Comparison of solution structures of dihydrofolate reductases and enzyme-ligand complexes using cross-reacting antibodies.
Polyclonal antibodies against dihydrofolate reductase (DHFR) from the human lymphoblastoid cell line WIL-2/M4 were used as probes to compare the antigenic structures in solution of native DHFRs obtained from a broad range of species and their complexes with substrate, cofactor, and folate antagonist inhibitors. All these antibodies could bind to the denatured human DHFR, indicating that they were specific for the primary structure of this enzyme. Denatured chicken liver and L1210 murine leukemic DHFRs competed for all of the antibodies that bound to the human enzyme, although less effectively than the denatured human enzyme, showing the presence of similar epitopes among the vertebrate enzymes. However, both direct binding and competition experiments showed low antibody cross-reactivities with native chicken liver (8%) and murine (10%) DHFRs, suggesting differences in the disposition of similar epitopes in these enzymes. The lactobacillus casei DHFR showed a low amount (less than 2%) of cross-reactivity with the antibodies while the same antibodies did not cross-react with the Escherichia coli enzyme. DHFR from soybean seedlings competed for a large proportion (70%) of the anti-human DHFR antibodies, indicating a close similarity in the antigenic structures of plant and animal DHFRs. Binary complexes of the L. casei, avian, murine, and human DHFRs with dihydrofolate, methotrexate (MTX), trimethoprim (TMP), NADPH, and NADP+ all showed significantly lower antibody binding capacity as compared with the corresponding free enzymes. Further, these ligands inhibited antibody binding to the enzyme to varying degrees.(ABSTRACT TRUNCATED AT 250 WORDS)
D000906:Antibodies; D000936:Antigen-Antibody Complex; D002460:Cell Line; D003429:Cross Reactions; D000939:Epitopes / Q000032:analysis; D006801:Humans; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D013762:Tetrahydrofolate Dehydrogenase / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D000939:Epitopes; D008024:Ligands; D013762:Tetrahydrofolate Dehydrogenase