Effect of uridine on de novo pyrimidine biosynthesis in rat hepatoma cells in culture.

Some of the factors responsible for the control of pyrimidine nucleotide biosynthesis in intact cells have been investigated using rat hepatoma cells grown in continuous culture. The addition of uridine (0.5 mM) to culture medium causes a decrease of approximately 70% in the rate of de nouo pyrimidine nucleotide biosynthesis, as determined by incorporation of WO 2. Maximum inhibition of W02 incorporation into pyrimidine nucleotides occurs within 60 min of addition of the uridine. After the uridine has been removed, the rate of WOZ incorporation recovers to normal levels with 8 hours. This recovery can be prevented by the addition of actinomycin D or cycloheximide to the medium. Uridine causes a decrease of approximately 70 % in the level of activity of orotate phosphoribosyltransferase; it does not significantly affect the activity of carbamyl-P synthetase, aspartate transcarbamylase, dihydroorotase, orotidine decarboxylase, or uridine kinase when these enzyme activities are measured in cell extracts. The decreased level of activity of orotate phosphoribosyltransferase is not due to a direct effect of uridine on the enzyme. The inhibition can be reversed by removal of uridine from the culture medium, but only if transcription and translation are not interfered with by the addition of actinomycin D or cycloheximide. Measurement of enzymes of the de nouo pyrimidine biosynthetic pathway (except for dihydroorotate dehydrogenase) under optimal conditions suggests that orotate phosphoribosyltransferase is ratelimiting in the biosynthesis of uridylic acid. The parallel effects of addition and removal of uridine on 14C0 2 incorporation into pyrimidine nucleotides and on orotate phosphoribosyltransferase activity support this suggestion.