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Three immunoidentical cytochromes P-450 from liver microsomes of phenobarbital-treated rats.

ABSTRACT: Three forms of cytochrome P-450 were purified to homogeneity from liver microsomes of Wistar-strain rats treated with phenobarbital. They had minimum mol.wts. of 52 000, 53 000 and 54 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and are designated as P-450(L), P-450(M) and P-450(H) respectively. They were shown to be immunoidentical by Ouchterlony double-diffusion analysis. Several criteria, such as isoelectric points, substrate specificities and sensitivities to tryptic digestion, however, indicated that these cytochromes are distinct isoenzymes of cytochrome P-450. Whereas P-450(L) was highly active on various substrates, P-450(H) had generally low catalytic activities, except on aminopyrine. The cytochromes purified by immunoaffinity chromatography using anti-P-450(L) showed a marked variation in their distribution depending on the strain and colony of rat. Limited tryptic digestion of P-450(H) gave one tryptic peptide showing the same mobility as P-450(L) by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and their primary structures were very similar. The result suggests a possibility that such limited proteolysis is involved in the post-translational modification of the cytochrome or its destruction.

SUBMITTER: Sakai H

PROVIDER: S-EPMC1152366 | biostudies-other | 1983 Oct

REPOSITORIES: biostudies-other

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Project description:The potent porphyrogen allylisopropylacetamide and related compounds decrease hepatic concentrations of cytochrome P-450. This decrease occurs particularly in phenobarbital-induced cytochrome P-450 and is caused by suicidal breakdown of the haem of cytochrome P-450. Quantitative rocket immunoelectrophoresis showed that the protein moiety of the major phenobarbital-inducible form of hepatic cytochrome P-450 was not diminished up to 1 h, but was markedly decreased (to 43% of that of the phenobarbital-treated control) at 20 h after allylisopropylacetamide treatment. In contrast, the concentration of total cytochrome P-450, measured spectrophotometrically, decreased to 30-40% of the control at both 1 and 20 h after allylisopropylacetamide. Cytochrome P-450-dependent demethylations of ethylmorphine and benzphetamine decreased to a similar extent. When liver homogenates from rats treated with allylisopropylacetamide 1 h before being killed were incubated with haem, functional holocytochrome P-450 could be reconstituted from the apoprotein. Incubation with haem increased spectrophotometrically measurable cytochrome P-450 to 69%, ethylmorphine demethylase to 64% and benzphetamine demethylase to 93% of the activities in rats treated with phenobarbital alone. At 20 h after allylisopropylacetamide treatment, however, little or no reconstitution of cytochrome P-450 occurred after incubation with haem. When liver homogenates were incubated with cobalt and protoporphyrin, and microsomal proteins were then subjected to polyacrylamide-gel electrophoresis, cobalt-protoporphyrin was found specifically associated with proteins of Mr 50 000-53 000. When homogenates from rats given allylisopropylacetamide for 1 h or 20 h were compared, it was found that the extent of this association was higher in livers from the rats containing more apocytochrome P-450, suggesting that cobalt-protoporphyrin had associated with the apocytochrome. The data provide insight into the association of haem with the protein moiety of cytochrome P-450 and factors affecting breakdown of this protein.

Project description:The induction of hepatic cytochromes P-450 by phenobarbital (PB) was studied in rat hepatocytes cultured for up to 5 weeks on Vitrogen-coated plates in serum-free modified Chee's medium then exposed to PB (0.75 mM) for an additional 4 days. Immunoblotting analysis indicated that P-450 forms PB4 (IIB1) and PB5 (IIB2) were induced dramatically (greater than 50-fold increase), up to levels nearly as high as those achieved in PB-induced rat liver in vivo. The newly synthesized cytochrome P-450 was enzymically active, as shown by the major induction of the P-450 PB4-dependent steroid 16 beta-hydroxylase and pentoxyresorufin O-dealkylase activities in the PB-induced hepatocyte microsomes (up to 90-fold increase). PB induction of these P-450s was markedly enhanced by the presence of dexamethasone (50 nM-1 microM), which alone was not an affective inducing agent, and was inhibited by greater than 90% by 10% fetal bovine serum. The PB response was also inhibited (greater than 85%) by growth hormone (250 ng/ml), indicating that this hormone probably acts directly on the hepatocyte when it antagonizes the induction of P-450 PB4 in intact rats. In untreated hepatocytes, P-450 RLM2 (IIA2), P-450 3 (IIA1) and NADPH P-450 reductase levels were substantially maintained in the cultures for 10-20 days. The latter two enzymes were also inducible by PB to an extent (3-4 fold elevation) that is comparable with that observed in the liver in vivo. Moreover, P-450c (IA1) and P-450 3 (IIA1) were highly inducible by 3-methylcholanthrene (5 microM; 48 h exposure) even after 3 weeks in culture. In contrast, the male-specific pituitary-regulated P-450 form 2c (IIC11) was rapidly lost upon culturing the hepatocytes, suggesting that supplementation of appropriate hormonal factors may be necessary for its expression. The present hepatocyte culture system exhibits a responsiveness to drug inducers that is qualitatively and quantitatively comparable with that observed in vivo, and should prove valuable for more detailed investigations of the molecular and mechanistic basis of the response to PB and its modulation by endogenous hormones.

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Three immunoidentical cytochromes P-450 from liver microsomes of phenobarbital-treated rats.

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