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 colt pyruvate d ehydrogenase complex (PDHc) and its pyruvate decarboxylating (EC 1,2,4 ,1; El) subunit is reported, 21 monoclonal antibodies were generated, and 20 were investigated, of which 9 were elicited to PDHc and 11 to p ure El subunit; 19 were of the IgG1 isotype and one of the IgG3 isotyp e. According to an enzyme immunoassay, all 20 of the monoclonal antibo dies bound the PDHc, and 17 bound the El subunit, According to Western blot analysis, 14 of the 19 monoclonal antibodies bound to the El sub unit, The monoclonal antibodies inhibited PDHc from 0 to > 98%. The si x monoclonal antibodies that displayed greater than 30% inhibition off . colt PDHc were unable to inhibit porcine heart PDHc nor did they bin d porcine heart PDHc according to dot blot analysis, Radiolabeling gav e binding constants ranging from 5 to 10 x 10(8) M(-1) on these six mo noclonal antibodies, with greater than 80% of maximal inhibition achie ved in less than 1 min, One of the six, 18A9, gave > 98% inhibition, r equired two antibodies/E1 subunit for maximum inhibition, and was show n to be a non-competitive inhibitor, Monoclonal antibody 15A9 was show n to counteract GTP-induced inhibition, while 1F2 influenced the confo rmation of El, allowing two antibodies, which did not previously bind El, to bind to it, A new mechanism-based kinetic assay is presented th at is specific for the El component of S-keto acid dehydrogenases. Thi s assay confirmed that the three most strongly inhibitory monoclonal a ntibodies specifically inhibited the El function while antibody 1F2 le d to enhanced activity, suggesting an induced conformational change in PDHc or in E1.