ANTIPEPTIDE IMMUNOGLOBULINS FROM RABBIT AND CHICKEN EGGS RECOGNIZE RECOMBINANT HUMAN DIHYDROOROTATE DEHYDROGENASE AND A 44-KDA PROTEIN FROMRAT-LIVER MITOCHONDRIA

Mitochondrially bound dihydroorotate dehydrogenase catalyses the fourt h sequential step in the de novo synthesis of uridine monophosphate. 3 12-bp and 983-bp regions of the human dihydroorotate dehydrogenase seq uence (1496 bp) were amplified by the polymerase chain reaction, and s ubcloned into the expression vector pQE 32. The identity of the PCR pr oducts was verified by dideoxynucleotide sequencing. Transformation of Escherichia coli strain M15 resulted in expression of 13-kDa and 36-k Da proteins with an affinity tag consisting of six consecutive histidi ne residues; these proteins could be purified by solubilisation in 8 M urea and by chromatography on a Ni2+-chelating resin. In immunoblotti ng analyses, the fusion proteins were recognised by polyclonal avian a nd mammalian anti-peptide immunoglobulins. These were generated agains t synthetic peptides corresponding to two amino acid sequences deduced from human and rat cDNA of dihydroorotate dehydrogenase. The peptides were synthesized as multiple copies on a branching lysyl matrix. Rabb its and laying hens were immunized with these peptides without conjuga tion to a carrier protein. Comparison of the anti-peptide immunogobuli ns produced from egg yolk and rabbit serum demonstrated that avian ant i-(dihydroorotate dehydrogenase) immunoglobulins may be considered a s uperior alternative to the mammalian equivalent; antibodies from both sources were applicable for all immunochemical purposes. Here, these a ntibodies were applied for identification of a 44-kDa protein from rat liver mitochondria, which was correlated with dihydroorotate dehydrog enase activity.