Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay

• Published in: Archives of biochemistry and biophysics Vol. 229; no. 2; pp. 519 - 531
• Main Authors: Seidel, Sharon L., Shawver, Laura K., Shires, Thomas K.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.1984
• Subjects: Animals; Carbon Monoxide - pharmacology; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - isolation & purification; Enzyme Induction; Enzyme-Linked Immunosorbent Assay; Kinetics; Microsomes, Liver - drug effects; Microsomes, Liver - metabolism; Molecular Weight; Oxidation-Reduction; Phenobarbital - pharmacology; Rats; Rats, Inbred Strains; Substrate Specificity
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/0003-9861(84)90183-8

The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl 4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form.