Selective inactivation by chloramphenicol of the major phenobarbital-inducible isozyme of dog liver cytochrome P-450

Abstract

Chloramphenicol (CAP) is a potent and effective mechanism-based inactivator of the major phenobarbital (PB)-inducible isozyme of dog liver cytochrome P-450 (PBD-2) in vitro. In a reconstituted system containing PBD-2, CAP causes a time- and NADPH-dependent irreversible loss of 7-ethoxycoumarin deethylase activity, with no loss of spectrally detectable cytochrome P-450. Inactivation is enhanced by cytochrome b5, and, in the presence of cytochrome b5, the concentration of CAP at which the rate constant for inactivation is half-maximal (Kl) and the maximal rate constant for inactivation (Kinact) are 5 microM and 1.2 min-1, respectively. CAP binds covalently to PBD-2 with a stoichiometry of 1 nmol of [14C]CAP bound/nmol of cytochrome P-450 inactivated. In addition, CAP is a selective inactivator of PBD-2. In intact liver microsomes from PB-treated dogs, CAP irreversibly inhibits androstenedione 16 alpha and 16 beta, but not 6 beta hydroxylation. Covalent binding of [14C]CAP to dog liver microsomes in vitro is increased 5.5 times by PB induction. This increase correlates well with the increased levels of immunochemically determined PBD-2 (5.8-fold) and 16 alpha and 16 beta hydroxylation of androstenedione (5.7- and 5.8-fold) in microsomes from PB-treated compared to control animals. Anti-PBD-2 IgG specifically inhibits by greater than 80% the covalent binding of [14C]CAP to microsomes from control and PB-treated dogs. Finally, in liver microsomes from PB-treated and control dogs, CAP appears to bind covalently to a single protein with the same molecular weight as PBD-2 as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography.