Anti-AMP antibody precipitation of multiply adenylylated forms of glutamine synthetase from Escherichia coli: a model relating both concentration and density of antigenic sites with the antibody-antigen interaction.
Sheep antibodies directed against an AMP-bovine serum albumin conjugate exhibit highly specific binding toward AMP. These antibodies bind to the AMP moiety of adenylylated glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] from Escherichia coli and to no other antigenic determinant on the protein. E coli glutamine synthetase can exist in variously modified (isomeric) forms that differ with respect to the number (0-12) and the distribution of identically adenylylated subunits [Ciardi, J. E., Cimino, F. & Stadtman, E. R. (1973) biochemistry 12, 4321-4330]. Using this enzyme, together with the AMP-specific antibodies, we have investigated the effects of the total concentration, population density, and topographical distribution of multiple identical antigenic determinants on the antigen-antibody interaction. Stopped flow fluorescence measurements show that the rate and extent of initial binding of the antibodies to the antigen are a function of the total concentration of AMP groups and are independent of the number of AMP groups der dodecamer. However, the rate of lattice formation increases with increasing epitope density, and the maximal amount of glutamine synthetase precipitated is directly proportional to the average number of adenylylated subunits per dodecamer. The data suggest that partially adenylylated enzyme preparations are composed of subpopulations of glutamine synthetase molecules that differ in their tendency to form precipitable aggregates, due presumably to differences in the topographical distribution of antigenic determinants on the surface of the enzyme. The enzyme species that form soluble immune complexes do so possibly due to intramolecular crosslinkage of the bivalent antibodies with adenylylated subunits to the exclusion of intermolecular crosslinkage.
D000249:Adenosine Monophosphate / Q000276:immunology*; D000936:Antigen-Antibody Complex; D000937:Antigen-Antibody Reactions; D004306:Dose-Response Relationship, Immunologic; D000939:Epitopes; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000276:immunology*; D012995:Solubility
D000936:Antigen-Antibody Complex; D000939:Epitopes; D000249:Adenosine Monophosphate; D005974:Glutamate-Ammonia Ligase