Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay
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 c...
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Published in | Archives of biochemistry and biophysics Vol. 229; no. 2; pp. 519 - 531 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Inc
01.01.1984
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Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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ISSN: | 0003-9861 1096-0384 |
DOI: | 10.1016/0003-9861(84)90183-8 |