Purification and properties of CTP synthetase from Ehrlich ascites tumor cells.

Abstract

CTP synthetase (UTP: ammonia ligase (ADP-forming), EC 6.3.4.2) was purified 200-fold from Ehrlich ascites tumor cells with about 50% purity as judged by polyacrylamide gel electrophoresis. The molecular weight estimated to be 118 000 by gel filtration. The optimal pH was 8.6. 2-Mercaptoethanol was required for optimal activity and stabilization of the enzyme. Magnesium was essential for the reaction and other divalent cations were ineffective. Ammonia could replace glutamine as the amino donor. When other substrates were at saturating concentrations, Michaelis-Menten kinetics were observed yielding Km values for UTP, ATP and glutamine of 0.18, 0.8 and 0.13 mM, respectively. With ATP at subsaturating concentration, the double-reciprocal plot for UTP saturation was sinusoidal and the Hill plot showed an n value of 1.3. The double-reciprocal plot for ATP saturation, when UTP was at subsaturating concentration, departed from Michaelis-Menten kinetics with an n value of 1.4. These data suggest the existence of cooperative interactions between enzyme and substrates at subsaturating concentrations of ATP or UTP. GTP was not essential, but it acted as an activator on the glutamine reaction; optimal activation was observed at 1 mM GTP. The affinity for glutamine was not affected by GTP.

• Authors: H Kizaki, T Sakurada, G Weber
• Journal: Biochimica et biophysica acta (Biochim Biophys Acta) Vol. 662 Issue 1 Pg. 48-54 (Nov 13 1981) ISSN: 0006-3002 [Print] Netherlands
• PMID: 7306558 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Amino Acids
• Cations, Divalent
• Sulfhydryl Reagents
• Guanosine Triphosphate
• Ligases
• Carbon-Nitrogen Ligases
• CTP synthetase

Topics

• Amino Acids (pharmacology)
• Animals
• Carbon-Nitrogen Ligases
• Carcinoma, Ehrlich Tumor (enzymology)
• Cations, Divalent (pharmacology)
• Electrophoresis, Polyacrylamide Gel
• Guanosine Triphosphate (pharmacology)
• Hydrogen-Ion Concentration
• Kinetics
• Ligases (isolation & purification)
• Molecular Weight
• Sulfhydryl Reagents (pharmacology)