Regulation of synthesis of phosphatidylcholine in mammalian cells can be controlled by CTP: phosphocholine cytidylyltransferase. This enzyme undergoes a reversible change in distribution from cytosolic to membrane-associated in cells made deficient in phosphatidylcholine. The proposed research is concerned with the structure and function of cytidylyltransferase in both its purified and cell-associated state. The quaternary structure of the purified enzyme will be determined by chemical cross-linking studies. The catalytic site and lipid-binding domains will be identified by affinity labeling techniques. The amino acid sequence of cytidylyltransferase will be determined by molecular cloning and sequencing a cDNA for the enzyme. The cDNA will also be used as a probe to examine tissue-specific expression of cytidylyltransferase as well as to screen genomic clones. These experiments will be performed with cytidylyltransferase isolated from rat liver as well as from rat brain. The subcellular location of cytidylyltransferase will be examined in control cultured cells and in cells that have been treated to deplete cellular phosphatidylcholine. Techniques to be used for the localization include immunofluorescence of intact cells and immunochemical analysis of membranes separated by gradient fractionation. Cytidylyltransferase localization will be examined in choline-starved cells, in which preliminary evidence indicates the enzyme is associated with the endoplasmic reticulum, as well as in phospholipase C-treated cells, where the enzyme does not appear to be in the endoplasmic reticulum. These experiments will thus examine in depth the structural aspects of cytidylyltransferase that allow it to be found associated with different cellular structures, as will identify those structures.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD010580-12
Application #
3311320
Study Section
Biochemistry Study Section (BIO)
Project Start
1977-07-01
Project End
1991-11-30
Budget Start
1989-12-01
Budget End
1990-11-30
Support Year
12
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Purdue University
Department
Type
Schools of Earth Sciences/Natur
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
MacDonald, J I; Kent, C (1993) Baculovirus-mediated expression of rat liver CTP:phosphocholine cytidylyltransferase. Protein Expr Purif 4:1-7
Wang, Y; MacDonald, J I; Kent, C (1993) Regulation of CTP:phosphocholine cytidylyltransferase in HeLa cells. Effect of oleate on phosphorylation and intracellular localization. J Biol Chem 268:5512-8
Watkins, J D; Wang, Y L; Kent, C (1992) Regulation of CTP:phosphocholine cytidylyltransferase activity and phosphorylation in rat hepatocytes: lack of effect of elevated cAMP levels. Arch Biochem Biophys 292:360-7
Watkins, J D; Kent, C (1992) Immunolocalization of membrane-associated CTP:phosphocholine cytidylyltransferase in phosphatidylcholine-deficient Chinese hamster ovary cells. J Biol Chem 267:5686-92
Jones, G A; Kent, C (1991) The role of protein kinase C in the stimulation of phosphatidylcholine synthesis by phospholipase C. Arch Biochem Biophys 288:331-6
Watkins, J D; Kent, C (1991) Regulation of CTP:phosphocholine cytidylyltransferase activity and subcellular location by phosphorylation in Chinese hamster ovary cells. The effect of phospholipase C treatment. J Biol Chem 266:21113-7
Watkins, J D; Kent, C (1990) Phosphorylation of CTP:phosphocholine cytidylyltransferase in vivo. Lack of effect of phorbol ester treatment in HeLa cells. J Biol Chem 265:2190-7
Morand, J N; Kent, C (1989) Localization of the membrane-associated CTP:phosphocholine cytidylyltransferase in Chinese hamster ovary cells with an altered membrane composition. J Biol Chem 264:13785-92
Kent, C; Evers, A; Haun, S S (1986) Diacylglycerol metabolism in phospholipase C-treated mammalian cells. Arch Biochem Biophys 250:519-25
Wright, P S; Kent, C (1986) Chinese hamster ovary cells depend on exogenous lipids to survive phospholipase C treatment. Arch Biochem Biophys 245:104-13

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