Cholesterol metabolism in the endoplasmic reticulum of rat liver
In work directed towards the purification of a liver microsomal cytochrome P-450 species capable of supporting cholesterol 7a-hydroxylase activity in a reconstituted system, the use of the detergent Renex 690 for the solubilisation of microsomal protein resulted in an unacceptable inhibition of cholesterol 7ct-hydroxylation. This confirmed previous work, showing that Nonidet P42 is the optimum choice of solubilising agent for this purpose. DEAE-cellulose chromatography of microsomal protein solubilised with Nonidet P42 was confirmed to be a suitable first step in the purification of liver microsomal cytochrome P-450, since there is a good separation of this species from NADPH cytochrome c reductase activity. However, the recovery and purification of total cytochrome P-450 was low. Dithiothreitol, 4-phenyl-imidazole, diethyldithiocarbamate and glycerol, by themselves or in combination with each other, were shown to be useful agents in liver microsomal cytochrome P-450 purification. It was further demonstrated that increasing the recovery of cytochrome P-450 gave a concomitant improvement in its purification. Chromatography on quaternary aminoethyl-Sephadex or on carboxymethylcellulose did not result in any purification of cytochrome P-450. Hydroxyapatite chromatography of cytochrome P-450 - containing fractions from the DEAE-cellulose eluate gave a further small purification of cytochrome P-450. The use of chemical donors of "active oxygen" in the reconstitution of cholesterol 7a-hydroxylase activity with liver microsomal cytochrome P-450 was shown to be limited by the rapid destruction of the cytochrome by these agents.