Regulation of Glutamine Synthetase Activity of Hepatoma Tissue Culture Cells by Glutamine and Dexamethasone

Richard G Kulka,Hanna Cohen

doi:10.1016/s0021-9258(19)43415-7

Abstract

Abstract Regulation of glutamine synthetase activity was studied in a glutamine-independent subclone of the hepatoma tissue culture cell line. In cells of this subclone glutamine synthetase activity was elevated by the synthetic corticosteroid hormone, dexamethasone, and depressed by glutamine. The increase in glutamine synthetase activity due to the addition of dexamethasone and that due to removal of glutamine were both inhibited by cycloheximide, indicating that both effects require de novo synthesis of protein. Actinomycin D completely inhibited the increase in glutamine synthetase activity due to addition of dexamethasone but only slightly inhibited the increase in glutamine synthetase activity due to lowering the glutamine concentration. These observations indicate that the action of the hormone requires the synthesis of ribonucleic acid, whereas the main effect of lowering the glutamine concentration is post-transcriptional. When glutamine was added to cells maintained in low glutamine plus dexamethasone, which have maximal glutamine synthetase activity, there was a rapid decay of enzyme activity. The initial rate of decay of glutamine synthetase activity increased with the concentration of glutamine in the medium, reaching a maximum at 5 mm glutamine. At this concentration of glutamine, glutamine synthetase activity decreased to half its initial value in about 2 hours. Actinomycin D slightly inhibited and cycloheximide markedly inhibited the disappearance of glutamine synthetase activity due to glutamine addition. NaF completely inhibited the decay of glutamine synthetase due to glutamine addition. The kinetics of decay of enzyme activity and the effects of inhibitors suggest that glutamine depresses glutamine synthetase activity by accelerating its inactivation or breakdown.

Highlights

Regulation of glutamine synthetase activity was studied in a glutamine-independent subclone of the hepatoma tissue culture cell line
These observations indicate that the action of the hormone requires the synthesis of ribonucleic acid, whereas the main effect of lowering the glutamine concentration is post-transcriptional
When glutamine was added to cells maintained in low glutamine plus dexamethasone, which have maximal glutamine synthetase activity, there was a rapid decay of enzyme activity

Summary

Methods

Glutamine synthetase activity was estimated by the glutamotransferase assay as described previously (11). Since enzymes other than glutamine synthetase have been reported to catalyze the glutamotransferase reaction (19), experiments were performed to determine whether synthetase and transferase activities in HTC cells vary in a similar manner. It was found that the synthetase and transferase activities changed in parallel, indicating that the transferase activity is due to glutamine synthetase. Tyrosine aminotransferase activity was determined by the method of Diamondstone (20) in cell extracts prepared as described previously (21). Protein was estimated by the method of Lowry et al (22) using bovine plasma albumin as standard. Aliquots (0.1 ml) of this cell suspension were placed on filter paper discs which were processed as described by Bollum (23). The filter discs were placed in vials containing 10 ml of scintillation fluid (0.3% 2,5-diphenyloxazole (PPO) and

Results

Discussion

Conclusion