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Title: Studies on the interaction of cholesterol analogues with cholesterol oxidising systems and membrane components
Author: Craig, I. F.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1979
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A series of cholesterol possessing side chains with either more or less carbon atoms than cholesterol were synthesised from hyodesoxycholic acid. These analogues were used in physical studies of the interaction between cholesterol and phospholipid and to investigate the substrate specificities of rat liver cholesterol 7a hydroxylase and adrenal mitochondrial cholesterol side chain cleavage enzyme. The effect of alteration of the non-polar side chain of cholesterol on the physical properties of cholesterol was investigated by incorporating them into liposomes prepared from precise molar proportions of cholesterol analogues and a series of saturated or unsaturated phospholipids. The interaction between cholesterol and phospholipid was then monitored by: (a) a series of spin labels including nitroxy octane, Tempo, 25 nitroxy cholestane and nitroxy cholestane, (b) stopped flow spectrophotometry of water permeability across the liposomal membrane and (c) by preparation of mixed monolayers on a Langmuir trough. The results of the liposomal 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 the monolayer studies. The role of cholesterol in natural membranes in particular the endoplasmic reticulum, has been investigated by alteration of membrane cholesterol by liposomes of variable composition. 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. The results showed that the metabolism of cholesterol is governed by its side chain, with the short side chain analogue being metabolised by the drug metabolising enzyme. A new GLC assay which measures the production of 7a hydroxy cholesterol has been developed and used to investigate the problem of substrate supply for cholesterol 7a hydroxylase. The involvement of a soluble protein isolated from the 100,000 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 SDS polyacrylmmide gel electrophoresis and its means of action investigated. 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 interaction with protein.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available