Characterization of a monoclonal antibody recognizing the E1α subunit of plant mitochondrial pyruvate dehydrogenase

Abstract

Summary We have isolated a monoclonal antibody that recognizes the E1α subunit of the plant mitochondrial pyruvate dehydrogenase complex. The antibody specifically recognizes the Ela subunit from maize seedling, pea leaf, and castor oil seed endosperm mitochondrial pyruvate dehydrogenases, but does not recognize the E1α subunit present in the plastid complexes from these plants. The pea mitochondrial pyruvate dehydrogenase complex was used for subsequent characterization of the antibody. Two-dimensional electrophoretic analysis of a phosphorylated pea mitochondrial pyruvate dehydrogenase complex preparation revealed that the monoclonal antibody recognizes all phosphorylated forms of the E1α subunit. Under these conditions, the only proteins recognized by the antibody are phosphorylated. Binding of the antibody to the pyruvate dehydrogenase complex inhibits both catalytic activity and phosphorylation of the E1α subunit, but does not significantly inhibit dephosphorylation. The monoclonal antibody recognizes mitochondrial E1α subunits from a variety of plant materials including monocot and dicot seedlings, and thermogenic and storage tissues. The antibody does not recognize the E1α subunit from rat liver or pig heart mitochondria, yeast, or bacteria. This highly specific antibody will be a useful tool for study of plant mitochondrial pyruvate dehydrogenase complexes.