title: Characterization of cytidylyltransferase enzyme activity through high performance liquid chromatography.
abstract: The cytidylyltransferases are a family of enzymes that utilize cytidine 5'-triphosphate (CTP) to synthesize molecules that are typically precursors to membrane phospholipids. The most extensively studied cytidylyltransferase is CTP:phosphocholine cytidylyltransferase (CCT), which catalyzes conversion of phosphocholine and CTP to cytidine diphosphocholine (CDP-choline), a step critical for synthesis of the membrane phospholipid phosphatidylcholine (PC). The current method used to determine catalytic activity of CCT measures production of radiolabeled CDP-choline from (14)C-labeled phosphocholine. The goal of this research was to develop a CCT enzyme assay that employed separation of non-radioactive CDP-choline from CTP. A C18 reverse phase column with a mobile phase of 0.1 M ammonium bicarbonate (98%) and acetonitrile (2%) (pH 7.4) resulted in separation of solutions of the substrate CTP from the product CDP-choline. A previously characterized truncated version of rat CCTα (denoted CCTα236) was used to test the HPLC enzyme assay by measuring CDP-choline product formation. The Vmax for CCTα236 was 3850 nmol/min/mg and K0.5 values for CTP and phosphocholine were 4.07 mM and 2.49 mM, respectively. The HPLC method was applied to glycerol 3-phosphate cytidylyltransferase (GCT) and CTP:2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase synthetase (CMS), members of the cytidylyltransferase family that produce CDP-glycerol and CDP-methylerythritol, respectively.
authors.affiliation: Department of Chemistry, Illinois State University, Normal, IL, 61790, USA.
authors.forename: James P
authors.initials: JP
authors.lastname: Brault
authors.affiliation: Department of Chemistry, Illinois State University, Normal, IL, 61790, USA. Electronic address: jfriese@ilstu.edu.
authors.forename: Jon A
authors.initials: JA
authors.lastname: Friesen
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis*; D019916:Choline-Phosphate Cytidylyltransferase / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D013293:Enterococcus faecalis / Q000201:enzymology*; D008089:Listeria monocytogenes / Q000201:enzymology*; D051381:Rats
chemical_list: D001426:Bacterial Proteins; D019916:Choline-Phosphate Cytidylyltransferase
keywords: Assay; Chromatography; Cytidylyltransferase; Enzyme; Phosphatidylcholine