Activities and kinetic mechanisms of native and soluble NADPH- cytochrome P450 reductase

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dc.contributor.author Venkateswarlu, K.
dc.contributor.author Lamb, David C.
dc.contributor.author Warrilow, Andrew G. S.
dc.contributor.author Kelly, Diane E.
dc.contributor.author Kelly, Steven L.
dc.date.accessioned 2009-11-02T16:40:57Z
dc.date.available 2009-11-02T16:40:57Z
dc.date.issued 2001-08-10
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
dc.identifier.issn 1090-2104
dc.identifier.other PURE: 126660
dc.identifier.other dspace: 2160/3389
dc.identifier.uri http://hdl.handle.net/2160/3389
dc.description Lamb, D. C., Warrilow, A. G. S., Venkateswarlu, K., Kelly, D. E., Kelly, S. L. (2001). Activities and kinetic mechanisms of native and soluble NADPH- cytochrome P450 reductase. Biochemical and Biophysical Research Communications, 286, (1), 48-54. Sponsorship: BBSRC en
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
dc.format.extent 7 en
dc.language.iso eng
dc.relation.ispartof Biochemical and Biophysical Research Communications en
dc.title Activities and kinetic mechanisms of native and soluble NADPH- cytochrome P450 reductase en
dc.type Text en
dc.type.publicationtype Article (Journal) en
dc.identifier.doi http://dx.doi.org/10.1006/bbrc.2001.5338
dc.contributor.institution Aberystwyth University en
dc.contributor.institution Institute of Biological, Environmental and Rural Sciences en
dc.description.status Peer reviewed en


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