Purification and properties of bovine liver holocarboxylase synthetase

Arch Biochem Biophys. 1994 Aug 15;313(1):8-14. doi: 10.1006/abbi.1994.1351.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biotin / metabolism
  • Carbon-Nitrogen Ligases*
  • Cattle
  • Cytosol / enzymology
  • Ligases / isolation & purification*
  • Ligases / metabolism*
  • Liver / enzymology*
  • Nucleotides / metabolism

Substances

  • Nucleotides
  • Biotin
  • Ligases
  • Carbon-Nitrogen Ligases
  • holocarboxylase synthetases