Initial Steps in Pyrimidine Synthesis in Ehrlich Ascites Carcinoma in Vitro: II. THE SYNTHESIS OF CARBAMYL PHOSPHATE BY A SOLUBLE, GLUTAMINE-DEPENDENT CARBAMYL PHOSPHATE SYNTHETASE

Abstract

Abstract A glutamine-dependent carbamyl phosphate synthetase, the occurrence of which was suggested by previous studies with intact cells, has been found in extracts of Ehrlich ascites carcinoma. The activity is detectable by measuring the incorporation of 14C-bicarbonate of high specific activity into citrulline when purified Streptococcus faecalis ornithine transcarbamylase and ornithine are added to the extract. The 14C-bicarbonate incorporation into citrulline requires (in addition to the tumor extract, ornithine transcarbamylase, and ornithine) magnesium, adenosine triphosphate, and glutamine or ammonia. The Km for glutamine is close to 10-5 m, whereas the Km for ammonium chloride is about 5 x 10-3 m. The incorporation is inhibited by glutamine analogues when either glutamine or ammonia is the substrate. A standard assay and a procedure to stabilize the enzyme are described. Other properties of the extracted enzyme are described. The enzyme is similar to the glutamine-dependent carbamyl phosphate synthetases from mushroom, Escherichia coli and yeast. The role of this enzyme in mammalian and avian pyrimidine synthesis is discussed.