Purification and Some Properties of Two Enzymes from Rat Liver Cytosol That Catalyze Carbonyl Reduction of 6-tert-Butyl-2,3-epoxy-5-cyclohexene-1,4-dione, a Metabolite of 3-tert-Butyl-4-hydroxyanisole
| Autor: | Tamio Mizutani, Mari Hashizaki, Kazuo Tajima, Shizuo Narimatsu, Kenji Yamamoto |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Male
Metabolite Aldo-Keto Reductases Biophysics Butylated Hydroxyanisole Biochemistry Catalysis Cofactor Substrate Specificity chemistry.chemical_compound Cytosol Aldehyde Reductase Benzoquinones Animals Isoelectric Point Enzyme Inhibitors Rats Wistar Molecular Biology chemistry.chemical_classification Gel electrophoresis Chromatography biology Carbonyl reduction Stereoisomerism Hydrogen-Ion Concentration Rats Enzyme Activation Alcohol Oxidoreductases Isoelectric point Enzyme Liver chemistry Sephadex biology.protein NAD+ kinase Oxidation-Reduction |
| Zdroj: | Archives of Biochemistry and Biophysics. 361:207-214 |
| ISSN: | 0003-9861 |
| Popis: | 6-tert-Butyl-2,3-epoxy-5-cyclohexene-1,4-dione (TBE), a metabolite of 3-tert-butyl-4-hydroxyanisole, was converted to 6-tert-butyl-2, 3-epoxy-4(R)-hydroxy-5-cyclohexen-1-one ((4R)-TBEH) and 6-tert-butyl-2,3-epoxy-4(S)-hydroxy-5-cyclohexen-1-one ((4S)-TBEH) by TBE-reducing enzymes in rat liver cytosol. Two TBE-reducing enzymes (TBE-R1 and TBE-R2) were purified 18- and 117-fold, respectively, to apparent homogeneity from rat liver cytosol using DEAE-Sephacel, Blue Sepharose CL-6B, hydroxylapatite, and Sephadex G-100 column chromatography. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both enzymes were monomeric. The purified TBE-R1 and TBE-R2 had molecular weights of 37 and 35 kDa and isoelectric points of 6.5 and 5.8, respectively. Both enzymes had an optimum pH of about 5.5 with TBE as substrate. TBE-R1 utilized NADH or NADPH equally as cofactor, and the Km values of NADH and NADPH for TBE with TBE-R1 were estimated to be 15 and 29 microM, respectively. On the other hand, TBE-R2 specifically utilized NADPH and the Km value for TBE was estimated to be 92 microM in the presence of NADPH. Both enzymes reduced aromatic aldehydes, ketones, and quinones at higher rates. In addition, TBE-R2 reduced and oxidized 3-ketosteroids at a higher rate in the presence of NAD(H) and/or NADP(H). Both enzyme activities were inhibited by quercitrin or p-chloromercuribenzoic acid, but little inhibition was observed with phenobarbital or pyrazole. Dicoumarol inhibited significantly TBE-R1 activity but not TBE-R2 activity. In the conversion of TBE to TBEH, TBE-R1 preferentially reduced TBE to (4R)-TBEH, whereas TBE-R2 preferred the reduction of TBE to (4S)-TBEH. |
| Databáze: | OpenAIRE |
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