18] Purification of a multifunction protein bearing carbamyl-phosphate synthase, aspartate transcarbamylase, and dihydroorotase enzyme activities from mutant hamster cells

Abstract

This chapter describes the purification of a multifunctional protein bearing carbamyl-phosphate synthase, aspartate transcarbamylase, and dihydroorotase enzyme activities from mutant hamster cells. Carbamyl-P synthase, aspartate transcarbamylase, and dihydroorotase, the first three enzymes of the pathway for the de novo synthesis of pyrimidine nucleotides, are partially purified in low yield from Drosophila melanogaster, bullfrog eggs, rat liver, hematopoietic mouse spleen, mouse Ehrlich ascites cells, and human lymphocytes and are extensively purified from mouse ascites hepatoma cells. In all cases, all three activities are associated with a single multi-enzyme complex of high molecular weight. The three enzymes are linked covalently as a multifunctional protein, providing a simple explanation for the coordinate control of their synthesis in a series of mutant cell lines that overaccumulate the complex to varying degrees. The entire procedure can be completed in less than 24 hours with a yield of about 40 mg of pure protein from about 15 ml of packed cells.