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Selective inactivation by chloramphenicol of the major phenobarbital-inducible isozyme of dog liver cytochrome P-450. 1987

P J Ciaccio, and D B Duignan, and J R Halpert
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721.

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.

UI MeSH Term Description Entries
D007527 Isoenzymes Structurally related forms of an enzyme. Each isoenzyme has the same mechanism and classification, but differs in its chemical, physical, or immunological characteristics. Alloenzyme,Allozyme,Isoenzyme,Isozyme,Isozymes,Alloenzymes,Allozymes
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008099 Liver A large lobed glandular organ in the abdomen of vertebrates that is responsible for detoxification, metabolism, synthesis and storage of various substances. Livers
D008297 Male Males
D008862 Microsomes, Liver Closed vesicles of fragmented endoplasmic reticulum created when liver cells or tissue are disrupted by homogenization. They may be smooth or rough. Liver Microsomes,Liver Microsome,Microsome, Liver
D009249 NADP Nicotinamide adenine dinucleotide phosphate. A coenzyme composed of ribosylnicotinamide 5'-phosphate (NMN) coupled by pyrophosphate linkage to the 5'-phosphate adenosine 2',5'-bisphosphate. It serves as an electron carrier in a number of reactions, being alternately oxidized (NADP+) and reduced (NADPH). (Dorland, 27th ed) Coenzyme II,Nicotinamide-Adenine Dinucleotide Phosphate,Triphosphopyridine Nucleotide,NADPH,Dinucleotide Phosphate, Nicotinamide-Adenine,Nicotinamide Adenine Dinucleotide Phosphate,Nucleotide, Triphosphopyridine,Phosphate, Nicotinamide-Adenine Dinucleotide
D010634 Phenobarbital A barbituric acid derivative that acts as a nonselective central nervous system depressant. It potentiates GAMMA-AMINOBUTYRIC ACID action on GABA-A RECEPTORS, and modulates chloride currents through receptor channels. It also inhibits glutamate induced depolarizations. Phenemal,Phenobarbitone,Phenylbarbital,Gardenal,Hysteps,Luminal,Phenobarbital Sodium,Phenobarbital, Monosodium Salt,Phenylethylbarbituric Acid,Acid, Phenylethylbarbituric,Monosodium Salt Phenobarbital,Sodium, Phenobarbital
D011506 Proteins Linear POLYPEPTIDES that are synthesized on RIBOSOMES and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of AMINO ACIDS determines the shape the polypeptide will take, during PROTEIN FOLDING, and the function of the protein. Gene Products, Protein,Gene Proteins,Protein,Protein Gene Products,Proteins, Gene
D002701 Chloramphenicol An antibiotic first isolated from cultures of Streptomyces venequelae in 1947 but now produced synthetically. It has a relatively simple structure and was the first broad-spectrum antibiotic to be discovered. It acts by interfering with bacterial protein synthesis and is mainly bacteriostatic. (From Martindale, The Extra Pharmacopoeia, 29th ed, p106) Cloranfenicol,Kloramfenikol,Levomycetin,Amphenicol,Amphenicols,Chlornitromycin,Chlorocid,Chloromycetin,Detreomycin,Ophthochlor,Syntomycin
D003573 Cytochrome b Group Cytochromes (electron-transporting proteins) with protoheme (HEME B) as the prosthetic group. Cytochromes Type b,Cytochromes, Heme b,Group, Cytochrome b,Heme b Cytochromes,Type b, Cytochromes,b Cytochromes, Heme,b Group, Cytochrome

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