De novo pyrimidine nucleotide biosynthesis in synchronized rat hepatoma (HTC) cells and mouse embryo fibroblast (3T3) cells

Abstract

Activity of carbamyl phosphate synthetase, aspartate transcarbamylase, and orotate phosphoribosyl transferase was measured in cultures of rat hepatoma (HTC) cells synchronized using Colcemid inhibition. All three enzymes had distinct peaks of activity during the S phase of the cell cycle and had very little activity during the G 2 and M phases. Whereas carbamyl phosphate synthetase and aspartate transcarbamylase activities increased rapidly during early G 1, orotate phosphoribosyl transferase activity did not increase rapidly until the end of G 1. The incorporation of 14CO 2 into acid-soluble pyrimidine nucleotides and RNA followed a similar pattern to that obtained for enzymes of de novo uridylic acid biosynthesis, with the maximum incorporation occurring during the S phase in HTC cells. 3T3 cells, synchronized by release from contact inhibition, also had the maximum incorporation of 14CO 2 into acid-soluble pyrimidine nucleotides and RNA during the S phase, although there was also a lesser peak of incorporation early in the G 1 phase. Neither culture incorporated 14CO 2 into DNA to any significant extent.