Microsomal fraction, cytochrome isoforms

The most important enzyme involved in bio transformation is cytochrome P-450, which catalyzes many phase 1 reactions. This enzyme is located primarily in the SER (microsomal fraction) of the cell and is especially abundant in liver cells. Cytochrome P-450 primarily catalyzes oxidation reactions and consists of many isoforms (isozymes). These isoenzymes have overlapping substrate specificities. The most important subfamily in humans is CYP3A4, although there is considerable variation in CYP3A4 expression between individuals. 

In vitro. The activity or absolute level of enzymes such as cytochrome P-450 and glucuronosyl transferase can be measured in cells, tissue fractions, or subcellular fractions (e.g., microsomes) and compared with those from control animals. The activity is measured by using a particular substrate for each of the isoforms of the enzyme (e.g., cytochrome P-450 or UDPGT) of interest. The total level of cytochrome P-450 could be determined by spectrophotometry using standard methods (e.g., carbon monoxide binding and difference spectra). Alternatively, the level of protein can be determined by gel electrophoresis and Western blotting, and this would allow the separation of different isoforms. 

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