Pyrimidine metabolism and sugar nucleotide synthesis in rat liver

Abstract

With radioactive precursors, the labelling kinetics of the soluble pyrimidine nucleotides and of RNA were measured in rat liver to determine the contribution of the metabolic flows through synthesis de novo and the salvage pathway. To separate and quantify all pyrimidine nucleotides, an h.p.l.c. technique was developed using anion-exchange chromatography and reversed-phase chromatography. The concentrations of cytidine nucleotides were in the range of 30-45 nmol/g wet weight, and the concentrations of the uridine phosphates and of the UDP-sugars were approx. 6 and 20 times higher respectively. After a single injection of [14C]orotic acid and of [3H]cytidine, the specific radioactivities were determined as a function of time. The 1'C/3H ratio was calculated and gave a good indication of the involvement of the different flows. It could be concluded that UTP derived from synthesis de novo and from the salvage pathway is not completely mixed before being utilized. The flow of the salvage pathway is relatively more directed to RNA synthesis in the nucleus and that of synthesis de novo to cytoplasmic processes. For CTP it could also be concluded that the flow of the salvage pathway was relatively more directed to RNA synthesis in the nucleus. Because of the nuclear localization of the enzyme CMP-NeuAc (N-acetylneuraminate) synthase, special attention was paid to CMP-NeuAc. However, a conclusion about a location about the synthesis of CMP-NeuAc could not unequivocally be drawn, because of the small differences in 14C/3H ratio and the different values for the CDP-lipids.

Pyrimidine nucleotide metabolism in rat hepatocytes: evidence for compartmentation of nucleotide pools

“…Elucidation of metabolic channelling in cytidine metabolism is therefore of particular relevance. We have previously reported our findings on the compartmentation of cytidine and uridine metabolism in rat liver in vivo [24,25]. In the present study we substantiate our findings using rat hepatocytes cultured in monolayer.…”

“…The RNA hydrolysate and washes were combined and excess HCl04 was removed with 2 vol. of tri-n-octylamine/ trichlorotrifluoroethane (1: 4, v/v) as described [24]. After centrifugation the aqueous upper phase was removed and the organic lower phase was washed once with 1 vol.…”

“…The absorbance was detected with a Kratos Spectroflow 757, operating at 280 nm, combined with a Hitachi D-2000 chromatointegrator. Separation of the soluble nucleotides was performed on a Whatman Partisphere SAX cartridge (4.6 mm x 125 mm; particle size 5 Itm), and was achieved with gradient elution as described [24]. The flow rate was 1.0 ml/min.…”

The effect of increasing nucleotide-sugar concentrations on the incorporation of sugars into glycoconjugates in rat hepatocytes

“…As part of our studies [see also Pels Rijcken et al (1990, 1993] processed during the subsequent transport to their final deson the latter mode of regulation, we determined the effect of tination. This process has appeared to be an essential prerequisite increases of the nucleotide-sugar pools in rat hepatocytes, as for obtaining biologically competent molecules at their proper brought about by pretreatment of the cells with uridine and location (Lodish, 1988).…”

Glutamine inhibits cytokine-induced apoptosis in human colonic epithelial cells via the pyrimidine pathway

“…Further experiments were conducted with uracil because intracellular concentrations of uracil exceed those of thymine (30). To determine the role of uracil during TRAIL-induced apoptosis, cultures were pretreated in serum-free medium for 24 h with increasing amounts of uracil (0, 0.05, 0.5, 1.0, 2.5, or 5.0 mM).…”