Methods were developed for the radioisotopic assay of argininosuccinate synthetase [L-citrulline: L-aspartate ligase (AMP-forming), EC 6.3.4.5] and argininosuccinase [L-argininosuccinate arginine-lyase, EC 4.3.2.1]. The assay of argininosuccinate synthetase was based on the separation of [14C]argininosuccinate formed from aspartate and [carbamoyl-14C]citrulline in the presence of ATP from the substrate citrulline. For this, the product was converted to its anhydride form by boiling for 30 min at pH 2.0 followed by application on a column of Dowex 50W (pyridine form). Argininosuccinic anhydride was eluted with 0.3 M pyridine acetate buffer, pH 4.25, while citrulline was eluted with 0.1 M pyridine acetate buffer, pH 3.80. The assay of argininosuccinase was based on the separation of [14C]argininosuccinic acid formed from arginine and [U-14C]fumaric acid from the substrate fumarate on a column of Dowex 50W(H+ form). The argininosuccinic acid was adsorbed on the column and eluted with 1 M pyridine solution, while fumarate was not adsorbed. The distributions of these two enzymes in various organs and cell fractions were reinvestigated using these methods.
The circadian rhythms of the urea concentrations in urine, serum, and liver and their generation mechanism were investigated. When rats were allowed to eat freely, the urea concentration and the total urea content of the urine were higher during the night than during the day-time. Consistent with these findings, the urea concentrations in the liver and serum had circadian rhythms with the highest values at 0200 hours and the lowest values at 1400 hours. The amplitude of the rhythm increased with increase in the dietary protein (casein) content. Of the five urea cycle enzymes in the liver, only argininosuccinate synthetase showed fluctuation in activity, and this had the same pattern as the circadian rhythms of urea concentrations. These findings suggest that the circadian rhythm of argininosuccinate synthetase in the liver might be directly responsible for the rhythms of change in urea concentrations in the liver, blood and urine. The circadian increase in enzyme activity was inhibited by cycloheximide, but not by actinomycin D.
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