Multiple forms of hepatic cytochrome P-450. Purification, characterisation and comparison of a novel clofibrate-induced isozyme with other major forms of cytochrome P-450

Abstract

In the present studies, a novel form of highly purified cytochrome P-450 (cytochrome P-452) isolated from the hepatic microsomes of clofibrate-pretreated rats has been compared to the major isozymes isolated from the hepatic microsomes of rats pretreated with phenobarbital (cytochrome P-450) and 2-naphthoflavone (cytochrome P-447) using a number of biochemical criteria.The results show that these three isozymes exhibit marked structural differences from each other as judged by a complete lack of immunochemical cross-reactivity between the isozymes and the heterologous rabbit serum antibodies using Ouchterlony double diffusion, and non-identity between the limited proteolytic digestion maps of the three isozymes obtained in the presence of chymotrypsin, papain and Staphylococcus aureus V, proteases. Furthermore, the three isozymes exhibited clear differences in their monomeric molecular weights determined on calibrated sodium dodecyl sulphate/polyacrylamide gel electrophoresis in gels of varying acrylamide concentration. Substantial differences were also observed in the substrate specificities of the isozymes, which were reflected in differences in the turnover rates and positional selectivities of the hemoproteins for some model substrates. In addition, the isozymes differed in their substrate binding affinities and their ability to interact with purified hepatic microsomal cytochrome b,, as judged using difference spectrophotometry. Finally, subtle differences were detected in the ultraviolet visible absorbance spectra of the hemoproteins in the ferric, ferrous, and carbonmonoxyferrous states.Taken collectively, the above data provides compelling evidence that fundamental differences exist between these cytochrome P-450 isozymes, further establishing the uniqueness of the major form of cytochrome P-450 induced by clofibrate pretreatment.Cytochrome P-450 is the terminal hemoprotein component of the hepatic microsomal electron-transfer chain which functions in the oxidation of a structurally diverse number of xenobiotics and endogenous compounds [I]. Considerable evidence has been presented in support of the existence of Much confusion exists regarding the terminology of cytochrome P-450 isozymes primarily because there is no uniform classification system which has been adopted to make unequivocal assignments of the similarity between isozymes isolated in different laboratories. Accordingly, the above three isozymes described in this paper have been classified according to the wavelength maxima of the ferrouscarbonmonoxy adducts. In the current paper, limited use is made of the term cytochrome P-450 in a general sense to collectively indicate all the cytochrome P-450 isozymes. Where appropriate, we make extended use of the term cytochrome P-450 to indicate that major isozyme induced by phenobarbital pretreatment in rat liver. This is regarded as necessary because to make a distinction for this latter isozyme and rename it would add further confusion to the literature. Similarly, throughout this paper, the terms c...

Preferential induction of the rat hepatic P450 I proteins by the food carcinogen 2‐amino‐3‐methyl‐imidazo[4,5‐f]quinoline

“…Antibodies to P450 I A1 were raised in sheer, mimed with 1 .0 mg and boosted with 0.25 mg plots whkh are viewed both in and perpendicular to the molecular plane. The following van der Waals radii were used to generate the molecular plots: carbon 1.6 nm, hydrogen 1.2 nm and nitrogen .5 nm, t-test, Cytochromes p450 I 3-methylcholanthrene and phenobarbital, respectively, have Statistical evaluation was carried out using the Student's of the antigen' [34]. Antisera to P450 I1 B1 were raised in rabbits and generously provided by Dr G. G. Gibson (University of Surrey).…”

Undernutrition enhances alcohol-induced hepatocyte proliferation in the liver of rats fed via total enteral nutrition

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Differential induction of peroxisomal and microsomal fatty‐acid‐oxidising enzymes by peroxisome proliferators in rat liver and kidney

“…5, tracks 5 and 7, respectively). In this context, it should be noted that the antibodies raised to cytochromes P-450,,, and P-450,,, recognise their homologous antigens both in Ouchterlony double immunodiffusion [42] and immunoprecipitation after mRNA translation in vitro (data not shown). Thus, we were relatively assured that the two bands observed on Western blotting (Fig.…”