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Title: Biochemical analysis of microsomal NADPH-cytochrome P450 oxidoreductase and carboxylesterase
Author: Smith, Graeme Cameron Murray
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1995
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The endoplasmic reticulum is a rich source of enzymes involved in the metabolism of xenobiotics/drugs. Two enzymes, NADPH-cytochrome P450 oxidoreductase and a carboxylesterase, have been studied. The microsomal flavoprotein NADPH-cytochrome P450 oxidoreductase functions as a 'shuttle' in the transfer of electrons from NADPH to the physiological acceptors cytochromes P450. Exogenous acceptors, such as paraquat and quinone containing compounds, can also be reduced. To assess the role of P450 reductase in free radical cytotoxicity via redox cycling of compounds, the rat enzyme was expressed in both S. typhimurium and E. coli. S. typhimurium expressing P450 reductase were found to be more sensitive to the cytotoxic effects of paraquat and menadione but not to the anticancer drug doxorubicin. Similarly, the growth inhibitory effects of paraquat and menadione were found to be enhanced in the P450 reductase expressing bacteria. Using superoxide dismutase and catalase as markers of the oxidative stress inducible regulons soxRS and oxyR, it was found that in E. coli there was no difference in the induction of these enzymes between the expressing and non-expressing strains. A human liver coboxylesterase was purified to electrophoretic homogenity after a two step procedure involving DEAE-cellulose and hydroxylapatite chromatography. The purified protein had an apparent molecular weight of 59 kDa. and was glycosylated with mannose residues. The enzyme had high specific activity to a variety of carboxylesterase substrates and was inhibited by the serine hydrolase inhibitor, phenylmethylsulphonyl fluoride, and the specific carboxylesterase inhibitor bis-4-nitrophenylphosphate.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available