Purification and characterization of a cytochrome P-450 isozyme catalyzing bunitrolol 4-hydroxylation in liver microsomes of male rats

T. Suzuki, S. Narimatsu, S. Fujita, Y. Masubuchi, S. Umeda, S. Imaoka, Y. Funae

Research output: Contribution to journalArticle

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Abstract

A cytochrome P-450 isozyme, P-450 bunitrolol (BTL), catalyzing bunitrolol 4-hydroxylation was partially purified from liver microsomes of adult male Sprague-Dawley rats by hydrophobic affinity chromatographic (ω-aminooctyl- Sepharose 4B) and high-performance liquid chromatographic (anion-exchange diethylaminoethyl-5PW) techniques. The specific content of the final preparation was 5.02 nmol/mg protein, which was 7.8-fold that of microsomes. It showed two protein bands of 49 and 32 kDa in sodium dodecylsulfate- polyacrylamide gel electrophoresis. N-Terminal 20 amino acid sequence of the protein of a higher molecular mass (49 kDa) isolated by an electroblotting technique is 94% homologous with that of CYP2D2. In a reconstituted system including NADPH-cytochrome P-450 reductase and an NADPH-generating system, the final preparation had the highest activity toward BTL and debrisoquine 4- hydroxylation among 12 isozymes of cytochrome P-450 examined. Kinetic parameters, K(M) and V(max) values, of P-450 BTL calculated for BTL 4- hydroxylation were 10.7 μM and 19.68 nmol/min/nmol P-450, respectively, whereas those values (mean ± SE) of rat liver microsomes were 0.84 ± 0.05 μM and 2.05 ± 0.11 nmol/min/nmol P-450. When preincubated with rat liver microsomes, the antibody against the final P-450 BTL preparation suppressed bunitrolol and debrisoquine 4-hydroxylase activities dose-dependently and almost completely. These results suggest that cytochrome P-450 BTL and its immunochemically related P-450 isozyme(s) play a major role in debrisoquine 4-hydroxylation as well as in BTL 4-hydroxylation in rat liver microsomes.

Original languageEnglish
Pages (from-to)367-373
Number of pages7
JournalDrug Metabolism and Disposition
Volume20
Issue number3
Publication statusPublished - 1992
Externally publishedYes

Fingerprint

Hydroxylation
Liver Microsomes
Liver
Cytochrome P-450 Enzyme System
Isoenzymes
Purification
Rats
Debrisoquin
NADPH-Ferrihemoprotein Reductase
Cytochrome P-450 CYP2D6
Proteins
bunitrolol
Microsomes
NADP
Molecular mass
Sepharose
Electrophoresis
Anions
Sprague Dawley Rats
Kinetic parameters

ASJC Scopus subject areas

  • Pharmacology
  • Toxicology

Cite this

Suzuki, T., Narimatsu, S., Fujita, S., Masubuchi, Y., Umeda, S., Imaoka, S., & Funae, Y. (1992). Purification and characterization of a cytochrome P-450 isozyme catalyzing bunitrolol 4-hydroxylation in liver microsomes of male rats. Drug Metabolism and Disposition, 20(3), 367-373.

Purification and characterization of a cytochrome P-450 isozyme catalyzing bunitrolol 4-hydroxylation in liver microsomes of male rats. / Suzuki, T.; Narimatsu, S.; Fujita, S.; Masubuchi, Y.; Umeda, S.; Imaoka, S.; Funae, Y.

In: Drug Metabolism and Disposition, Vol. 20, No. 3, 1992, p. 367-373.

Research output: Contribution to journalArticle

Suzuki, T, Narimatsu, S, Fujita, S, Masubuchi, Y, Umeda, S, Imaoka, S & Funae, Y 1992, 'Purification and characterization of a cytochrome P-450 isozyme catalyzing bunitrolol 4-hydroxylation in liver microsomes of male rats', Drug Metabolism and Disposition, vol. 20, no. 3, pp. 367-373.
Suzuki, T. ; Narimatsu, S. ; Fujita, S. ; Masubuchi, Y. ; Umeda, S. ; Imaoka, S. ; Funae, Y. / Purification and characterization of a cytochrome P-450 isozyme catalyzing bunitrolol 4-hydroxylation in liver microsomes of male rats. In: Drug Metabolism and Disposition. 1992 ; Vol. 20, No. 3. pp. 367-373.
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abstract = "A cytochrome P-450 isozyme, P-450 bunitrolol (BTL), catalyzing bunitrolol 4-hydroxylation was partially purified from liver microsomes of adult male Sprague-Dawley rats by hydrophobic affinity chromatographic (ω-aminooctyl- Sepharose 4B) and high-performance liquid chromatographic (anion-exchange diethylaminoethyl-5PW) techniques. The specific content of the final preparation was 5.02 nmol/mg protein, which was 7.8-fold that of microsomes. It showed two protein bands of 49 and 32 kDa in sodium dodecylsulfate- polyacrylamide gel electrophoresis. N-Terminal 20 amino acid sequence of the protein of a higher molecular mass (49 kDa) isolated by an electroblotting technique is 94{\%} homologous with that of CYP2D2. In a reconstituted system including NADPH-cytochrome P-450 reductase and an NADPH-generating system, the final preparation had the highest activity toward BTL and debrisoquine 4- hydroxylation among 12 isozymes of cytochrome P-450 examined. Kinetic parameters, K(M) and V(max) values, of P-450 BTL calculated for BTL 4- hydroxylation were 10.7 μM and 19.68 nmol/min/nmol P-450, respectively, whereas those values (mean ± SE) of rat liver microsomes were 0.84 ± 0.05 μM and 2.05 ± 0.11 nmol/min/nmol P-450. When preincubated with rat liver microsomes, the antibody against the final P-450 BTL preparation suppressed bunitrolol and debrisoquine 4-hydroxylase activities dose-dependently and almost completely. These results suggest that cytochrome P-450 BTL and its immunochemically related P-450 isozyme(s) play a major role in debrisoquine 4-hydroxylation as well as in BTL 4-hydroxylation in rat liver microsomes.",
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AU - Narimatsu, S.

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AU - Funae, Y.

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