Graduate Thesis Or Dissertation
 

Analysis of cytochrome P-450 isozymes from rat liver and studies on the mechanism of induction

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  • In order to determine the total number of cytochrome P-450 isozymes present in a given species following various treatments, mature male rats (Sprague-Dawley) were injected intraperitoneally with the classic mixed-function oxidase inducers phenobarbital (PB), pregnenolone- 16a-carbonitrile (PCN), 3-methylcholanthrene (3-MC), or the appropriate control vehicle. Hepatic microsomes were solubilized and P-450 separated from the majority of other microsomal proteins by hydrophobic chromatography on w-aminooctyl agarose (AOA). The P-450 isozymes were then separated by either sequential chromatography on hydroxyapatite and DEAE at 4°C or by DEAE at room temperature. The degree of P-450 purity during the procedure was monitored by determining the specific content (nmoles P- 450 per mg protein) and by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Isoelectric focusing (IF) was also used to determine the homogeneity of final preparations. P-450, partially purified by AOA, was resolved into three to six peaks by DEAE. The pattern of elution obtained differed widely between the four groups. Although the specific content of most peaks was quite high, only the major P-450 isozyme from the PB induced group appeared homogenous. SDS-PAGE and IF, although useful in determining the homogeneity of a particular preparation, were too limited in resolving power to distinguish isozymes from two or more preparations. A recently developed model for the molecular mechanism by which chemicals induce the synthesis of P-450 was examined by analyzing the binding of the above inducers (radiolabeled) to cytosol from uninduced rats. Using modifications of techniques for analyzing ligand-receptor binding, the existence of a specific, high affinity receptor for ³H-3MC was demonstrated and the binding parameters compared to published results. The existence of high affinity receptors for ¹⁴C-PCN and ³H-3-MC was suggested, but the low radiospecific activity of the former and the very small percent bound of the latter, prevented any significant conclusions.
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