Separation of cytochrome P-450 containing vesicles from the midgut microsomal fraction of Manduca sexta

1. Microsomes prepared from midgut tissue of Manduca sexta by differential centrifugation are a heterogeneous population of vesicles. 2. Upon centrifugation of microsomes in a sucrose gradient of 30%, 15% and 10%, specific activity of cytochrome P-450 catalyzed O-demethylation of p-nitroanisole (a marker enzyme for endoplasmic reticulum) was increased 2.5 to 3.5-fold in the 15% fraction. 3. Specific activities of alkaline phosphatase and leucine aminopeptidase (marker enzymes for brush border membranes) were increased 3.1 to 5.7-fold in the pellet. 4. Differential centrifugation is a useful step in the purification of cytochrome P-450 enzymes.