Effect of washing the hepatic microsomal fraction in sucrose solutions and in sucrose solution containing edta upon the metabolism of foreign compounds

Washing the hepatic microsomal fraction of the rat in 0·25 M sucrose containing 0·05 M Tris buffer (pH 7·4) resulted in the metabolism and binding to cytochrome P-450 of Type I substrates, although not of a Type II substrate. The levels of microsomal cytochrome b₅, cytochrome P-450 and NADPH-cytochrome c reductase was unchanged, whilst the activity of NADPH-cytochrome P-450 reductase was reduced. The inhibitors of lipid peroxidation EDTA and Mn²⁺ added to the washing medium prevented the decrease in the metabolism of Type I substrates. It is suggested that the effects of washing may be related to the increase in the level of peroxidised microsomal lipid which could lead to a selective destruction of the Type I binding site. EDTA added to the washing medium also produced an increase above control values, in the metabolism and binding of both Type I and Type II substrates, which may be related to the apparent increase in the amount of microsomal cytochrome P-450. Washing the microsomal fraction almost completely abolished the ability of acetone to enhance aniline hydroxylation. It is concluded that the effects of acetone are not due to the displacement of endogenous substrates bound to the microsomal fraction.