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

Kazuo Tajima, Mari Hashizaki, Kenji Yamamoto, Shizuo Narimatsu, Tamio Mizutani

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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 K(m) values of NADH and NADPH for TBE with TBE- R1 were estimated to be 15 and 29 μM, respectively. On the other hand, TBE- R2 specifically utilized NADPH and the K(m) value for TBE was estimated to be 92 μM 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.

Original languageEnglish
Pages (from-to)207-214
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume361
Issue number2
DOIs
Publication statusPublished - Jan 15 1999

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Metabolites
Binding energy
Liver
Cytosol
Purification
Rats
NADP
Enzymes
NAD
p-Chloromercuribenzoic Acid
Ketosteroids
Dicumarol
Quinones
Isoelectric Point
Durapatite
Phenobarbital
Ketones
Aldehydes
Sodium Dodecyl Sulfate
Gel Chromatography

Keywords

  • Cytosol
  • Inhibitors
  • Metabolism
  • Purification
  • Rat liver
  • Reductases
  • Stereoselectivity
  • Steroids
  • Substrate specificity

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

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. / Tajima, Kazuo; Hashizaki, Mari; Yamamoto, Kenji; Narimatsu, Shizuo; Mizutani, Tamio.

In: Archives of Biochemistry and Biophysics, Vol. 361, No. 2, 15.01.1999, p. 207-214.

Research output: Contribution to journalArticle

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abstract = "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 K(m) values of NADH and NADPH for TBE with TBE- R1 were estimated to be 15 and 29 μM, respectively. On the other hand, TBE- R2 specifically utilized NADPH and the K(m) value for TBE was estimated to be 92 μM 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.",
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T1 - 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

AU - Tajima, Kazuo

AU - Hashizaki, Mari

AU - Yamamoto, Kenji

AU - Narimatsu, Shizuo

AU - Mizutani, Tamio

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N2 - 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 K(m) values of NADH and NADPH for TBE with TBE- R1 were estimated to be 15 and 29 μM, respectively. On the other hand, TBE- R2 specifically utilized NADPH and the K(m) value for TBE was estimated to be 92 μM 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.

AB - 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 K(m) values of NADH and NADPH for TBE with TBE- R1 were estimated to be 15 and 29 μM, respectively. On the other hand, TBE- R2 specifically utilized NADPH and the K(m) value for TBE was estimated to be 92 μM 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.

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KW - Inhibitors

KW - Metabolism

KW - Purification

KW - Rat liver

KW - Reductases

KW - Stereoselectivity

KW - Steroids

KW - Substrate specificity

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