Studies on cholesterol 7α-Hydroxylase
Hattersley, Neil G.
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Attempts were made to solubilize the enzyme cholesterol 7α-hydroxylase from native rat liver microsomes and from rat liver microsomal acetone and butanol powders. Mechanical techniques such as freezing and thawing, repeated homogenisation and sonication, and also the use of hydrolytic enzymes such as Phospholipase A and those contained in pancreatin and Naja naja venom, all failed to solubilize the enzyme. Solubilizing agents such as urea, n-butanol, sodium deoxycholate and cholate, cetyltrimethylammonium bromide, and all the non-ionic detergents tested, with the exception of Nonidet P40 and P42, failed to release into solution cholesterol 7α -hydroxylase activity or greatly inhibited this enzyme. Nonidet P42 solubilized microsomes were applied to a column of DEAE-cellulose, and chromatography separated cytochrome P-450, cytochrome b5 and NADPH-cytochrome c oxidoreductase from each other. Fractions eluted from DEAE-cellulose contained very little or no cholesterol 7α-hydroxylase activity, but on recombination of the cytochrome P-450 fraction with a fraction containing NADPH-cytochrome c oxidoreductase, cholesterol 7α-hydroxylase activity was reconstituted. The interdependence of cytochrome P-450 and NADPH-cytochrome c oxidoreductase and the effect of cytochrome b5 was investigated in the reconstituted cholesterol 7α-hydroxylase system. Further attempts have been made to increase the purity of cytochrome P-450 and NADPH-cytochrome c oxidoreductase, and these partially purified fractions were recombined and tested for their ability to support the 7α-hydroxylation of cholesterol. Some chemical and biochemical properties of Nonidet P42 solubilized rat liver microsomes and rat liver microsomal acetone and butanol powders have also been investigated to characterise the system. The effect of modifications to the cholesterol side chain on cholesterol 7α-hydroxylase activity has been observed. Studies on the substrate specificity of cholesterol 7α—hydroxylase have revealed that this enzyme is very sensitive to small changes in the side chain of the sterol.