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: Y Chiba, Y Suzuki, Y Aoki, Y Ishida, K Narisawa
• Journal: Archives of biochemistry and biophysics (Arch Biochem Biophys) Vol. 313 Issue 1 Pg. 8-14 (Aug 15 1994) ISSN: 0003-9861 [Print] United States
• PMID: 8053691 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

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

Topics

• Animals
• Biotin (metabolism)
• Carbon-Nitrogen Ligases
• Cattle
• Cytosol (enzymology)
• Ligases (isolation & purification, metabolism)
• Liver (enzymology)
• Nucleotides (metabolism)