Comparison of dihydropyrimidine dehydrogenase from human, rat, pig and cow liver. Biochemical and immunological properties.
Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the catabolic pathway of pyrimidines and has an important role in cancer chemotherapy with fluoropyrimidine drugs. Recently, we purified and characterized this enzyme from human liver and raised a rabbit polyclonal antibody against the purified human enzyme (Lu et al., J Biol Chem 267: 17102-17109, 1992). In the present study, using this purification procedure, DPD was purified to homogeneity from three other mammalian species, i.e. pig, rat, and cow. Comparison of the biochemical properties of these purified enzymes was conducted. Molecular masses of DPD from human, pig, rat, and cow liver were: 210, 204, 210, and 216 kDa, respectively. DPD from all four species appeared to be composed of two subunits. The apparent pI values were 4.6, 4.8, 4.85, and 5.25, respectively. Kinetic studies with uracil, thymine, 5-fluorouracil, and NADPH were carried out with the purified DPD preparation, suggesting species differences in kinetic parameters. Amino acid composition of these purified enzymes also demonstrated slight species differences. In the present study, a rabbit polyclonal antibody against rat liver DPD was raised. Using polyclonal antibodies against human and rat liver DPD, immunoblotting demonstrated cross-reactivity among the four species. In summary, purification and comparison of DPD from different mammalian species will provide a basis for further biochemical and molecular studies of this enzyme.
Lu
Z H
ZH
Department of Pharmacology, University of Alabama at Birmingham 35294.
Zhang
R
R
Diasio
R B
RB
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002417:Cattle; D003429:Cross Reactions; D042943:Dihydrouracil Dehydrogenase (NADP); D006801:Humans; D007526:Isoelectric Point; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D011817:Rabbits; D051381:Rats; D013045:Species Specificity; D013552:Swine
D000596:Amino Acids; D000906:Antibodies; D010088:Oxidoreductases; D042943:Dihydrouracil Dehydrogenase (NADP)