TY - JOUR
T1 - Characterization of a lipid activated CTP:phosphocholine cytidylyltransferase from Drosophila melanogaster
AU - Helmink, Beth Ann
AU - Friesen, Jon A.
N1 - Funding Information:
This work was supported by National Institutes of Health grant R15 GM064512 and Illinois State Pretenure Faculty Initiative Grant to J.A.F., as well as an Illinois State University Honors program fellowship to B.A.H. and Industrial Research Grant funding to B.A.H. from Abbott Pharmaceutical Corporation through the Department of Chemistry at Illinois State University.
PY - 2004/7/5
Y1 - 2004/7/5
N2 - 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×-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 Vmax value of 1352±159 nmol CDP-choline/min/mg, a Km 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×-His-tagged enzyme has similar properties with a Vmax value of 2254±253 nmol CDP-choline/min/mg, a Km 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 phosphatidylserine (PS) and phosphatidylinositol (PI) were incorporated into vesicles, and PC alone or PC vesicles containing phosphatidylethanolamine were the least efficient enzyme activators.
AB - 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×-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 Vmax value of 1352±159 nmol CDP-choline/min/mg, a Km 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×-His-tagged enzyme has similar properties with a Vmax value of 2254±253 nmol CDP-choline/min/mg, a Km 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 phosphatidylserine (PS) and phosphatidylinositol (PI) were incorporated into vesicles, and PC alone or PC vesicles containing phosphatidylethanolamine were the least efficient enzyme activators.
KW - CCT
KW - CTP:phosphocholine cytidylyltransferase
KW - CTP:phosphoethanolamine cytidylyltransferase
KW - Cytidylyltransferase
KW - Drosophila melanogaster
KW - ECT
KW - GCT
KW - glycerol-3-phosphate cytidylyltransferase
KW - Lipid activation
KW - PC
KW - Phosphatidylcholine
KW - Spodoptera frugiperda
UR - http://www.scopus.com/inward/record.url?scp=3042651464&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=3042651464&partnerID=8YFLogxK
U2 - 10.1016/j.bbalip.2004.04.008
DO - 10.1016/j.bbalip.2004.04.008
M3 - Article
C2 - 15238222
AN - SCOPUS:3042651464
SN - 1388-1981
VL - 1683
SP - 78
EP - 88
JO - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
JF - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
IS - 1-3
ER -