Role of the CYP2D subfamily in metabolism-dependent covalent binding of propranolol to liver microsomal protein in rats.
In vitro covalent binding of a chemically reactive metabolite of propranolol to microsomal macromolecules, which is presumed to cause inhibition of its own metabolism in rats, was diminished in liver microsomes from rats pretreated with propranolol. Covalent binding was suppressed by the addition of an antibody against P450BTL, which is a cytochrome P450 (P450) isozyme belonging to the CYP2D subfamily. SDS-PAGE of microsomal proteins after incubation with [3H]propranolol and NADPH indicated that the binding was non-selective but prominent at the molecular mass of approx. 50 kDa, corresponding to those of the P450 protein. The radioactivity peak was markedly but not completely diminished by the addition of reduced glutathione. In a reconstituted system containing P450BTL, NADPH-cytochrome P450 reductase (fp2) and dilauroylphosphatidylcholine, propranolol 4-, 5- and 7-hydroxylase activities decreased time dependently following preincubation with propranolol in the presence of NADPH, indicating time-dependent inactivation of P450BTL. The covalent binding of a reactive metabolite of [3H]propranolol to the proteins was also observed in this system. SDS-PAGE showed that among the three proteins in the reconstituted system, fp2 and P450BTL consisting of two polypeptides with molecular masses of 49 and 32 kDa, the binding was specific for a polypeptide corresponding to the P450 isozyme with a molecular mass of 49 kDa. In addition, the ratio of the amount of covalently bound radiolabelled materials to that of P450BTL which was estimated from each impaired propranolol hydroxylase activity under the same reconstitutional conditions was calculated to be approx. 1.0. These findings indicate that propranolol is a mechanism-based inactivator of a cytochrome P450 isozyme(s) belonging to the CYP2D subfamily.
Masubuchi
Y
Y
Laboratory of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Narimatsu
S
S
Hosokawa
S
S
Suzuki
T
T
D000595:Amino Acid Sequence; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D011433:Propranolol / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D011506:Proteins; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D011433:Propranolol; D006899:Mixed Function Oxygenases