The Use of Liposomes in the Study of Drug Metabolism: A Method to Incorporate the Enzymes of the Cytochrome P450 Monooxygenase System into Phospholipid, Bilayer Vesicles
Although lipids are essential for the optimal activity of the cytochromes P450 monooxygenase system, relatively little is known about the membrane environment in which these enzymes function. One approach used to mimic the structural arrangement of lipids and enzymes within the endoplasmic reticulum is to physically incorporate the cytochromes P450 and their redox partners in a vesicle bilayer of phospholipids. Several methods have been devised for this purpose. This chapter describes a method in which the P450 monooxygenase system is incorporated by first, solubilizing the enzymes and lipid with sodium glycocholate. After the protein and lipid aggregates are dispersed, the detergent is removed by adsorption using BioBeads SM-2 resin which leads to the formation of bilayer vesicles of phospholipid containing incorporated cytochrome P450 and NADPH cytochrome P450 reductase. This procedure requires relatively a short preparation time, provides concentrated reconstituted systems that can be used in a wide range of applications, allows for several enzyme samples to be prepared simultaneously so that different conditions can be compared, and results in minimal loss of active enzyme.
Key wordsPhospholipid vesicles Cytochromes P450 Reconstituted systems Drug metabolism
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