Topographical separation of the catalytic sites of asparagine synthetase explored with monoclonal antibodies.
Monoclonal antibodies which inhibited the enzymatic activity of bovine pancreatic asparagine synthetase were mapped to two topographically separate regions of the enzyme surface using competitive binding assays. Three antibodies which all inhibited glutamine- and NH3-dependent synthesis of asparagine bound to a common antigenic region. A fourth monoclonal antibody which interfered with glutamine binding or cleavage but not with NH3-dependent synthesis of asparagine was mapped to a separate region of the enzyme surface. These findings suggest a topographical separation between the aspartyl-AMP and glutamine-binding sites of bovine pancreatic asparagine synthetase. Three noninhibitory antibodies exhibited conformation-dependent binding and were mapped to a third region of the enzyme. Binding assays were used to demonstrate extensive cross-reaction of these antibodies with asparagine synthetases isolated from bovine liver and sheep pancreas. Substantial cross-reactions were also seen with the enzyme isolated from rat liver or pancreas, a human tumor cell line, and a mouse tumor cell line. Of the four antibodies that inhibited glutamine- and NH3-dependent synthesis of asparagine from ruminant species, only one bound to and inhibited the enzyme from rat liver or mouse cells, which suggests significant structural differences between the ruminant and rodent enzymes.
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D001217:Aspartate-Ammonia Ligase / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D002460:Cell Line; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D051379:Mice; D010179:Pancreas / Q000201:enzymology; D010449:Peptide Mapping; D051381:Rats; D012756:Sheep; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D005973:Glutamine; D001224:Aspartic Acid; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000469:Alkaline Phosphatase; D008025:Ligases; D001217:Aspartate-Ammonia Ligase