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

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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 V8 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 b5, 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.

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