Complementary DNA cloning and characterization of cytochrome P450 2D29 from Japanese monkey liver

Hiroyuki Hichiya, Chie Takemi, Daisuke Tsuzuki, Shigeo Yamamoto, Kazuo Asaoka, Satoshi Suzuki, Tetsuo Satoh, Sumio Shinoda, Hiroyuki Kataoka, Shizuo Narimatsu

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

A cDNA was cloned from Japanese monkey liver mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) using oligonucleotide primers based on the marmoset cytochrome P450 2D19 (CYP2D19) nucleotide sequence. The full-length cDNA encoded a 497 amino acid protein (designated CYP2D29) that is 96, 91, and 88% homologous to human CYP2D6, cynomolgus monkey CYP2D17, and marmoset monkey CYP2D19, respectively. Yeast cells (Saccharomyces cerevisiae AH-22 strain) transfected with pGYR1 vectors containing the CYP2D29 cDNA were cultured, and microsomal fractions were obtained. Reduced carbon monoxide-difference spectra and western blot analysis using polyclonal antibodies raised against rat CYP2D2 demonstrated that in yeast cell microsomal fractions, the level of CYP2D29 holoenzyme was similar to that of CYP2D6 holoenzyme. However, western blot analysis indicated that the level of CYP2D29 in Japanese monkey liver microsomes might be much higher than that of CYP2D6 in human liver microsomes. Japanese monkey liver microsomes exhibited much higher activities than did human liver microsomes, expressed as nmol/min/mg protein, for debrisoquine (DB) 4-hydroxylation and bufuralol (BF) 1″-hydroxylation (typical reactions catalyzed by CYP2D6), whereas recombinant CYP2D29 activity, expressed as nmol/min/nmol CYP, was similar to that of CYP2D6 for DB and BF hydroxylation. In kinetic analyses, the Km value of CYP2D29 for DB 4-hydroxylation was much lower than that of Japanese monkey liver microsomes, whereas the Km value of CYP2D6 for DB 4-hydroxylation was similar to that of human liver microsomes. In contrast, Km values for BF 1″-hydroxylation were similar for Japanese monkey and human liver microsomes and yeast cell microsomal fractions expressing recombinant CYP2D29 or CYP2D6. These results suggest that the properties of Japanese monkey CYP2D29 are similar to those of human CYP2D6, but their populations and/or some other factors in liver microsomes may cause the difference in microsomal DB 4-hydroxylase activities between Japanese monkeys and humans.

Original languageEnglish
Pages (from-to)1101-1110
Number of pages10
JournalBiochemical Pharmacology
Volume64
Issue number7
DOIs
Publication statusPublished - Oct 1 2002

Fingerprint

Cytochrome P-450 CYP2D6
Cloning
Liver Microsomes
Liver
Organism Cloning
Macaca
Hydroxylation
Complementary DNA
Debrisoquin
Yeast
Callithrix
Holoenzymes
Yeasts
Cells
Cytochrome P-450 Enzyme System
Western Blotting
Japanese monkey cytochrome P450 2D29
DNA Primers
Macaca fascicularis
Polymerase chain reaction

Keywords

  • Bufuralol
  • CYP2D29
  • CYP2D6
  • Debrisoquine
  • Japanese monkey
  • Saccharomyces cerevisiae

ASJC Scopus subject areas

  • Pharmacology

Cite this

Hichiya, H., Takemi, C., Tsuzuki, D., Yamamoto, S., Asaoka, K., Suzuki, S., ... Narimatsu, S. (2002). Complementary DNA cloning and characterization of cytochrome P450 2D29 from Japanese monkey liver. Biochemical Pharmacology, 64(7), 1101-1110. https://doi.org/10.1016/S0006-2952(02)01287-X

Complementary DNA cloning and characterization of cytochrome P450 2D29 from Japanese monkey liver. / Hichiya, Hiroyuki; Takemi, Chie; Tsuzuki, Daisuke; Yamamoto, Shigeo; Asaoka, Kazuo; Suzuki, Satoshi; Satoh, Tetsuo; Shinoda, Sumio; Kataoka, Hiroyuki; Narimatsu, Shizuo.

In: Biochemical Pharmacology, Vol. 64, No. 7, 01.10.2002, p. 1101-1110.

Research output: Contribution to journalArticle

Hichiya, H, Takemi, C, Tsuzuki, D, Yamamoto, S, Asaoka, K, Suzuki, S, Satoh, T, Shinoda, S, Kataoka, H & Narimatsu, S 2002, 'Complementary DNA cloning and characterization of cytochrome P450 2D29 from Japanese monkey liver', Biochemical Pharmacology, vol. 64, no. 7, pp. 1101-1110. https://doi.org/10.1016/S0006-2952(02)01287-X
Hichiya, Hiroyuki ; Takemi, Chie ; Tsuzuki, Daisuke ; Yamamoto, Shigeo ; Asaoka, Kazuo ; Suzuki, Satoshi ; Satoh, Tetsuo ; Shinoda, Sumio ; Kataoka, Hiroyuki ; Narimatsu, Shizuo. / Complementary DNA cloning and characterization of cytochrome P450 2D29 from Japanese monkey liver. In: Biochemical Pharmacology. 2002 ; Vol. 64, No. 7. pp. 1101-1110.
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AU - Asaoka, Kazuo

AU - Suzuki, Satoshi

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N2 - A cDNA was cloned from Japanese monkey liver mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) using oligonucleotide primers based on the marmoset cytochrome P450 2D19 (CYP2D19) nucleotide sequence. The full-length cDNA encoded a 497 amino acid protein (designated CYP2D29) that is 96, 91, and 88% homologous to human CYP2D6, cynomolgus monkey CYP2D17, and marmoset monkey CYP2D19, respectively. Yeast cells (Saccharomyces cerevisiae AH-22 strain) transfected with pGYR1 vectors containing the CYP2D29 cDNA were cultured, and microsomal fractions were obtained. Reduced carbon monoxide-difference spectra and western blot analysis using polyclonal antibodies raised against rat CYP2D2 demonstrated that in yeast cell microsomal fractions, the level of CYP2D29 holoenzyme was similar to that of CYP2D6 holoenzyme. However, western blot analysis indicated that the level of CYP2D29 in Japanese monkey liver microsomes might be much higher than that of CYP2D6 in human liver microsomes. Japanese monkey liver microsomes exhibited much higher activities than did human liver microsomes, expressed as nmol/min/mg protein, for debrisoquine (DB) 4-hydroxylation and bufuralol (BF) 1″-hydroxylation (typical reactions catalyzed by CYP2D6), whereas recombinant CYP2D29 activity, expressed as nmol/min/nmol CYP, was similar to that of CYP2D6 for DB and BF hydroxylation. In kinetic analyses, the Km value of CYP2D29 for DB 4-hydroxylation was much lower than that of Japanese monkey liver microsomes, whereas the Km value of CYP2D6 for DB 4-hydroxylation was similar to that of human liver microsomes. In contrast, Km values for BF 1″-hydroxylation were similar for Japanese monkey and human liver microsomes and yeast cell microsomal fractions expressing recombinant CYP2D29 or CYP2D6. These results suggest that the properties of Japanese monkey CYP2D29 are similar to those of human CYP2D6, but their populations and/or some other factors in liver microsomes may cause the difference in microsomal DB 4-hydroxylase activities between Japanese monkeys and humans.

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