The mode of action of Leflunomide, an immunomodulatory drug used in rheumatoid arthritis, is debated. This study, using 14 C-labeled de novo purine and pyrimidine synthesis precursors, proves conclusively that the prime target in proliferating human T-lymphocytes is pyrimidine biosynthesis at the level of dihydroorotic- 14 C]Glycine studies confirmed that restriction of de novo purine synthesis occurred secondary to inhibition of proliferation since this was reversed by uridine rescue, except at 100 M Leflunomide. 100 M Leflunomide markedly depleted ATP and GTP pools also, which would have serious consequences for ATP-dependent enzymes essential to the immune response, thereby explaining non-pyrimidinerelated effects reported for Leflunomide at 100 M and above.

The trifunctional protein CAD, which contains the first three enzyme activities of pyrimidine nucleotide biosynthesis (carbamyl phosphate synthetase II, aspartate transcarbamylase and dihydro-orotase), is phosphorylated stoichiometricaily by cyclic AMP-dependent protein kinase. Phosphorylation activates the ammonia-dependent carbamyl phosphate synthetase activity of the complex by reducing the apparent Km for ATP. This effect is particularly marked in the presence of the allosteric feedback inhibitor, UTP, when the apparent Km is reduced by >4-fold. Inhibition by physiological concentrations of UTP is substantially relieved by phosphorylation. Cyclic AMP-dependent protein kinase phosphorylates two serine residues on the protein termed sites 1 and 2, and the primary structures of tryptic peptides containing these sites have been determined: Site 1: Arg-Leu-Ser(P)-Ser-Phe-Val-Thr-Lys Site 2: lle-His-Arg-Ala-Ser(P)-Asp-Pro-Gly-Leu-Pro-Ala-Glu-Glu-Pro-Lys During the phosphorylation reaction, activation of the carbamyl phosphate synthetase shows a better correlation with occupancy of site 1 rather than site 2. Both phosphorylation and activation can be reversed using purified preparations of the catalytic subunits of protein phosphatases 1-and -2A, and inactivation also correlates better with dephosphorylation of site 1 rather than site 2. We believe this to be the first report that a key enzyme in nucleotide biosynthesis is regulated in a significant manner by reversible covalent modification. The physiological role of this phosphorylation in the stimulation of cell proliferation by growth factors and other mitogens is discussed.

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We report the identification of a hitherto unknown nucleotide that is present in micromolar concentrations in the erythrocytes of healthy subjects and accumulates at levels comparable with the ATP concentration in erythrocytes of patients with chronic renal failure. The unknown nucleotide was isolated and identified by liquid chromatography with UV and tandem mass detection, . We suggest that 4PYTP formation in the erythrocytes is a hitherto unknown process aimed at sequestering potentially toxic 4PYR in a form that could be safely transported and subsequently released and excreted during passage of erythrocytes through the kidney.

Orotate (OA) is well-known as a precursor in biosynthesis of pyrimidines; in mammals it is released from the mitochondrial dihydroorotate dehydrogenase (DHODH) for conversion to UMP by the cytoplasmic UMP synthase enzyme. OA is also a normal part of the diet, being found in milk and dairy products, and it is converted to uridine for use in the pyrimidine salvage pathway predominantly in liver, kidney and erythrocytes. Early research into nutrition identified orotate as "vitamin B13," and its use as a complex with organic cations or metal ions was promulgated in body-building, and in assisting therapies of metabolic syndromes. It has recently been established that the amelioration of gout by dairy products arises from the competition of orotate and urate at the hURAT1 transporter. The orotic aciduria that arises in children with defective UMP synthase can be rescued by oral uridine therapy, since UMP is the end-product and also a feedback inhibitor of the de novo pathway. In contrast, Miller (dysmorphology) syndrome is connected with defects in DHODH, and hence in the supply of OA, and cannot be helped by uridine. Other models of dysmorphisms are connected with enzymes early in the pyrimidine de novo pathway. We conclude that the OA molecule is itself required for the regulation of genes that are important in the development of cells, tissues and organisms.

Nicotinic acid-adenine dinucleotide phosphate (NAADP) is fast emerging as a new intracellular Ca2+-mobilizing messenger. In sea urchin egg homogenates, binding of NAADP to its receptor is not readily reversible; hence, prior incubation with low concentrations of NAADP is more effective in inhibiting subsequent binding of radiolabelled NAADP than incubating the preparation with the two ligands simultaneously [Patel, Churchill and Galione (2000) Biochem. J. 352, 725-729]. We extend this finding to show that NAADP is more effective still in inhibiting the subsequent radioligand binding at lower homogenate concentrations, an effect again quite probably due to the non-reversible nature of the receptor-ligand interaction. Enhanced sensitivity of the preparation to NAADP afforded by simple manipulation of the experimental conditions has been applied to determine low levels of NAADP in acid extracts from human red blood cells, rat hepatocytes and Escherichia coli without interference from NADP breakdown. Our improved method for the quantification of NAADP should prove useful in the further assessment of its signalling role within cells.