title: Purification and properties of bovine liver holocarboxylase synthetase.
abstract: Holocarboxylase synthetase was purified 18,000-fold from bovine liver cytosol by a sequence of ammonium sulfate fractionation, alumina C gamma fractionation, DEAE-Sepharose CL-6B, EAH-Sepharose 4B, Sephacryl S-200 HR, Bio-Gel HTP, and Phenyl-Superose HR 5/5 chromatographies. Holocarboxylase synthetase activity was assayed using apopropionyl-CoA carboxylase from a patient with holocarboxylase synthetase deficiency as a substrate. Apopropionyl-CoA carboxylase was easily prepared from cultured lymphoblasts from this patient. Enzyme activity coincided with a 64,000-Da protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Additionally, the molecular mass of the native enzyme was estimated to be 60,000 Da by gel filtration on Sephacryl S-200 HR. These results suggest that purified holocarboxylase synthetase from bovine liver cytosol is a monomeric enzyme. Its Km value for biotin was estimated to be 13 nM.
authors.affiliation: Department of Biochemical Genetics, Tohoku University School of Medicine, Sendai, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Chiba
authors.forename: Y
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Y
authors.initials: Y
authors.lastname: Aoki
authors.forename: Y
authors.initials: Y
authors.lastname: Ishida
authors.forename: K
authors.initials: K
authors.lastname: Narisawa
mesh_terms: D000818:Animals; D001710:Biotin / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases; D002417:Cattle; D003600:Cytosol / Q000201:enzymology; D008025:Ligases / Q000302:isolation & purification* / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D009711:Nucleotides / Q000378:metabolism
chemical_list: D009711:Nucleotides; D001710:Biotin; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C023312:holocarboxylase synthetases