Selective inactivation by chloramphenicol of the major phenobarbital-inducible isozyme of dog liver cytochrome P-450.
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.
D000818:Animals; D001345:Autoradiography; D002701:Chloramphenicol / Q000494:pharmacology*; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis; D003573:Cytochrome b Group / Q000378:metabolism; D015786:Cytochromes b5; D004285:Dogs; D004586:Electrophoresis; D004790:Enzyme Induction / Q000187:drug effects; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology*; D011506:Proteins / Q000378:metabolism
D065607:Cytochrome P-450 Enzyme Inhibitors; D003573:Cytochrome b Group; D007527:Isoenzymes; D011506:Proteins; D009249:NADP; D002701:Chloramphenicol; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital