A library of monoclonal antibodies to Escherichia coli K-12 pyruvate dehydrogenase complex. A biochemical analysis and their ability to inhibit the enzyme complex.
A library of monoclonal antibodies to K-12 Escherichia coli pyruvate dehydrogenase complex (PDHc) and its pyruvate decarboxylating (EC 1.2.4.1; E1) subunit is reported. 21 monoclonal antibodies were generated, and 20 were investigated, of which 9 were elicited to PDHc and 11 to pure E1 subunit; 19 were of the IgG1 isotype and one of the IgG3 isotype. According to an enzyme immunoassay, all 20 of the monoclonal antibodies bound the PDHc, and 17 bound the E1 subunit. According to Western blot analysis, 14 of the 19 monoclonal antibodies bound to the E1 subunit. The monoclonal antibodies inhibited PDHc from 0 to > 98%. The six monoclonal antibodies that displayed greater than 30% inhibition of E. coli PDHc were unable to inhibit porcine heart PDHc nor did they bind porcine heart PDHc according to dot blot analysis. Radiolabeling gave binding constants ranging from 5 to 10 x 10(8) M-1 on these six monoclonal antibodies, with greater than 80% of maximal inhibition achieved in less than 1 min. One of the six, 18A9, gave > 98% inhibition, required two antibodies/E1 subunit for maximum inhibition, and was shown to be a non-competitive inhibitor. Monoclonal antibody 15A9 was shown to counteract GTP-induced inhibition, while 1F2 influenced the conformation of E1, allowing two antibodies, which did not previously bind E1, to bind to it. A new mechanism-based kinetic assay is presented that is specific for the E1 component of 2-keto acid dehydrogenases. This assay confirmed that the three most strongly inhibitory monoclonal antibodies specifically inhibited the E1 function while antibody 1F2 led to enhanced activity, suggesting an induced conformational change in PDHc or in E1.
McNally
A J
AJ
Roche Diagnostics Systems, Inc., Somerville, New Jersey 08876-1760, USA.
Motter
K
K
Jordan
F
F
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000918:Antibody Specificity; D001665:Binding Sites; D001667:Binding, Competitive; D004926:Escherichia coli / Q000201:enzymology* / Q000276:immunology*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006825:Hybridomas / Q000276:immunology; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D011487:Protein Conformation; D011767:Pyruvate Decarboxylase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology; D011768:Pyruvate Dehydrogenase Complex / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology*; D011869:Radioligand Assay; D013045:Species Specificity; D013552:Swine
D000911:Antibodies, Monoclonal; D011768:Pyruvate Dehydrogenase Complex; D006160:Guanosine Triphosphate; D011767:Pyruvate Decarboxylase