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.
|Number of pages||7|
|Journal||Drug Metabolism and Disposition|
|Publication status||Published - 1992|
ASJC Scopus subject areas
- Pharmaceutical Science