Purification of homogeneous glutamine-dependent carbamyl phosphate synthetase from ascites hepatoma cells as a complex with aspartate transcarbamylase and dihydroorotase.
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.
D000581:Amidohydrolases / Q000302:isolation & purification*; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000302:isolation & purification*; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000302:isolation & purification*; D006528:Carcinoma, Hepatocellular / Q000201:enzymology*; D066298:In Vitro Techniques; D008113:Liver Neoplasms / Q000201:enzymology*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification; D009374:Neoplasms, Experimental / Q000201:enzymology; D010770:Phosphotransferases / Q000302:isolation & purification*; D051381:Rats
D009097:Multienzyme Complexes; D001221:Aspartate Carbamoyltransferase; D010770:Phosphotransferases; D000581:Amidohydrolases; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)