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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Mycotoxin Prevention and Applied Microbiology Research » Research » Publications at this Location » Publication #238506

Title: Identification and Characterization of the Nuclear Isoform of Drosophila melanogaster CTP:Phosphocholine Cytidylyltransferase

item TILLEY, DANA - Illinois State University
item EVANS, CHADRICK - Illinois State University
item Larson, Troy
item EDWARDS, KEVIN - Illinois State University
item FRIESEN, JON - Illinois State University

Submitted to: Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/20/2008
Publication Date: 10/16/2008
Citation: Tilley, D.M., Evans, C.R., Larson, T.M., Edwards, K.A., Friesen, J.A. 2008. Identification and Characterization of the Nuclear Isoform of Drosophila melanogaster CTP:Phosphocholine Cytidylyltransferase. Biochemistry. 47:11838-11846.

Interpretive Summary: This study determined the location of two enzymes, CCT1 and CCT2, with Drosophila cells through the use of fluorescent tags. CCT1 had be previously uncharacterized and was found to localize to the nucleus, while CCT2 was found to be cytoplasmic. It was shown that the activity of CCT1 was also enhanced in the presence of fatty acids. This study complements previous work on CCT2. By determining the location of the enzymes within the cell, this research also provides direction for future research regarding the overall function of the enzymes.

Technical Abstract: CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the conversion of phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline for the eventual synthesis of phosphatidylcholine (PC). The enzyme is regulated by reversible association with cellular membranes, with the rate of catalysis increasing following membrane association. Two isoforms of CCT appear to be present in higher eukaryotes, including Drosophila melanogaster, which contains the tandem genes Cct1 and Cct2. Before this study, the CCT1 isoform had not been characterized and the cellular location of each enzyme was unknown. In this investigation, the cDNA encoding the CCT1 isoform from D. melanogaster has been cloned and the recombinant enzyme purified and characterized to determine catalytic properties and the effect of lipid vesicles on activity. CCT1 exhibited a Vmax of 23904 nmol of CDP-choline min**-1 mg**-1 and apparent Km values for phosphocholine and CTP of 2.29 and 1.21 mM, respectively, in the presence of 20 µM PC/oleate vesicles. Cytidylyltransferases require a divalent cation for catalysis, and the cation preference of CCT1 was found to be as follows: Mg**2+ > Mn**2+ = Co**2+ > Ca**2+ = Ni**2+ > Zn**2+. The activity of the enzyme is stimulated by a variety of lipids, including phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, diphosphatidylglycerol, and the fatty acid oleate. Phosphatidylethanolamine and phosphatidic acid, however, did not have a significant effect on CCT1 activity. The cellular location of both CCT1 and CCT2 isoforms was elucidated by expressing green fluorescent fusion proteins in cultured D. melanogaster Schneider 2 cells. CCT1 was identified as the nuclear isoform, while CCT2 is cytoplasmic.