PT  - JOURNAL ARTICLE
AU  - T Suzuki
AU  - S Narimatsu
AU  - S Fujita
AU  - Y Masubuchi
AU  - S Umeda
AU  - S Imaoka
AU  - Y Funae
TI  - Purification and characterization of a cytochrome P-450 isozyme catalyzing bunitrolol 4-hydroxylation in liver microsomes of male rats.
DP  - 1992 May 01
TA  - Drug Metabolism and Disposition
PG  - 367--373
VI  - 20
IP  - 3
4099  - http://dmd.aspetjournals.org/content/20/3/367.short
4100  - http://dmd.aspetjournals.org/content/20/3/367.full
SO  - Drug Metab Dispos1992 May 01; 20
AB  - 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 (omega-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, KM and Vmax values, of P-450 BTL calculated for BTL 4-hydroxylation were 10.7 microM and 19.68 nmol/min/nmol P-450, respectively, whereas those values (mean +/- SE) of rat liver microsomes were 0.84 +/- 0.05 microM 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.