A series of cholesterol analogues possessing side chains with
either more or less carbon atoms than cholesterol were synthesised
from hyodesoxycholic acid and fully characterised by nuclear magnetic
resonance, mass spectrometry, and infra red analysis. These analogues
were used in (a) physical studies of cholesterol phospholipid interactions
and (b) experiments to investigate the substrate specificities of
cholesterol 7a"hydroxylase from rat liver and the adrenal mitochondrial
cholesterol side chain cleavage enzyme.
(a) The physical properties of cholesterol and a series of cholesterol
side chain analogues were investigated by incorporating them into
liposomes prepared from lipid films containing the sterol and a series
of saturated or unsaturated phospholipids in the required molar
proportions. A series of spin labels (3-nitroxy octane, Tempo, 25
nitroxy cholestane, and 3 nitroxy cholestane) were incorporated into the
sterol phospholipid liposomes and the effect of each analogue on each
spin label mobility was observed. In addition the effect of each
analogue on water permeability across the liposomal membrane was determined.
The results of both sets of experiments showed that the cholesterol molecule
with its iso-octane side chain was optimally adapted for maximal interaction
with phospholipid. This specificity was not observed in monolayer
studies of cholesterol phospholipid interactions.
(b) The involvement of the cholesterol side chain in cholesterol
oxidation by the liver microsomal enzyme, cholesterol 7a~hydroxylase
was determined using an established assay based on the oxidation of
added radioactive cholesterol. Using phenobarbitone pretreated rats
it was shown that the metabolism of short side chain analogues was
stimulated whereas 7a~hydroxylase activity was unaffected. Thus it is
postulated that the side chain of cholesterol determines its metabolic
fate and that the shorter side chain analogues are metabolised by the
inducible hepatic drug metabolising enzyme system. A new G.L.C.
assay which measures the production of 7a-hydroxy cholesterol
from endogenous microsomal cholesterol has been developed and
used to investigate the problem of substrate supply for the 7ahydroxylase
enzyme. This involved the addition of cholesterol in
detergents in sonicated and unsonicated liposomes containing different
molar proportions of sterol and in organic solvents to a cholesterol
depleted form of the enzyme.
The involvement of a soluble protein isolated from the 100,000 x g
supernatant of rat liver by ammonium sulphate fractionation, gel
filtration and ion exchange chromatography has also been demonstrated.
The molecular weight of the protein has been determined by S.D.S.
polyacrylamide gel electrophoresis, and its means of action investigated.
The cholesterol side chain cleavage enzyme of rat adrenal
mitochondria has been shown to possess an absolute requirement for
cholesterol as substrate by use of a radio-immuno-assay that measures
pregnenolone production from the exogenous cholesterol side chain
The results of both the physical measurements and the enzyme
assays show that the side chain of cholesterol plays a major role
in determining the extent of the interaction between cholesterol and
phospholipids and the specificity of cholesterol protein interaction