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In nitric oxide (NO)-generating cells, the citrulline/NO cycle is an important pathway in controlling the arginine supply because NO is generated from arginine by the activation of NO synthase. Argininosuccinate synthetase (AS ; EC 6.3.4.5) is a rate-limiting enzyme in the citrulline/NO cycle, which catalyses the condensation of citrulline and aspartate to form argininosuccinate, the immediate precursor of arginine. In this report, we examined AS activity in the parotid glands of rat, rabbit, mouse, and guinea pig. AS activity was detected in all species, and, in the rabbit parotid gland, most of the activity was observed in the cytosolic fraction. In the parotid glands of postnatal rabbits, AS activity increased prior to NOS activity. These results suggest that AS contributes to NO generation in the parotid gland.

Thirteen continuous lines of Syrian hamster epidermal cells were isolated following direct treatment of epidermal cells with N-methyl-N′-nitro-N-nitrosoguanidine, or from epidermal cells cultured from animals treated transplacentally with 7,12-dimethylbenz(a)anthracene or dimethylnitrosamine. The epidermal origin of the lines was evidenced by the presence of desmosomes with radiating tonofilaments, keratin fibers, and the ability of the cells to use citrulline in place of arginine corresponding with their high argininosuccinate synthetase activity.

All 5 enzymes of the urea cycle have been determined on liver samples obtained from rats during late foetal and neonatal life. Since no argininosuccinate synthetase activity could be detected before

Argininosuccinate synthetase activity in amniotic fluid cells from a fetus at risk for citrullinaemia was low compared to the activity in amniotic fluid cells from a normal fetus, but five times the activity in fibroblasts from a patient with citrullinaemia. These enzyme values indicated indicated a normal or heterozygous fetus. Chromosome analysis of the amniotic fluid cells from the fetus at risk, however, showed an unusual X/20 translocation. As we could not guarantee the delivery of a normal child, the parents chose to have a therapeutic abortion. Argininosuccinate synthetase activity in the liver and kidney of the aborted fetus was in the normal and heterozygous range respectively, confirming the prenatal diagnosis. The activity in the father's fibroblasts was low, less than 10% of normal. The difficulty of interpreting the results of prenatal diagnosis in such a family and the importance of studying parental cells are discussed.

Urea cycle enzymes of rats were altered by feeding protein-free 15% casein, or 70% casein diets. Cells isolated from the livers of these rats were incubated with 10 mM ammonia and varying levels of ornithine (2 or 10 mM) and lactate (0-10 mM). Conditions providing the greatest rate of urea synthesis depended on the diet consumed and on whether rats were fed or starved 24 hours before cell isolation. Maximum rates of urea synthesis, though not different for the fed and starved groups, increased with the protein content of the diet and were equal to the maximum theoretical rates, as determined by argininosuccinate synthetase activity. This indicates that with optimal conditions, the activity of argininosuccinate synthetase is the rate-limiting factor in the synthesis of urea in isolated hepatocytes.

The activities and kinetic properties of argininosuccinate synthetase in cultured skin fibroblasts of three citrullinemic patients with qualitative or quantitative abnormalities of hepatic argininosuccinate synthetase were examined. The cultured skin fibroblasts of a citrullinemic patient with a low hepatic argininosuccinate synthetase activity showed low activity and abnormal kinetics similar to those of the hepatic enzyme. This suggests a similar genetic origin of the fibroblast argininosuccinate synthetase as of the hepatic enzyme. However, we found normal argininosuccinate synthetase activity in cultured fibroblasts of two citrullinemic patients who had a low hepatic enzyme activity resulting from decrease in the amount of enzyme protein but with normal kinetic properties. This suggests that part of the quantitative abnormality of hepatic argininosuccinate synthetase of citrullinemic patients may be due to an abnormal organ-specific gene expression in the liver.