title: Isolation and characterization of buckwheat (Fagopyrum esculentum M.) chalcone synthase and its polyclonal antibodies.
abstract: 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.
mesh_terms: D000217:Acyltransferases / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D000917:Antibody Formation; D002523:Edible Grain / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005779:Immunodiffusion; D007700:Kinetics; D008027:Light; D011817:Rabbits
chemical_list: D000906:Antibodies; D000217:Acyltransferases; C020141:flavanone synthetase