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Abstract
Bovine pancreatic asparagine synthetase was studied with regard to the ammonia-dependent production of asparagine. Asparagine was found to be a partial competitive inhibitor with respect of ammonia, except when MgATP was nonsaturating, then it became linear competitive. This is consistent with a kinetic mechanism where ammonia is bound followed by MgATP which causes asparagine release.
Monoclonal antibodies were produced to asparagine synthetase and used in immunoaffinity chromatography to purify the enzyme 1159-fold. Two of the antibodies inhibited glutamine and ammonia dependent activities but not glutaminase, and another inhibited glutamine dependent and glutaminase activities but not ammonia dependent. This is discussed in terms of multiple substrate binding domains in asparagine synthetase.
The monoclonal antibodies which inhibited asparagine synthetase were mapped to two topographically separate regions of the enzyme surface using competitive binding assays. The results of these assays suggest a topographical separation between an asparatyl-AMP and glutamine binding sites. The non-inhibitory antibodies could be mapped to a third distinct site. Binding studies showed extensive cross-reaction of these antibodies with enzyme from bovine liver and sheep pancreas, and to a lesser degree with rat liver or pancreas, a human tumor cell line, and a mouse tumor cell line. Only one of the four antibodies inhibitory toward the ruminants was inhibitory toward rodent, suggesting significant structural differences between the enzymes.
The presence of dimer and monomer forms of asparagine synthetase and factors affecting the relative amounts of each was studied. The inhibition of the enzyme by substrate analogs DON and CONV and their effect on subunit composition was also studied. Among other things the analogs were found to shift the enzyme to the dimer form and CONV caused it to become uv invisible as well. When Mabs were allowed to bind to enzyme previously inactivated by the analogs the binding of Mab 5A6 was prevented, giving further proof that 5A6 recognizes the glutamine binding site of the enzyme.
