Characterization of a phenobarbital-inducible dog liver cytochrome P450 structurally related to rat and human enzymes of the P450IIIA (steroid-inducible) gene subfamily.
A cytochrome P450 called PBD-1 isolated from liver microsomes of an adult male Beagle dog treated with phenobarbital (PB) is structurally and functionally similar to members of the P450IIIA gene subfamily in rat and human liver microsomes. The sequence of the first 28 amino-terminal residues of PBD-1 is identical in 15 and 20 positions, respectively, to the P450IIIA forms P450p from rat and P450NF (and HLp) from human. Upon immunoblot analysis, anti-PBD-1 IgG recognizes PCNa (P450p) and PCNb (PB/PCN-E) from rat, P450NF from human, and two proteins in liver microsomes from both untreated and PB-treated dogs. Similarly, anti-PCNb IgG cross-reacts with PBD-1 and with at least one protein in microsomes from untreated dogs and two proteins in microsomes from PB-treated dogs. P450IIIA-form marker steroid 6 beta-hydroxylase activities increase 2.5-fold upon PB-treatment of dogs and are selectively inhibited by anti-PBD-1 IgG. NADPH-dependent triacetyloleandomycin (TAO) complex formation and erythromycin demethylase, also marker activities for P450IIIA forms from rats and humans, increase 4- and 5-fold in dog liver microsomes upon PB treatment, whereas immunochemically reactive PBD-1 is induced 3-fold. In microsomes from PB-treated dogs, 5 mg anti-PBD-1 IgG/nmol P450 inhibits greater than 75 and 50% of TAO complex formation and erythromycin demethylase activity, respectively. TAO complex formation is not inhibited by chloramphenicol, a selective inhibitor of the major PB-inducible dog liver cytochrome P450, PBD-2. These data suggest that PBD-1 or another immunochemically related form is responsible for a major portion of macrolide antibiotic metabolism by microsomes from PB-treated dogs and for steroid 6 beta-hydroxylation by microsomes from both untreated and PB-treated dogs. Major species differences were noted, however, in the apparent Km for 6 beta-hydroxylation of androstenedione by liver microsomes from untreated rats (24 microM), humans (380 microM), and untreated dogs (4700 microM).
Ciaccio
P J
PJ
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721.
Halpert
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D002701:Chloramphenicol / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000302:isolation & purification; D004285:Dogs; D004790:Enzyme Induction / Q000187:drug effects; D015151:Immunoblotting; D007074:Immunoglobulin G; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000494:pharmacology; D010634:Phenobarbital / Q000494:pharmacology*; D051381:Rats; D013053:Spectrophotometry; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D014217:Troleandomycin / Q000378:metabolism / Q000494:pharmacology
D065607:Cytochrome P-450 Enzyme Inhibitors; D007074:Immunoglobulin G; D009249:NADP; D002701:Chloramphenicol; D003577:Cytochrome P-450 Enzyme System; D014217:Troleandomycin; D013250:Steroid Hydroxylases; C022657:steroid hormone 6-beta-hydroxylase; D010634:Phenobarbital