A fast and novel assay of CTP synthetase. Evidence for hysteretic properties of the mammalian enzyme.
A novel assay of CTP synthetase was developed which allows the processing of large numbers of samples. The amount of glutamate produced by CTP synthetase was determined with glutamate dehydrogenase and the NAD analogue acetyl-pyridine-adenine dinucleotide was used to shift the initial unfavourable equilibrium towards the formation of a-ketoglutarate. The amount of glutamate determined with the assay was comparable to that of CTP. The assay proved to be linear with time up to 90 min and protein concentrations up to 520 micrograms/ml. However, at low protein concentrations as well as at early time points a lag phase was observed. This hysteretic phenomenon of CTP synthetase may be due to the association-dissociation equilibrium that exists between the various polymerisation states of the enzyme.
Van Kuilenburg
A B
AB
Academic Medical Centre, Division of Pediatrics, Amsterdam, The Netherlands.
Elzinga
L
L
Van den Berg
A A
AA
Slingerland
R J
RJ
Van Gennip
A H
AH
D000818:Animals; D019731:Carbon-Nitrogen Ligases; D002417:Cattle; D003067:Coenzymes; D005969:Glutamate Dehydrogenase; D005971:Glutamates / Q000032:analysis / Q000378:metabolism; D018698:Glutamic Acid; D007202:Indicators and Reagents; D007700:Kinetics; D008025:Ligases / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000031:analogs & derivatives; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D003067:Coenzymes; D005971:Glutamates; D007202:Indicators and Reagents; D009243:NAD; D018698:Glutamic Acid; C005941:3-acetylpyridine adenine dinucleotide; D005969:Glutamate Dehydrogenase; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase