Ligand-induced structural constraints in human dihydrofolate reductase revealed by peptide-specific antibodies.
Peptides from human dihydrofolate reductase (DHFR) generated by cyanogen bromide cleavage and corresponding to residues 15-52, 53-111, 112-125, and 140-186 (carboxyl terminus) were purified and used to immunize rats. Titration of the immune sera against denatured human DHFR by solid-phase immunoassay showed that peptides 15-52 and 140-186 were relatively highly immunogenic, unlike the native enzyme which is most immunogenic in the sequence 53-111. The antisera were specific for the corresponding peptides used for immunization. Antibodies to peptides 15-52, 53-111, and 140-186 cross-reacted with native human DHFR in solution in competition assays. However, the binding of nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and the inhibitors folate and methotrexate, both in binary and in ternary complexes with the enzyme, caused a striking reduction in binding of antibody. Using a sensitive radioactive assay, it was found that antisera to peptides 15-52 and 140-186, both of which exhibited a high antibody titer, caused significant inhibition of DHFR. Because peptide 140-186 does not include any active-site residues, it is concluded that at least in this case all the antibodies bound to regions outside the active site. Since comparison of the X-ray structures of the chicken liver DHFR holoenzyme with the apoenzyme reveals no changes in secondary structural elements (alpha-helices and beta-sheets), the reduction in antibody binding to DHFR-ligand complexes must not involve epitopes within these structures.(ABSTRACT TRUNCATED AT 250 WORDS)
D000906:Antibodies; D000936:Antigen-Antibody Complex; D003429:Cross Reactions; D003488:Cyanogen Bromide; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D003488:Cyanogen Bromide