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dc.contributor.author K. en_US
dc.contributor.author David C. en_US
dc.contributor.author Andrew G. S. en_US
dc.contributor.author Diane E. en_US
dc.contributor.author Steven L. en_US
dc.date.accessioned 2009-11-02T16:40:57Z
dc.date.available 2009-11-02T16:40:57Z
dc.date.issued 2001-08-10 en_US
dc.identifier http://dx.doi.org/10.1006/bbrc.2001.5338 en_US
dc.identifier.citation Venkateswarlu , K , Lamb , D C , Warrilow , A G S , Kelly , D E & Kelly , S L 2001 , ' Activities and kinetic mechanisms of native and soluble NADPH- cytochrome P450 reductase ' Biochemical and Biophysical Research Communications , pp. 48-54 . , 10.1006/bbrc.2001.5338 en_US
dc.identifier.other PURE: 126660 en_US
dc.identifier.other dspace: 2160/3389 en_US
dc.identifier.uri http://hdl.handle.net/2160/3389
dc.description.abstract Native yeast NADPH–cytochrome P450 oxidoreductase (CPR; EC 1.6.2.4) and a soluble derivative lacking 33 amino acids of the NH2-terminus have been overexpressed as recombinant proteins in Escherichia coli. The presence of a hexahistidine sequence at the N-terminus allowed protein purification in a single step using nickel-chelating affinity chromatography. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis confirmed the predicted molecular weights of the proteins and indicated a purity of >95%. Protein functionality was demonstrated by cytochrome c reduction and reconstitution of CYP61-mediated sterol Δ22-desaturation. Steady-state kinetics of cytochrome c reductase activity revealed a random Bi-Bi mechanism with NADPH donating electrons directly to CPR to produce a reduced intermediary form of the enzyme. The kinetic mechanism studies showed no difference between the two yeast CPRs in mechanism or after reconstitution with CYP61-mediated 22-desaturation, confirming that the retention of the NH2-terminable membrane anchor is functionally dispensable. en_US
dc.format.extent 7 en_US
dc.relation.ispartof Biochemical and Biophysical Research Communications en_US
dc.title Activities and kinetic mechanisms of native and soluble NADPH- cytochrome P450 reductase en_US
dc.contributor.pbl Aberystwyth University en_US
dc.contributor.pbl Institute of Biological, Environmental and Rural Sciences en_US


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