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Dihydroorotate dehydrogenase activity assays in cell extracts

JB Jonna Bouwknegt
CK Charlotte C. Koster
AV Aurin M. Vos
RO Raúl A. Ortiz-Merino
MW Mats Wassink
ML Marijke A. H. Luttik
MB Marcel van den Broek
PH Peter L. Hagedoorn
JP Jack T. Pronk
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Dihydroorotate dehydrogenase assays were performed at 30 °C in potassium phosphate buffer, (100 mM, pH 7.5) using a Hitachi U-3010 UV/Visible spectrophotometer (Chiyoda, Tokyo, Japan). Formation of orotate or reduction of NAD(P)+ was monitored by measuring absorbance at 300 nm (ε = 3.05 mM−1 cm−1; [14]) or 340 nm (ε = 6.22 mM−1 cm−1; [107]) respectively, upon addition of 1 mM dihydroorotate to a temperature-equilibrated reaction mixture containing buffer, cell extract and/or either of the electron acceptors fumarate (1 mM), decylubiquinone (QD, 0.1 mM, dissolved in dimethylsulfoxide), nicotinamide adenine dinucleotide (NAD+, 1 mM), nicotinamine adenine dinucleotide phosphate (NADP+, 1 mM), flavine adenine dinucleotide (FAD, 20 μM), flavin mononucleotide (FMN, 20 μM) or the artificial electron acceptor phenazine methosulfate (PMS, 0.1 mM). Enzyme assays were performed on two separately prepared cell extracts. Reduction potentials of tested electron acceptors mentioned in the text are relative to the standard hydrogen electrode.

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