{"id":{"repo_id":"edinburgh","oai_identifier":"oai:era.ed.ac.uk:1842/18561"},"canonical_url":"https://search.dev.ndltd.org/etd/edinburgh/oai:era.ed.ac.uk:1842/18561","repository":{"repo_id":"edinburgh","name":"University of Edinburgh","base_url":"https://era.ed.ac.uk/server/oai/request"},"display":{"title":"Cholesterol metabolism in the endoplasmic reticulum of rat liver","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.","abstract_html":"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.","abstract_has_math":false,"creators":["Rea, P. W. H"],"institution":"The University of Edinburgh","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":1979,"date_issued":"1979","date_published":"1979","updated_at":"2026-07-24T02:13:57Z","subjects":[],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/1842/18561","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Rea, P. W. 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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."]},{"key":"dc:title","label":"Title","values":["Cholesterol metabolism in the endoplasmic reticulum of rat liver"]}]}],"canonical_facts":{"dc:creator":["Rea, P. W. H"],"dc:date.accessioned":["2016-12-06T10:44:11Z"],"dc:date.available":["2016-12-06T10:44:11Z"],"dc:date.issued":["1979"],"dc:description.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."],"dc:identifier.uri":["http://hdl.handle.net/1842/18561"],"dc:publisher":["The University of Edinburgh"],"dc:title":["Cholesterol metabolism in the endoplasmic reticulum of rat liver"],"dc:type":["Thesis or Dissertation"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["PhD Doctor of Philosophy"]},"updated_at":"2026-07-24T02:13:57Z"}