Purification from hamster cells of the multifunctional protein that initiates de novo synthesis of pyrimidine nucleotides.
Carbamyl-P synthetase (EC 2.7.2.9), aspartate transcarbamylase (EC 2.1.3.2), and dihydro-orotase (EC 3.5.2.3), the first three enzymes of the de novo pathway for synthesis of pyrimidine nucleotides, have been co-purified as a single oligomeric protein from a mutant line of hamster cells selected for its ability to resist N-(phosphonacetyl)-L-aspartate (PALA), a potent and specific inhibitor of aspartate transcarbamylase. All three enzymes overaccum,late in the mutant cells (Kempe, T.D., Swyryd, E.A., Bruist, M., and Stark, G.R. (1976) Cell 9, 541-550) and the oligomer represents nearly 10% of the total cellular protein. Tens of milligrams of oligomer have been purified to homogeneity by a simple and rapid procedure, with recovery of about 50% of all three activities. The pure protein contains only one size of polypeptide, Mr approximately 200,000, as revealed by electrophoresis in danaturing gels. All three enzyme activities are associated with this polypeptide, indicating that it is multifunctional. Further evidence for a multifunctional protein is provided by titration of the oligomer with radioactive PALA, which reveals that the number of PALA binding sites approximately equals the number of polypeptide chains. The isolated multifunctional protein is a mixture of trimers and hexamers.
D001221:Aspartate Carbamoyltransferase / Q000302:isolation & purification; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000302:isolation & purification; D002460:Cell Line; D004080:Dihydroorotase / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D011489:Protein Denaturation; D011742:Pyrimidine Nucleotides / Q000096:biosynthesis*; D014461:Ultracentrifugation
D009097:Multienzyme Complexes; D011742:Pyrimidine Nucleotides; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)