A simple radioassay for dihydroorotate dehydrogenase |
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Authors: | GW Smithers Annette M Gero WJ OSullivan |
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Institution: | School of Biochemistry, University of New South Wales, Kensington, New South Wales, 2033, Australia |
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Abstract: | A simple radioassay for dihydroorotate dehydrogenase (DHO-DHase; EC 1.3.3.1) has been developed. l-carboxy-14C]Dihydroorotate was prepared from carboxy-14C]orotic acid using DHO-DHase derived from Zymobacterium oroticum and was purified by elution from DEAE-Sephadex A-25 with 0.2 m ammonium formate, pH 7. DHO-DHase activity in human spleen mitochondria was determined by the release of 14CO2 from the carboxy-14C-labeled l-dihydroorotate, the reaction being coupled with added orotate phosphoribosyltransferase and orotidylate decarboxylase. An apparent Km value of ~5 μm for l-dihydroorotate was established using the radioassay. This value correlated well with results from other methods. |
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