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Conitinuous breakdown and replenishment of the macromolecular constituents of animal cells was recognized some time ago in the pioneering experiments of Schoenheimer and his colleagues,' but the significance of degradative processes in regulation of metabolism has become apparenit only recently. Schimke, Sweeney, and Berlin have demonstrated2 that the physiological level of the enzyme tryptophan pyrrolase can be elevated either by stimulation of its synthesis or by inhibition of its degradation, the former process being initiated by glueocorticoid hormones and the latter by the substrate of the enzyme, tryptophan. In. a recent report' from our laboratory, it was shown that degradation of the tyrosine transaminase of liver was blocked when protein synthesis was inhibited by agents such as cycloheximide or puromycin.; under these conditions, the transaminase level was stabilized by conconmitant inhibition. of both synthesis and degradation of the enzyme. At that time it was suggested3 that certain "paradoxical" effects of inhibitors of RNA or protein, synthesis on enzyme levels might result from differential inhibition. of the cellular processes involved in forming an enzyme and of those required for its removal. In the present study, we have analyzed the roles of synthesis and degradation. in the elevation of tyrosine transaminase, which follows the addition of actinomycin D to hormonally induced eell cultures. We find that transaminase synthesis, initially high due to induction by hydrocortisone, is progressively inhibited by the antibiotic. That the transarninase level rises during this interval, despite inhibition of its synthesis, reflects a marked inhibition of degradation of the enzyme. Materials and Methods.-Hepatoma cultures of 'the Reuber H-354 and hepatoma tissue culture (HTC)5 cell lines were grown in monolayer in 250-ml Falcoil plastic flasks conltaining 10 ml of Eagle's basal medium (BME) enriched fourfold with amino acids and vitamins and supplemented with 20% fetal calf serum and 5% calf serum. Penicillin G (100 units/ml) and streptomycin sulfate (100 ,g/ml) were added for routine culturilng. Occasionally, antibiotics have been omitted for several passages and the medium cultured for bacterial contamination. Checks for pleuropuieumonia-like (PPLO) coitatamination also have beein carried out using the method of Barile, Yaguchi, and Eveland6 but with horse serum in place of whole blood in the plating agar. No contaminlation by bacteria or mycoplasmas has been detected. All tissue culture materials were purchased from Grand Island Biological, Co. except penicillin G and streptomycin sulfate, which were obtained from Squibb. During logarithmic growth, both cell lines had a doubling time of approximately 24 hr. Inoculation of cells at a concentration of 1.3 X 105 cells/ml resulted in cultures which enter stationary phase on about day 9. All experilments were performed using 9to 12day (stationary phase) moiiolayer cultures in serum-free IX BME (i.e., unenriched). Reuber H-35 and HTC cells do not grow in serumn-free BME, but will resume growth on readdition of serum after as long as 6 days in its absence. Hydrocortisone (Calbiochem) was dissolved in a minimal volume of ethanol and diluted

"Superinduction" of tyrosine transaminase in hepatoma cell cultures: differential inhibition of synthesis and turnover by actionomycin D.