Cholesterol metabolism in the endoplasmic reticulum of rat liver
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Date
1979Author
Rea, P. W. H
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Abstract
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.