NR2 showed highly restricted specificity for NADPH. The K_m and V_<max> values of NR1 and NR2 for naloxone were 1.0 and 0.06 mM, and 76 and 162 munits/mg, respectively. In addition to naloxone, naltrexone and dihydromorphinone served as good substrates for NR2 but were poor substrates for NR1. Both enzymes reduced aliphatic and aromatic aldehydes, cyclic and aromatic ketones, and quinones at higher rates. The two enzymes catalyzed the dehydrogenation of 17β-hydroxysteroids with low K_m values, and NR2 showed an additional 3α-hydroxysteroid dehydrogenase activity. Amino acid sequence data of NR1 (99% of whole protein) and NR2 (66%) showed that both enzymes belong to the aldo-keto reductase (AKR) superfamily and can be classified into the AKR1C subfamily. These findings therefore indicate that they are new members of the AKR superfamily and may be involved physiologically in the steroid metabolism as well as in the detoxification of xenobiotic carbonyl compounds.

• 1999-10-15

著者

• Yamano Shigeru

  Faculty of Pharmaceutical Sciences, Fukuoka University

• Todaka Takashi

  Faculty Of Engineering Oita University

• Yamano S

  Faculty Of Pharmaceutical Sciences Fukuoka University

• Yamano Shigeru

  Faculty Of Pharmaceutical Sciences Fukuoka University

• Todaka Takashi

  Fukuoka Univ. Fukuoka Jpn

• TOKI Satoshi

  Faculty of Pharmaceutical Sciences, Fukuoka University

• ICHINOSE Fumio

  Faculty of Pharmaceutical Sciences, Fukuoka University

• Ichinose Fumio

  Faculty Of Pharmaceutical Sciences Fukuoka University

• Toki Satoshi

  Faculty Of Pharmaceutical Sciences Fukuoka University

• Yamano Shigeru

  Faculty of Pharmaceutical Science, Fukuoka University

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