Alteration in structure of multifunctional protein from Chinese hamster ovary cells defective in pyrimidine biosynthesis.
A combined genetic, biochemical, and immunological approach has clarified structural relationships involving the first three enzymes of de novo pyrimidine biosynthesis. A procedure involving antibody and protein A-Sepharose was used to isolate the enzymes carbamoyl-phosphate synthase [ATP:carbamate phosphotransferase (dephosphorylating, amido-transferring), EC 2.7.2.9], aspartate transcarbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), and dihydro-orotase (L-5,6-dihydroorotate amidohydrolase, EC 3.5.2.3) from Chinese hamster ovary cell CHO-K1, the uridine-requiring auxotroph Urd(-)A, and selected Urd(-)A revertants. The enzymes of Urd(-)A and the Urd(-)A revertants were significantly altered in activity, native structure, and molecular weight from those of CHO-K1. The results presented permit the conclusion that (i) these three enzymes reside in a single multifunctional 220,000-dalton polypeptide; (ii) the aspartate transcarbamoyltransferase activity is located on a portion ( approximately 20,000 daltons) at one end of the polypeptide; (iii) this portion may also be required for monomers to aggregate into the multimeric from present in mammalian cells; (iv) the mutations in Urd(-)A and the Urd(-)A revertants lie in the structural gene for this multifunctional protein; and (v) increased sensitivity to proteases could account for the alterations in the structure of these enzymes in the mutants.
D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D004080:Dihydroorotase / Q000378:metabolism*; D005260:Female; D046911:Macromolecular Substances; D008970:Molecular Weight; D009154:Mutation; D010053:Ovary; D010770:Phosphotransferases / Q000378:metabolism*; D011743:Pyrimidines / Q000096:biosynthesis*
D046911:Macromolecular Substances; D011743:Pyrimidines; D001221:Aspartate Carbamoyltransferase; D010770:Phosphotransferases; D000581:Amidohydrolases; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)