title: Determination of CTP synthetase activity in crude cell homogenates by a fast and sensitive non-radiochemical assay using anion-exchange high-performance liquid chromatography.
abstract: 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.
authors.affiliation: Academic Medical Center, University of Amsterdam, Division of Clinical Chemistry, Netherlands.
authors.forename: A B
authors.initials: AB
authors.lastname: Van Kuilenburg
authors.forename: L
authors.initials: L
authors.lastname: Elzinga
authors.forename: A C
authors.initials: AC
authors.lastname: Verschuur
authors.forename: A A
authors.initials: AA
authors.lastname: Van den Berg
authors.forename: R J
authors.initials: RJ
authors.lastname: Slingerland
authors.forename: A H
authors.initials: AH
authors.lastname: Van Gennip
mesh_terms: 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
chemical_list: D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase