Date of Award
Master of Science (MS)
Department of Chemistry
The cytidylyltransferases are a family of enzymes that utilize cytidine 5â?? triphosphate (CTP) to synthesize molecules that are precursors to membrane phospholipids. There are four well known enzymes: CTP: phosphoethanolamine cytidylyltransferase (ECT), CTP: glycerol-3-phosphate cytidylyltransferase (GCT), 2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase synthetase (CMS), and CTP: phosphocholine cytidylyltransferase (CCT). Previously, a radioisotope tagging method was employed to study cytidylyltransferase catalysis. Using CCT as a model, a method utilizing high performance liquid chromatography (HPLC) was developed to replace the radioisotope scintillation technique. The development of this new HPLC method is cheaper, more efficient, and more accurate than the previously established method.
The targets of separation are the substrate CTP and the product cytidine diphosphate-choline (CDP-choline) of the reaction catalyzed by CCT. A solvent system based on previous literature which separated nucleotides and nucleosides was
successfully developed using standard solutions of CTP and CDP-choline. Once successful separation was achieved, it was applied to CCT.
The cytidylyltransferase used to test the HPLC enzyme assay was a truncated version of CCT denoted CCT236. The full length CCT has 3 domains, an N-terminal catalytic domain, a membrane lipid activation domain, and a C-terminal phosphorylated domain (Friesen et al. 1999). The full length enzyme is activated by membrane association, specifically with lipids. The truncated version only has the N-terminal catalytic domain so it does not need lipid association. An enzymatic assay was performed using the CCT236 isoform that was subjected to the newly developed HPLC method that successfully separated the reactant (CTP) from the product (CDP-choline). This method was then applied to GCT and CMS for separation as well. Herein the results of the application of this new HPLC separation method to members of the cytidylyltransferase family are presented.
Brault, James, "Characterization of Cytidylyltransferase Enzyme Activity Through High Performance Liquid Chromatography" (2015). Theses and Dissertations. 490.