Purification and characterization of dihydropyrimidine dehydrogenase from human liver.
Although dihydropyrimidine dehydrogenase has been purified to varying degrees from several species, very little is known about the human enzyme. The importance of this enzyme has recently been shown with cancer chemotherapy, particularly in patients with genetic deficiency of this enzyme. In the present study, this enzyme was purified 7800-fold to homogeneity from human liver by introducing several novel methods including chromatofocusing, HPLC gel filtration, reversed-phase HPLC for the enzyme assay. Purified human enzyme has a molecular mass of 210 +/- 5 kDa and appears to be composed of two subunits. The apparent pI is pH 4.6 (+/- 0.2). The human enzyme contains approximately four flavin nucleotide molecules (two each of FAD and FMN) and 33 iron atoms per molecule of enzyme. Kinetic studies with uracil, thymine, 5-fluorouracil, and NADPH were carried out. Amino acid composition and the N-terminal amino acid sequence of this enzyme were reported. A rabbit polyclonal antibody was raised and shown to be specific for the human liver enzyme. In conclusion, in the present manuscript, we report not only a novel procedure for purification of dihydropyrimidine dehydrogenase from human liver but also new data on its properties compared to other species, which will provide a basis for further biochemical and molecular studies of this enzyme.
Lu
Z H
ZH
Department of Pharmacology, University of Alabama, Birmingham 35294.
Zhang
R
R
Diasio
R B
RB
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D042943:Dihydrouracil Dehydrogenase (NADP); D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D013552:Swine
D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP)