Purification of homogeneous glutamine-dependent carbamyl phosphate synthetase from ascites hepatoma cells as a complex with aspartate transcarbamylase and dihydroorotase.

Abstract

Glutamine-dependent carbamyl phosphate synthetase [EC 2.7.2.9] was purified 1,300-fold from rat ascites hepatoma cells (AH 13) as a multienzyme complex with aspartate transcarbamylase[EC 2.1.3.2] and dihydroorotase[EC 3.5.2.3], using dimethyl sulfoxide, glycerol, and dithiothreitol as stabilizers. The purified complex was essentially homogeneous on agarose-acrylamide composite gel electrophoresis and analytical ultracentrifugation. Its molecular weight was estimated to be about 870,000 by sedimentation equilibrium studies. After alkylation with iodoacetamide or reduction with 0.6% dithiothreitol at 100 degrees, the complex gave a single band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate in a position corresponding to a molecular weight of 210,000. These results indicate that the complex consists of four subunits of similar size.

• Authors: M Mori, M Tatibana
• Journal: Journal of biochemistry (J Biochem) Vol. 78 Issue 1 Pg. 239-42 (Jul 1975) ISSN: 0021-924X [Print] England
• PMID: 172492 (Publication Type: Journal Article)

Chemical References

• Multienzyme Complexes
• Aspartate Carbamoyltransferase
• Phosphotransferases
• Amidohydrolases
• Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)

Topics

• Amidohydrolases (isolation & purification)
• Animals
• Aspartate Carbamoyltransferase (isolation & purification)
• Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) (isolation & purification)
• Carcinoma, Hepatocellular (enzymology)
• In Vitro Techniques
• Liver Neoplasms (enzymology)
• Molecular Weight
• Multienzyme Complexes (isolation & purification)
• Neoplasms, Experimental (enzymology)
• Phosphotransferases (isolation & purification)
• Rats