Purfication and characterization of two forms of 2, 3, 4, 7, 8-pentachlorodibenzofuran-inducible cytochrome P-450 in hamster liver

Nobuynki Koga, Noritaka Ariyoshi, Hiroshi Nakashima, Hidetoshi Yoshimura

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Two forms of cytochrome P-450 (P-450) from liver microsomes of hamsters treated with 2,3,4,7,8-pentachlorodibenzofuran (PenCDF), which possesses the potent acute toxicity and 3-methylcholanthrene (MC)-type inducing ability of liver microsomal monooxygenases in animals, were purified and characterized. These P-450 forms, designated as hamster P-450H and hamster P-450L, had the molecular masses of 52 and 50 kDa, respectively, and showed the absorption maximum of CO-reduced difference spectra at 446 nm. The absolute spectra of their oxidized forms indicated that hamster P-450H was in high-spin state and hamster P-450L was in low-spin state. A part of PenCDF injected into hamster was tightly bound to purified hamster P-450H at a ratio of 0. 107 nmol PenCDF/nmol P-450. In a reconstituted system, both hamster P-450H and hamster P-450L showed relatively low catalytic activities for 3-hydroxylation of benzo[α]pyrene and O-deethylations of both 7-ethoxyresorufin and 7-ethoxycoumarin, while they both catalyzed lα- and 2α-hydroxylations of testosterone effectively to a similar extent. Addition of cytochrome b5 to a reconstituted system accelerated the formation of 7α-hydroxytestosterone 5. 3-fold with hamster P-450L and 2. 2-fold with hamster P-450H. In addition, hamster P-450H catalyzed estradiol 2-hydroxylation at a high rate but hamster P-450L did not. Immunochemical studies using antiserum to each P-450 form revealed that hamster P-450H and hamster P-450L differ from each other and comprise about 61 and 31% of the total P-450 in PenCDF-treated microsomes, respectively, indicating that these are PenCDF-inducible and major forms of P-450 in PenCDF-treated hamsters. Similarly to PenCDF, inducers such as MC, 3, 4, 5, 3', 4' -pentachlorobiphenyl, and isosafrole also preferentially induced hamster P-450H rather than hamster P-450L, but β-naphthoflavone preferentially increased hamster P-450L. Phenobarbital, pregnenolone 16α-carbonitrile and ethanol did not affect the contents of these forms at all. Analyses of NH2-terminal amino acid sequences demonstrated that hamster P-450H and hamster P-450L correspond to rat P-450d and rat P-450a, respectively.

Original languageEnglish
Pages (from-to)826-833
Number of pages8
JournalJournal of Biochemistry
Volume107
Issue number6
Publication statusPublished - Jun 1990
Externally publishedYes

Fingerprint

Hydroxylation
Cricetinae
Liver
Cytochrome P-450 Enzyme System
Rats
Pyrene
Molecular mass
3,4,5,3',4'-pentachlorobiphenyl
Methylcholanthrene
Toxicity
Amino acids
Catalyst activity
Animals
Fold
Ethanol
Proteins
Estradiol
Aryl Hydrocarbon Hydroxylases
Cytochromes b5
Amino Acid Sequence

ASJC Scopus subject areas

  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Physiology (medical)
  • Radiology Nuclear Medicine and imaging
  • Molecular Biology
  • Biochemistry

Cite this

Purfication and characterization of two forms of 2, 3, 4, 7, 8-pentachlorodibenzofuran-inducible cytochrome P-450 in hamster liver. / Koga, Nobuynki; Ariyoshi, Noritaka; Nakashima, Hiroshi; Yoshimura, Hidetoshi.

In: Journal of Biochemistry, Vol. 107, No. 6, 06.1990, p. 826-833.

Research output: Contribution to journalArticle

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abstract = "Two forms of cytochrome P-450 (P-450) from liver microsomes of hamsters treated with 2,3,4,7,8-pentachlorodibenzofuran (PenCDF), which possesses the potent acute toxicity and 3-methylcholanthrene (MC)-type inducing ability of liver microsomal monooxygenases in animals, were purified and characterized. These P-450 forms, designated as hamster P-450H and hamster P-450L, had the molecular masses of 52 and 50 kDa, respectively, and showed the absorption maximum of CO-reduced difference spectra at 446 nm. The absolute spectra of their oxidized forms indicated that hamster P-450H was in high-spin state and hamster P-450L was in low-spin state. A part of PenCDF injected into hamster was tightly bound to purified hamster P-450H at a ratio of 0. 107 nmol PenCDF/nmol P-450. In a reconstituted system, both hamster P-450H and hamster P-450L showed relatively low catalytic activities for 3-hydroxylation of benzo[α]pyrene and O-deethylations of both 7-ethoxyresorufin and 7-ethoxycoumarin, while they both catalyzed lα- and 2α-hydroxylations of testosterone effectively to a similar extent. Addition of cytochrome b5 to a reconstituted system accelerated the formation of 7α-hydroxytestosterone 5. 3-fold with hamster P-450L and 2. 2-fold with hamster P-450H. In addition, hamster P-450H catalyzed estradiol 2-hydroxylation at a high rate but hamster P-450L did not. Immunochemical studies using antiserum to each P-450 form revealed that hamster P-450H and hamster P-450L differ from each other and comprise about 61 and 31{\%} of the total P-450 in PenCDF-treated microsomes, respectively, indicating that these are PenCDF-inducible and major forms of P-450 in PenCDF-treated hamsters. Similarly to PenCDF, inducers such as MC, 3, 4, 5, 3', 4' -pentachlorobiphenyl, and isosafrole also preferentially induced hamster P-450H rather than hamster P-450L, but β-naphthoflavone preferentially increased hamster P-450L. Phenobarbital, pregnenolone 16α-carbonitrile and ethanol did not affect the contents of these forms at all. Analyses of NH2-terminal amino acid sequences demonstrated that hamster P-450H and hamster P-450L correspond to rat P-450d and rat P-450a, respectively.",
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T1 - Purfication and characterization of two forms of 2, 3, 4, 7, 8-pentachlorodibenzofuran-inducible cytochrome P-450 in hamster liver

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N2 - Two forms of cytochrome P-450 (P-450) from liver microsomes of hamsters treated with 2,3,4,7,8-pentachlorodibenzofuran (PenCDF), which possesses the potent acute toxicity and 3-methylcholanthrene (MC)-type inducing ability of liver microsomal monooxygenases in animals, were purified and characterized. These P-450 forms, designated as hamster P-450H and hamster P-450L, had the molecular masses of 52 and 50 kDa, respectively, and showed the absorption maximum of CO-reduced difference spectra at 446 nm. The absolute spectra of their oxidized forms indicated that hamster P-450H was in high-spin state and hamster P-450L was in low-spin state. A part of PenCDF injected into hamster was tightly bound to purified hamster P-450H at a ratio of 0. 107 nmol PenCDF/nmol P-450. In a reconstituted system, both hamster P-450H and hamster P-450L showed relatively low catalytic activities for 3-hydroxylation of benzo[α]pyrene and O-deethylations of both 7-ethoxyresorufin and 7-ethoxycoumarin, while they both catalyzed lα- and 2α-hydroxylations of testosterone effectively to a similar extent. Addition of cytochrome b5 to a reconstituted system accelerated the formation of 7α-hydroxytestosterone 5. 3-fold with hamster P-450L and 2. 2-fold with hamster P-450H. In addition, hamster P-450H catalyzed estradiol 2-hydroxylation at a high rate but hamster P-450L did not. Immunochemical studies using antiserum to each P-450 form revealed that hamster P-450H and hamster P-450L differ from each other and comprise about 61 and 31% of the total P-450 in PenCDF-treated microsomes, respectively, indicating that these are PenCDF-inducible and major forms of P-450 in PenCDF-treated hamsters. Similarly to PenCDF, inducers such as MC, 3, 4, 5, 3', 4' -pentachlorobiphenyl, and isosafrole also preferentially induced hamster P-450H rather than hamster P-450L, but β-naphthoflavone preferentially increased hamster P-450L. Phenobarbital, pregnenolone 16α-carbonitrile and ethanol did not affect the contents of these forms at all. Analyses of NH2-terminal amino acid sequences demonstrated that hamster P-450H and hamster P-450L correspond to rat P-450d and rat P-450a, respectively.

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