Carbamyl phosphate-aspartate transcarbamylase activity in tumors.

The enzyme carbamyl phosphate-aspartate transcarbamylase catalyzes the transfer of the carbamyl group from carbamyl phosphate (CP) to L-aspartate ($). Preliminary experiments with a series of six relatively advanced azo dye-induced hepatomas indicated that hepatoma tissue had a higher CP-aspartate transcarbamylase activity than did liver from normal animals. Experiments are reported here in which CP-aspartate trans-carbamylase activity was measured in portions of macroscopically normal liver and portions of well developed hepatoma nodules from the same rat. In addition, the activity of the enzyme in Ehrlich ascites tumor cells and mouse liver and brain is reported. MATERIALS AND METHODS Animals.mYoung adult, albino Sprague-Daw-ley rats were fed 0.06 per cent 3'-methyl-4-dimeth-ylaminoazobenzene with 18 per cent casein in a purified diet for 3 months (3). The animals were then kept on the diet without the azo dye for anOther ~ months. At the time of the experiments the rats showed well developed hepatic tumors. Ehr-lich ascites tumor cells were grown in mice for a period of 4 days. The freshly harvested cells were pooled and centrifuged at low speed. The resulting supernatant fluid was then centrifuged at high speed (approximately 10,000 X g) for 30 minutes. The latter was considered to represent ascitic fluid. The ascites cells and the ascites fluid were treated as indicated under "Estimation of enzyme activity." Substrates.-Carbamyl phosphate-C 14 (C14P) was prepared as described in a previous study (~). A solution of C14p containing 1.0 ~mole and hay-ing an activity of 13,320 counts/rain in 0.1 ml. was prepared and kept at-20 ~ C., under which conditions the preparation is stable for weeks. L-Aspartic acid was a commercial preparation. Preparation of the enzyme from liver and hepa-toma tissue.-The animals were killed by decapita-tion and exsanguinated as completely as possible. The liver was rapidly removed and placed on crushed ice. As soon as possible, representative portions of macroscopically "normal" liver (hereafter called adjacent liver tissue) and portions of tumor tissue were dissected and placed in ice-cold isotonic NaC1 solution. Care was taken to avoid the inclusion of necrotic portions. The selected fragments were blotted and weighed. Portions of tissue weighing from 0.5 to 2.5 gm. were suspended in 9 ml. of ice-cold isotonic KCI solution and homogenized in a glass homogenizer. The ho-mogenates were then centrifuged for 45 minutes at 9,300 r.p.m. (average centrifugal force, 8,500 g). The supernatant solution, which contains all the enzyme (~), was used as the source of …