Vol. 137, No. 2, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS June 13. 1986 Pages 884-891 CHARACTERIZATION OF MONOCLONAL ANTIBODIES DIRECTED AGAINST PYRUVATE OXIDASE FROM ESCHERICHIA COLI: MODULATION OF ANTTRODY-INDUCED INHTBITION BY ENZYME CONFORMATION Carlos A. Barassi? Robert G. Kranzb and Robert B. Gennis* Departments of Chemistry and Biochemistry, University of Illinois, 505 South Mathews Street, Urbana, Illinois 61801 Received May 2, 1986 Monoclonal antibodies have been prepared against pyruvate oxidase, a flavoprotein dehydrogenase isolated from Escherchia ~011, six monoclonals were obtained, but only one was found to bind to the native form of the enzyme. This monoclonal, 111, was a potent inhibitor. Although this antibody inhibited the unactivated and lipid-activated forms of the enzyme, it had much less of an inhibitory effect on the protease-actf vated form of the enzyme, although the antibody still bound to this form. Hence, the coupling between antibody binding and the conformation at the active site can itself be modulated by the conformation of the protein. Q 1986 Academic Press, Inc. Pyruvate oxidase is a flavoprotein dehydrogenase associated with the aerobic respiratory chain of Escherichia coli (1,2). The enzyme catalyzes the oxidativc decarboxyation of pyruvate to acetate plus CO2 and reduces ubiquinone-8 in the cytoplasmic membrane (3). The enzyme is aCurrent address: Facultad de Ciencias Agrarias, Universidad National de Mar de1 Planta, Argentina. bCurrent address: Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637. *To whom correspondence should be addressed. Abbreviations used: PIPES, piperazine-N,N’-bis (2-ethanesulfonic acid), mAb, monoclonal antibody; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; MES, 2-(N-morpholine) ethanesulfonic acid. 0006-291X/86 $1.50 Copyright 0 I986 by Academic Press, Inc. Ail rights of reproduction in any form reserved. 884