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Inhibition studies of crystallized rat liver argininosuccinate synthetase [EC 6.3.4.5] are described. 1. L-Argininosuccinate, L-histidine, and L-tryptophan inhibited the enzyme activity at saturating amounts of the substrates. 2. L-Norvaline, L-argininosuccinate, L-arginine, L-isoleucine, and L-valine competitively inhibited the enzyme activity at a low concentration of L-citrulline, with Ki values of 1.3 x 10(4) M, 2.5 X 10(-4) M, 6.7 X 10(-4) M, 6.3 X 10(-4) M, and 6.0 x 10(-4) M, respectively. 3. L-Argininosuccinate and L-arginine competitively inhibited the enzyme activity at a low concentration of L-aspartate, with Ki values of 9.5 x 10(-4) M and 1.2 x 10(-3) M, respectively. 4. The modes of inhibition by L-histidine were mixed-noncompetitive, uncompetitive, and noncompetitive types with respect to L-citrulline, L-aspartate, and ATP, respectively. 5. When the enzyme was preincubated with L-citrulline, the enzyme activity was slightly increased in the presence of a low concentration of L-histidine in the assay mixture. 6. The conformation of the enzyme was markedly changed by the addition of L-histidine as judged from the CD spectrum. This change was partially reversed by incubation with L-citrulline.

1. To study the role of group-specific protease in enzyme degradation, alternation of its activity under various physiological conditions was examined. 2. Studies on the distribution of group-specific protease in various organs of rats showed high activity in skeletal muscle and the muscle layer of small intestine, and rather low activity in liver. The activity varied in different muscles, but red muscle tended to have higher activity than white muscle. Activity was much lower in the muscles of the stomach and colon than in those of the small intestine. 3. Group-specific protease in skeletal muscle increased under various dietary conditions (starvation, protein-free diet or high protein diet), but the activities in the muscle layer of the small intestine and liver were not greatly influenced by dietary conditions. None of the hormones tested (i.e. hydrocortisone, glucagon, insulin, growth hormone and estrogen) influenced the activity of group-specific protease in liver. 4. The level of group-specific protease in skeletal muscle was increased markedly fifteen days after denervation, with a reciprocal decrease in the level of muscle phosphorylase, which is a good substrate of the protease. 5. Liver protease activity appeared in the late suckling period. The activity in skeletal muscle was high at the time of birth and attained the adult level 3 weeks after birth. The activity in the muscle layer of the small intestine did not change after birth. Thus the mechanism for evoking these three specific proteases during development are apparently different. The activity of liver protease began to decrease approximately 12 h after partial hepatectomy and reached a minimum after about 72 h. Recovery of the protease activity was very slow and activity had not returned to the normal value 7 days after the operation. This observation seems to be consistent with the fact that there is little or no protease activity in liver in the neonatal period.

Properties of argininosuccinate synthetases (ASS), including immunological properties and heat stability, from young and old rats were quite similar. However the degradation rates of ASS from young and old rats were found to be different as shown in the experiment using the double labeling technique. Three forms of this enzyme from young and old rat livers were separated by DEAE-Sephadex A-50 column chromatography, and they were called ASS 1, 2 and 3 in order of elution. The degradation rate of ASS 1 from young and old rat livers was very similar, but the rate of degradation of ASS 2 form old rat livers was greater than that from young rat livers.

The rates of synthesis and degradation of arginosuccinate synthetase in rat liver under various dietary conditions were determined. The relative rate of the enzyme synthesis in the livers of rats fed on 70% casein diet was 4.0 times greater than that for rats fed on 5% casein diet. The rate constants of degradation (Kd of argininosuccinate synthetase were estimated to be 0.15 and 0.16 day-1 under 70% and 5% casein feeding, respectively. When the dietary conditions were changed acutely from 70% to 5% casein diet or vice versa, the rates of the enzyme synthesis decreased or increased, respectively, and the rates of enzyme degradation were also affected. The change from 5% to 70% casein diet caused a transient decrease in the rate of degradation. After the enzyme activity had achieved a new steady-state level, the enzyme degradation proceeded at the normal steady rate. On the other hand, the change from 70% to 5% casein diet caused a transient increase in the rate of degradation. Thus, the only factor regulating the amount of enzyme in rat liver is the rate of enzyme synthesis under the steady-state conditions. However, the rates of both enzyme synthesis and degradation are involved in the regulation of the amount of enzyme during dietary transition.