Conformation-specific monoclonal antibodies to glutamine synthetase in Escherichia coli.
Glutamine synthetase from Escherichia coli is composed of 12 identical subunits and exists in various forms: unadenylylated, adenylylated, divalent cation bound (taut), and divalent cation free (relaxed). The relaxed dodecamer readily dissociates into monomers upon exposure to 1 M urea or pH 8.0. Glutamine synthetase can be inactivated irreversibly by oxidizing a particular histidine residue or by incubating with methionine sulfoximine and ATP. In order to establish hybridoma monoclones that produce antibodies capable of differentiating between different conformers of glutamine synthetase, homogeneous antibodies produced from 7 clones (10-76-1, 48-76-1, 68-2-1, 57-142-2, 72-104-1, 68-3-2, 57-8-1) were characterized for their binding specificity and effects on glutamine synthetase activity. Two antibodies (10-76-1, 48-76-1) bind only to the monomeric form, two antibodies (57-142-2, 68-3-2) bind only to the dodecameric forms (taut or relaxed) and the three others (68-2-1, 72-104-1, 57-8-1) bind to both forms. At a low antibody concentration, 68-3-2 binds preferentially to taut glutamine synthetase over oxidized glutamine synthetase. None of the 7 antibodies differentiates between unadenylylated and adenylylated form. Nevertheless, the gamma-glutamyltransferase activities of the resulting antibody-glutamine synthetase complexes were influenced variably depending upon the state of adenylylation and the divalent cation.
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000276:immunology*; D006639:Histidine / Q000378:metabolism; D007525:Isoelectric Focusing; D008027:Light; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D011487:Protein Conformation; D012542:Scattering, Radiation
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D006639:Histidine; D005974:Glutamate-Ammonia Ligase; D008274:Magnesium