Nucleotide cofactor-binding-domain-specific antibodies show immunologic relatedness among unrelated proteins that bind phosphoryl compounds.
The immunologic relatedness of various cofactor-binding sites of enzymes requiring different nucleotide cofactors was examined. Chicken antibodies specific for NADPH- or CoA-binding domains were raised using an NADPH- or CoA-requiring enzyme as an immunogen. Antibodies specific for either NADPH- or CoA-binding domains were isolated by immunoaffinity chromatography of the respective antisera using unrelated NADPH- or CoA-requiring enzymes as affinity ligands. The reactivities of the NADPH- and CoA-binding-site-specific antibodies with a variety of enzymes that required different cofactors was shown on Western blots of SDS-PAGE of the enzymes. Variable cross-reactivities were observed among all nucleotide-cofactor requiring enzymes with each specific cofactor-domain-antibody population. Numerous proteins not physiologically associated with nucleotide cofactors, including acyl carrier protein, were completely unreactive. Proteins that bound phosphoryl compounds either as substrates or cofactors showed varying degrees of reactivity with each population of specific antibodies. These included aldolase, ribulose-1,5-bisphosphate carboxylase/oxygenase, ribonuclease A, carbonic anhydrase and triosephosphate isomerase. The immunologic cross-reactivity suggested that these proteins share a common structural feature, probably a primary structure epitope, since the proteins had been subjected to denaturing polyacrylamide gel electrophoresis. A candidate for this common structural feature is a glycine-rich sequence comprising a phosphate binding loop.
Tucker
M M
MM
Department of Biochemistry, James H. Quillen College of Medicine, East Tennessee State University, Johnson City 37614-0581.
Worsham
L M
LM
Ernst-Fonberg
M L
ML
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001665:Binding Sites; D015153:Blotting, Western; D002645:Chickens; D003065:Coenzyme A / Q000276:immunology / Q000378:metabolism*; D003429:Cross Reactions; D004798:Enzymes / Q000276:immunology / Q000378:metabolism*; D005260:Female; D009249:NADP / Q000276:immunology / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*
D000906:Antibodies; D004798:Enzymes; D009943:Organophosphorus Compounds; D009249:NADP; D003065:Coenzyme A