Characterization of a lipid activated CTP:phosphocholine cytidylyltransferase from Drosophila melanogaster

Biochimica Et Biophysica Acta
Beth Ann Helmink, Jon A Friesen

Abstract

CTP:phosphocholine cytidylyltransferase (CCT) is an enzyme critical for cellular phosphatidylcholine (PC) synthesis, converting phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline. We have isolated a cDNA encoding an isoform of CCT from Drosophila melanogaster and expressed the recombinant native and 6 x -His-tagged forms using a baculovirus expression system in Spodoptera frugiperda (Sf9) insect cells. Immunoblot using anti-phospho amino acid antibodies reveals the enzyme is phosphorylated on serine and threonine residues, but not tyrosine. The purified native enzyme exhibits a V(max) value of 1352+/-159 nmol CDP-choline/min/mg, a K(m) value of 0.50+/-0.09 mM for phosphocholine, and a K' (Hill constant) value of 0.72+/-0.10 mM for CTP. The 6 x -His-tagged enzyme has similar properties with a V(max) value of 2254+/-253 nmol CDP-choline/min/mg, a K(m) value of 0.63+/-0.13 mM for phosphocholine and a K' for CTP equal to 0.81+/-0.20 mM. Each form of the enzyme was activated to a similar extent by synthetic PC vesicles containing 50 mol% oleate. The efficiency of lipid activation was greatest using PC vesicles containing diphosphatidylglycerol (DPG), significantly less efficient activation was seen when phosphatidylser...Continue Reading

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Citations

Jul 11, 2006·Biochemical and Biophysical Research Communications·Katsuhito NagaiSadaki Fujimoto
Jan 10, 2016·Biochimica Et Biophysica Acta·Rosemary B Cornell
Sep 26, 2009·Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology·Sheng LiDavid W Borst
May 22, 2007·Progress in Lipid Research·Athanasios Lykidis
Mar 8, 2005·Biochimica Et Biophysica Acta·Claudia Kent
Nov 13, 2004·The Journal of Biological Chemistry·Suzanne Jackowski, Paolo Fagone
Apr 3, 2020·Journal of Molecular Biology·Rosemary B Cornell

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