Purification of a phenobarbital-inducible UDP-glucuronosyltransferase isoform from dog liver which catalyzes morphine and testosterone glucuronidation

Kazuta Oguri, Akiko Kurogi, Kou Ichi Yamabe, Mitsuko Tanaka, Kunihiro Yoshisue, Yuji Ishii, Hidetoshi Yoshimura

Research output: Contribution to journalArticle

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Abstract

A morphine UDP-glucuronosyltransferase (UGT) which could belong to the UGT2B subfamily was isolated from liver microsomes of a male beagle dog treated with phenobarbital. Glucuronidation toward morphine in the dog liver microsomes was increased threefold by the treatment. The microsomes were solubilized with Emulgen 911 and applied on a column of hemisuccinate derivative of Sepharose 4B column which has been developed in our laboratory. An isoform of UGT in the eluate was purified further by chromatofocusing and UDP-hexanolamine-affinity chromatography. A purified enzyme, UGT(DOG-PB), was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional electrophoresis and exhibited a subunit molecular weight of 50 kDa. This isoform showed activities toward the 3-hydroxyl group of morphine, 4-hydroxybiphenyl, 4-nitrophenol, 4-methylumbelliferone, and testosterone, but not toward chloramphenicol and the 6-hydroxyl group of morphine. The substrate specificity of UGT(DOG-PB) is similar to that of stably expressed UGT2B1 which is considered a phenobarbital-inducible morphine UGT in the rat except that UGT(DOG-PB) is capable of glucuronidating 4-nitrophenol but not chloramphenicol. The NH2-terminus until the 30th residue of UGT(DOG-PB) is highly homologous to UGT2B subfamily, and the NH2- terminal 15 residues of UGT(DOG-PB) are completely identical to those of UGT2B1, UGT2B8, and UGT2B15. This is the first report describing the UGT isoform of dog and the purification of morphine UGT which may belong to UGT2B subfamily.

Original languageEnglish
Pages (from-to)159-166
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume325
Issue number2
DOIs
Publication statusPublished - Jan 15 1996

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Glucuronosyltransferase
Phenobarbital
Liver
Morphine
Purification
Testosterone
Protein Isoforms
Dogs
Liver Microsomes
Chloramphenicol
Electrophoresis
Hydroxyl Radical
Hymecromone
Nonoxynol
Affinity chromatography
Uridine Diphosphate
Electrophoresis, Gel, Two-Dimensional
Substrate Specificity
Microsomes
Affinity Chromatography

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Purification of a phenobarbital-inducible UDP-glucuronosyltransferase isoform from dog liver which catalyzes morphine and testosterone glucuronidation. / Oguri, Kazuta; Kurogi, Akiko; Yamabe, Kou Ichi; Tanaka, Mitsuko; Yoshisue, Kunihiro; Ishii, Yuji; Yoshimura, Hidetoshi.

In: Archives of Biochemistry and Biophysics, Vol. 325, No. 2, 15.01.1996, p. 159-166.

Research output: Contribution to journalArticle

Oguri, Kazuta ; Kurogi, Akiko ; Yamabe, Kou Ichi ; Tanaka, Mitsuko ; Yoshisue, Kunihiro ; Ishii, Yuji ; Yoshimura, Hidetoshi. / Purification of a phenobarbital-inducible UDP-glucuronosyltransferase isoform from dog liver which catalyzes morphine and testosterone glucuronidation. In: Archives of Biochemistry and Biophysics. 1996 ; Vol. 325, No. 2. pp. 159-166.
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abstract = "A morphine UDP-glucuronosyltransferase (UGT) which could belong to the UGT2B subfamily was isolated from liver microsomes of a male beagle dog treated with phenobarbital. Glucuronidation toward morphine in the dog liver microsomes was increased threefold by the treatment. The microsomes were solubilized with Emulgen 911 and applied on a column of hemisuccinate derivative of Sepharose 4B column which has been developed in our laboratory. An isoform of UGT in the eluate was purified further by chromatofocusing and UDP-hexanolamine-affinity chromatography. A purified enzyme, UGT(DOG-PB), was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional electrophoresis and exhibited a subunit molecular weight of 50 kDa. This isoform showed activities toward the 3-hydroxyl group of morphine, 4-hydroxybiphenyl, 4-nitrophenol, 4-methylumbelliferone, and testosterone, but not toward chloramphenicol and the 6-hydroxyl group of morphine. The substrate specificity of UGT(DOG-PB) is similar to that of stably expressed UGT2B1 which is considered a phenobarbital-inducible morphine UGT in the rat except that UGT(DOG-PB) is capable of glucuronidating 4-nitrophenol but not chloramphenicol. The NH2-terminus until the 30th residue of UGT(DOG-PB) is highly homologous to UGT2B subfamily, and the NH2- terminal 15 residues of UGT(DOG-PB) are completely identical to those of UGT2B1, UGT2B8, and UGT2B15. This is the first report describing the UGT isoform of dog and the purification of morphine UGT which may belong to UGT2B subfamily.",
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