Determination of CTP synthetase activity in crude cell homogenates by a fast and sensitive non-radiochemical assay using anion-exchange high-performance liquid chromatography.
A non-radiochemical assay procedure for CTP synthetase was developed in which CTP is detected at 280 nm after separation with anion-exchange HPLC. A complete separation of all nucleoside triphosphates was achieved within 11 min and the minimum amount of CTP which could be accurately determined proved to be 5 pmol. Therefore, our assay procedure is ten-fold more sensitive compared to the frequently used radiochemical assays. The assay was linear with time and protein concentration, although at low protein concentration a lag phase was observed. An amount of 2 x 10(6) cells was already sufficient to determine the specific activity of CTP synthetase in HL-60 cells, lymphocytes and in lymphoblasts obtained from pediatric patients suffering from acute lymphoblastic leukemia.
Van Kuilenburg
A B
AB
Academic Medical Center, University of Amsterdam, Division of Clinical Chemistry, Netherlands.
Elzinga
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L
Verschuur
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AC
Van den Berg
A A
AA
Slingerland
R J
RJ
Van Gennip
A H
AH
D019731:Carbon-Nitrogen Ligases; D002478:Cells, Cultured; D002648:Child; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006801:Humans; D008025:Ligases / Q000032:analysis*; D008214:Lymphocytes / Q000201:enzymology*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000201:enzymology*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase