title: Binding properties of the components of branched-chain 2-oxo acid dehydrogenase complex on ELISA.
abstract: Binding characteristics among three catalytic components of rat liver branched-chain 2-oxo acid dehydrogenase complex (BCKADH) were investigated by ELISA. Dihydrolipoamide dehydrogenase (E3) was bound to solid-phase dihydrolipoamide acyltransferase (E2). The binding curve was hyperbolic giving a calculated half-maximal binding concentration of 167 ng/ml for E3. Specificity of the binding of E3 to E2 was certified by a competition experiment measuring binding of biotin-labeled E3 in the presence of unlabeled E3. The decarboxylase component (E1), which is the other catalytic component of the complex, prevented the E3 binding to E2. The specific binding between E2 and E3 was verified in the opposite direction with immobilized E3. E2 also bound to solid-phase E1 in a specific manner, and addition of E3 prevented the E2 binding to E1. No binding between E1 and E3 was observed. Thus, E1 and E3 prevented each other's binding to E2, suggesting that E1 and E3 may recognize overlapped binding sites on the E2 polypeptide or that they may, at least in part, sterically interact with each other on their binding to E2. The reconstitution experiment of the complex also supported such a mutually exclusive binding of E1 and E3 to the E2 core.
authors.affiliation: Department of Biochemistry, Fukushima Medical College.
authors.forename: T
authors.initials: T
authors.lastname: Mori
authors.forename: Y
authors.initials: Y
authors.lastname: Oshima
authors.forename: H
authors.initials: H
authors.lastname: Kochi
mesh_terms: D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats
chemical_list: D009097:Multienzyme Complexes; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)