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Characterization and Partial Purification of a Factor Supporting NADPH-Dependent Lipid Peroxidation.

Movassagh, Mehregan. (1998) Characterization and Partial Purification of a Factor Supporting NADPH-Dependent Lipid Peroxidation. Doctoral thesis, University of Surrey (United Kingdom)..

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Abstract

The NADPH-dependent lipid peroxidation in rat liver microsomal membrane was studied by using an effective and sensitive "reconstituted lipid peroxidation assay", which closely resembled the microsomal system. The NADPH-dependent lipid peroxidation was monitored by Thiobarbituric Acid Reactive Substances (TBARS) formation in the reconstituted assay system, initially using a 1.2%-0.4% (w/v) differentially cholate solubilized fraction (CCMs), which had a 3-fold increase in relative specific activity compared to microsomes. The lipid peroxidation activity in the reconstituted assay was demonstrated to be dependent on the presence of NADPH, NADPH-Cytochrome P-450 reductase enzyme, CCMs and extracted microsomal membrane phospholipids. The optimal concentrations of these reagents in the reconstituted assay, were determined. It was shown that the factor supporting NADPH-lipid peroxidation activity was heat labile, lending support to the possible protein nature of this factor. By using Gas Chromatography, it was demonstrated that arachidonate and docosahexanoate fatty acids were the preferred substrates in the assay. Both phosphate and cholate detergent inhibited lipid peroxidation activity. The lipid peroxidation active fraction was extremely labile. Solubilization with alternative detergents such as 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), or addition of protease inhibitors such as phenylmethylsulfonylfluoride, did not improve its labile nature. However, addition of bovine serum albumin did improve stability but then further purification steps were required. Nevertheless, some degree of purification of the lipid peroxidation activity was achieved. An 11-, 10-, 5- and 4- fold purification was achieved (compared to microsomes) using 75%-45% (w/v) ammonium sulphate precipitation method, diethylaminoethyl-, hydroxyapatite- and laurate-sepharose- chromatographies, respectively. All the post chromatography eluate fractions with measurable lipid peroxidation activity, had an increased level of “background” TBARS. This resulted in an overall decrease in their lipid peroxidation activity. Although an electrophoretically homogeneous preparation was not produced, partial progress has been made, providing a basis for future purification and characterisation of this enzyme system.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Movassagh, Mehregan.
Date : 1998
Additional Information : Thesis (M.Phil.)--University of Surrey (United Kingdom), 1998.
Depositing User : EPrints Services
Date Deposited : 06 May 2020 14:15
Last Modified : 06 May 2020 14:17
URI: http://epubs.surrey.ac.uk/id/eprint/856075

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