Bovine pancreatic asparagine synthetase explored with substrate analogs and specific monoclonal antibodies.
Several substrate analogs were tested for their ability to inhibit bovine pancreatic asparagine synthetase. Of the substrate analogs tested both 6-diazo-5-oxo-L-norleucine (DON) and 5-chloro-4-oxo-L-norvaline (CONV) were shown to inhibit the enzyme strongly. DON inhibited the glutaminase and glutamine-dependent asparagine synthetase activities and CONV inhibited the ammonia-dependent activity as well. Both of these inhibitors appeared to be relatively tight binding since desalting failed to remove the inhibition. The inactivation of bovine pancreatic asparagine synthetase by DON is accompanied by a shift from a 47,000 molecular weight monomer to a 96,000 molecular weight dimer as observed by HPLC gel filtration chromatography. This DON-induced shift is prevented by the presence of the substrate glutamine. A monoclonal antibody known to inhibit specifically the ammonia-dependent and glutamine-dependent asparagine synthetase activities but not glutaminase (monoclonal antibody 2B4) binds to both the monomer and the dimer forms of untreated enzyme, as well as to the dimer form of the DON-inactivated enzyme. On the other hand, a monoclonal antibody known to inhibit specifically the glutaminase and glutamine-dependent activities and not the ammonia-dependent asparagine synthetase (monoclonal antibody 5A6) binds to both forms of untreated enzyme but cannot bind to the DON-inactivated enzyme. These data are used to describe the relation of regions of the active site of asparagine synthetase in relation to antibody binding sites.
Department of Pathology, University of California, La Jolla 92093.
P M
PM
Mehlhaff
S M
SM
Schuster
D000641:Ammonia / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001217:Aspartate-Ammonia Ligase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D002850:Chromatography, Gel; D003980:Diazooxonorleucine / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D000939:Epitopes; D005973:Glutamine / Q000378:metabolism; D046911:Macromolecular Substances; D015394:Molecular Structure; D010179:Pancreas / Q000201:enzymology; D011485:Protein Binding
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D003980:Diazooxonorleucine; D005973:Glutamine; D000641:Ammonia; D001217:Aspartate-Ammonia Ligase