Isolation and characterization of buckwheat (Fagopyrum esculentum M.) chalcone synthase and its polyclonal antibodies.

Chalcone synthase was isolated from illuminated buckwheat (Fagopyrum esculentum M.) hypocotyls and purified to electrophoretic homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using (NH)4SO4 fractionation, gel filtration on AcA 44, ion exchange chromatography on DEAE-Bio-Gel, and HPLC on hydroxylapatite. The properties of the enzyme were pH optimum, 8.0; Mr approximately 83,000 +/- 1000; Mr subunit, approximately 41,500 +/- 500; isoelectric point, pH 5.2; Km, 1 X 10(-6)M for malonyl-CoA, and 0.6 X 10(-6) M for p-coumaryl-CoA. Buckwheat chalcone synthase used p-coumaryl-CoA as substrate and also utilized caffeyl-CoA and ferulyl-CoA at 20 and 80%, respectively, of the rate of p-coumaryl-CoA in the chalcone synthase reaction. Antibodies against the buckwheat chalcone synthase were developed in a New Zealand white rabbit and characterized for specificity by enzyme-linked immunosorbent assay, Ouchterlony double immunodiffusion, and Western blotting.