title: Purification and properties of 3-hydroxyacyl coenzyme A dehydrogenase-binding protein from rat liver mitochondria.
abstract: Mitochondria isolated from rat liver were freeze-thawed and washed with 0.1 M potassium phosphate, pH 7.4. Most of the 3-hydroxyacyl coenzyme A (CoA) dehydrogenase activities were removed and this mitochondrial membrane fraction could bind exogenous 3-hydroxyacyl-CoA dehydrogenase. 3-Hydroxyacyl-CoA dehydrogenase-binding protein was extracted from the washed membrane fraction with a buffer containing 2% Triton X-100 and 2% sodium taurodeoxycholate. The binding protein was purified by Ultrogel AcA 34 gel chromatography, calcium phosphate gel-cellulose chromatography and 3-hydroxyacyl-CoA dehydrogenase affinity chromatography. The molecular mass of the purified binding protein was estimated to be 140 kDa by gel filtration and its subunit molecular mass was determined as 60 kDa by SDS-PAGE suggesting that the protein is a homodimer. The binding protein and 3-hydroxyacyl-CoA dehydrogenase formed a complex at low ionic strength and the stoichiometry revealed that 1 mol of the binding protein bound 2 mol of 3-hydroxyacyl-CoA dehydrogenase. Purified 3-hydroxyacyl-CoA dehydrogenase-binding protein interacted with 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase but did not bind other mitochondrial beta-oxidation enzymes. The pH optimum of the binding activity was from pH 6 to 7 and the binding activity was diminished by increasing the concentration of salt in the medium.
authors.affiliation: Department of Biochemistry, Shinshu University School of Medicine, Nagano. sfuruta@gipac.shinshu-u.ac.jp
authors.forename: S
authors.initials: S
authors.lastname: Furuta
authors.forename: T
authors.initials: T
authors.lastname: Hashimoto
mesh_terms: D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D008930:Mitochondria, Liver / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats
chemical_list: D011506:Proteins; D015094:3-Hydroxyacyl CoA Dehydrogenases