Use of monoclonal antibodies in the purification and characterization of 1-aminocyclopropane-1-carboxylate synthase, an enzyme in ethylene biosynthesis

Abstract

1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14), extracted from tomato pericarp tissue, was purified 6500-fold by conventional and high-performance liquid chromatography. Two-dimensional gel electrophoresis of this preparation indicated that ACC synthase activity was associated with a protein band at 50 kDa, a value consistent with size determinations by gel filtration. Monoclonal antibodies against ACC synthase were obtained from murine hybridoma cell lines. These antibodies recognized the native enzyme, as shown with an immunoprecipitation assay. A monoclonal IgG immunoaffinity gel was used to isolate, from a relatively crude enzyme preparation, a single protein, which migrated at 50 kDa in a NaDodSO(4)/polyacrylamide gel. In vivo labeling of wounded tomato pericarp tissue with [(35)S]methionine followed by immunoaffinity purification of ACC synthase yielded a radioactive protein of 50 kDa. We conclude that the 50-kDa protein represents ACC synthase in extracts of wounded tomato pericarp tissue.