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
