Production of a monocot-specific monoclonal antibody against acetohydroxyacid synthase and its use in the purification and characterization of the enzyme
Singh, B.K. | Lumanglas, A. | Wang, B.S.
Acetohydroxyacid synthase [AHAS; acetolactate pyruvate-lyase (carboxylating). EC 4.1.3.18], the first enzyme unique to the biosynthesis of valine, leucine, and isoleucine, is a known target for several different chemical classes of herbicides. Antibodies required for immunological characterization of the enzyme have not been generated by the conventional method of antibody production using purified protein. Monoclonal antibodies were raised against AHAS from corn by using as immunogen a synthetic peptide representing this enzyme. This antibody immunoprecipitated the enzyme activity from corn. On a Western blot, a protein band with a molecular weight of 65,000 was detected in crude extracts of corn. Furthermore, a monoclonal antibody immunoaffinity gel was used to isolate a single protein from crude enzyme preparations that migrated at Mr, 65,000 in an SDS/polyacrylamide gel. The molecular weight of this protein band is the molecular weight predicted for a plant AHAS from a cloned gene sequence. These results strongly suggest that the Mr 65,000 protein represents AHAS in corn extracts. Interestingly, the monoclonal antibody specifically recognized the enzyme from monocots and did not crossreact with AHAS from any dicot species tested. Identification of this monoclonal antibody that distinguishes monocot and dicot AHAS is significant because of a very high degree of amino acid conservation (85%) between AHAS proteins from different species.
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