Characterization of cytidylyltransferase enzyme activity through high performance liquid chromatography

• Published in: Analytical biochemistry 2016-10, Vol.510, p.26-32
• Main Authors: Brault, James P., Friesen, Jon A.
• Format: Article
• Language: English
• Subjects: Animals; Assay; Bacterial Proteins - analysis; Choline-Phosphate Cytidylyltransferase - analysis; Chromatography; Chromatography, High Pressure Liquid - methods; Cytidylyltransferase; Enterococcus faecalis - enzymology; Enzyme; Listeria monocytogenes - enzymology; Phosphatidylcholine; Rats
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2016.07.018

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 14C-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. •Separation of CTP and CDP-choline is accomplished using HPLC.•An alternative to the radioisotope-based cytidylyltransferase assay is presented.•Kinetic analysis of cytidylyltransferases is simple and inexpensive using the HPLC assay.•Multiple cytidylyltransferase family members can be assayed using the HPLC assay.