Summary
The synergistic effects of potential amino donors were studied in the assay of CTP synthetase in extracts of Chinese hamster fibroblasts. We found that L-glutamine was not effective as the sole amino donor, but combinations of L-glutamine with NH4HCO3, L-arginine or potassium phosphate did result in the conversion of UTP to CTP. L-arginine or potassium phosphate were also not effective when used alone, and NH4HCO3 was only slightly effective. Our studies demonstrate that the individual synergistic combinations were not additive; multiple combinations of components decreased rather than increased the formation of CTP.
The synergistic combinations of L-glutamine with either NH4HCO3 or L-arginine had an absolute requirement for ATP; when ATP and PEP were absent no conversion of UTP to CTP occurred. The presence of GTP in a reaction mixture slightly increased the formation of CTP when L-glutamine and NH4HCO3 were used and substantially increased CTP formation when L-glutamine and L-arginine were used.De novo CTP synthesis was greatly reduced when nonradioactive CTP was added to an assay mixture, suggesting feedback inhibition.
A TLC procedure has been developed that allows for the direct separation of UTP and CTP without requiring prior conversion to the mononucleotide or nucleoside level.
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Abbreviations
- β-ME:
-
β-mercaptoethanol
- PEI:
-
polyethyleneimine
- PEP:
-
phosphoenolpyruvate
- TLC:
-
thin layer chromatography.
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McLaren, J.D., Chu, E.H.Y. Apparent synergism between amino donors for CTP synthesis in Chinese hamster fibroblasts. Mol Cell Biochem 57, 167–175 (1983). https://doi.org/10.1007/BF00849193
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DOI: https://doi.org/10.1007/BF00849193