Open AccessAlteration in structure of multifunctional protein from Chinese hamster ovary cells defective in pyrimidine biosynthesis
Author(s) -
Jeffrey N. Davidson,
David Patterson
Publication year - 1979
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.76.4.1731
Subject(s) - aspartate carbamoyltransferase , pyrimidine metabolism , chinese hamster ovary cell , biochemistry , enzyme , structural gene , biosynthesis , pteridine , dihydroorotate dehydrogenase , biology , mutant , microbiology and biotechnology , amidohydrolase , chemistry , gene , receptor , allosteric regulation , purine
A combined genetic, biochemical, and immunological approach has clarified structural relationships involving the first three enzymes ofde 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 (≈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.