The Reconstitution of Hepatic Microsomal Mixed Function Oxidase Activity with Fractions Derived from Weanling Rats Fed Different Levels of Protein

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Abstract
Weanling rats were fed for 10 days either a purified diet containing 5% casein ad libitum (5C), a purified diet containing 20% casein ad libitum (20C), or a commercial cereal based stock diet (SD). All rats were administered sodium phenobarbital (80 mg/kg) in 0.9% saline for the last 4 to 6 days of the experiment in order to induce sufficient quantities of microsomes to obtain the individual mixed function oxidase (MFO) enzyme components of lipid, cytochrome P-450, and cytochrome c reductase. After fractionation and isolation of these three components derived from the three dietary treatment groups, they were recombined in various combinations so that the contributions of specific activities and interactions between components could be studied with respect to benzphetamine N-demethylation. The order of reconstituted activities for the three dietary groups, where each assay contained the components from the same diet, was SD > 20C > 5C; the differences were similar to those previously seen with the MFO activity of intact microsomes. The lipid fractions containing the requisite phosphatidylcholine were interchangeable between dietary groups, even though it is known that dietary protein level can modify fatty acyl substitution and alter in vitro MFO activity. Both P-450 and cytochrome c reductase substitution with the counterpart components from another diet greatly modified benzphetamine N-demethylation of the reconstituted system. Because these substitutions were made on an equivalent activity basis, the modification in reconstituted activities were the result of altered interaction between these two essential components. The effect on the interaction was diet-specific; both the substitution of 20C components with 5C components and the substitution of SD components with either 20C or 5C components depressed the efficiency of the interaction. The implications of these observations for MFO rate-limitations are discussed and it is suggested that the primary effect of diet on “specific” MFO activities previously observed may have been due to an effect on the interaction between P-450 and cytochrome c reductase, rather than on the specific activities of the individual components.