Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells: III. Association of carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase in mutants of cultured Chinese hamster cells

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Abstract
Carbamyl phosphate synthetase (EC 2.7.2.9), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3), the first three enzymes in de novo pyrimidine synthesis in Chinese hamster ovary cell strain Kl (CHO-Kl), cosediment through a glycerol gradient. When an extract from Urd− A, a pyrimidine-requiring auxotroph reduced in all three activities, is run on a glycerol gradient, the enzyme activities appear in two peaks higher in the gradient, a peak of aspartate transcarbamylase separated from a peak of carbamyl phosphate synthetase and dihydroorotase. Revertants of Urd− A have increased activity of all three enzymes and give glycerol gradient patterns similar to either CHO- Kl or Urd− A. The gradient pattern for Urd− A and some of its revertants can be mimicked by treating the CHO- Kl cell extract with trypsin. Hybrids made between a CHO-Kl purine- requiring auxotroph (Ade− C) and a Urd− A revertant gave a glycerol gradient pattern which is a composite of the CHO- Kl and revertant patterns. A model is presented for the structure of this multifunctional protein.

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