Characteristics of a microsomal dechlorination system

Microsomes were solubilized and the components of the mixed function oxidase system were isolated. With a reconstituted system containing partially purified cytochrome P 450, NADPH cytochrome c reductase, NADPH, and phospholipid, dechlorination of 1,1,2 trichloroethane was observed. Addition of cytochrome b₅, NADH cytochrome b₅ reductase, and cyanide sensitive factor to this system resulted in additional activity, but the cytochrome b₅ system by itself would not dechlorinate. The activity of the complete system was not altered by the presence of the cyanide sensitive factor. Whole microsomes also were studied and the kinetics of the dechlorination reaction were determined. The K(m) was 3.32 mM; V(max) was 1.09 nmoles of substrate dechlorinated per minute per milligram of protein. The addition of NADH to the NADPH system significantly increased both K(m) and V(max). The microsomal enzyme system required molecular oxygen; the presence of the cell supernatant fraction produced an increase in the V(max) of dechlorination.