Monoclonal antibodies specific for bovine pancreatic asparagine synthetase. Production and use in structural studies.
Thirteen stable hybridoma cell lines producing monoclonal antibodies specific for asparagine synthetase were established and one monoclonal antibody was chosen to produce an immunoaffinity resin for the purification of asparagine synthetase. Bovine pancreatic asparagine synthetase was purified to a specific activity of 395 nmol of Asn produced/min/mg. Electrophoresis of the affinity-purified enzyme in sodium dodecyl sulfate polyacrylamide gels resulted in a single Mr = 54,000 polypeptide. Prior cross-linking with dimethyl suberimidate resulted in a band at Mr = 52,500 (monomer) and two additional bands at Mr = 97,000 and 98,000 (dimers), suggesting the possibility of a heterogeneous enzyme population with slight differences in subunit composition. The ratio of Gln-dependent and NH3-dependent asparagine synthetase activities was constant for immunoaffinity-purified enzyme, but the ratios of glutaminase activity to synthetase activities varied, suggesting separate aspartate and glutamine binding sites. The monoclonal antibodies were tested as inhibitors of the Gln-dependent and NH3-dependent asparagine synthetase activities as well as for inhibition of the glutaminase activity of the enzyme. Two antibodies inhibited Gln- and NH3-dependent synthesis of asparagine, but did not affect the glutaminase activity of immunoaffinity-purified asparagine synthetase. A third monoclonal antibody inhibited Gln-dependent synthesis of asparagine and glutaminase activity, but activated NH3-dependent asparagine synthetase activity. These data are discussed in terms of multiple substrate binding domains within the asparagine synthetase molecule.
Pfeiffer
N E
NE
Mehlhaff
P M
PM
Wylie
D E
DE
Schuster
S M
SM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D001217:Aspartate-Ammonia Ligase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification*; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D004120:Dimethyl Suberimidate; D005972:Glutaminase / Q000276:immunology / Q000302:isolation & purification; D005973:Glutamine / Q000378:metabolism; D006825:Hybridomas / Q000276:immunology; D008025:Ligases / Q000302:isolation & purification*; D010179:Pancreas / Q000201:enzymology*; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D005973:Glutamine; D004120:Dimethyl Suberimidate; D001224:Aspartic Acid; D005972:Glutaminase; D008025:Ligases; D001217:Aspartate-Ammonia Ligase