Purification and characterization of a cytochrome P-450 isozyme catalyzing bunitrolol 4-hydroxylation in liver microsomes of male rats.
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.
Department of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
T
T
Suzuki
S
S
Narimatsu
S
S
Fujita
Y
Y
Masubuchi
S
S
Umeda
S
S
Imaoka
Y
Y
Funae
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002384:Catalysis; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006900:Hydroxylation; D007527:Isoenzymes / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D011412:Propanolamines / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D000906:Antibodies; D007527:Isoenzymes; D011412:Propanolamines; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C071864:bunitrolol 4-hydroxylase