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Purification and regulation of CTP:phosphocholine cytidylyltransferase

University of British Columbia

CTP:phosphocholine cytidylyltransferase was purified to homogeneity using a procedure involving aggregation of enzyme with exogenous lipid and selective dissociation with detergents and chromatography on conventional and FPLC columns. SDS-PAGE of purified enzyme showed a single protein of molecular weight ~42 Kd. Native-PAGE showed the protein to migrate at a molecular weight of ~90 Kd, indicating that the native enzyme may be a dimer. IEF-PAGE revealed the enzyme to have a pI of ~5.8. 2-D-PAGE showed the enzyme to have at least two isoforms. Attempts to generate polyclonal antibody to the purified enzyme in rabbits were unsucessful. Even after two booster injections, the antibody titre was still weak indicating that the enzyme may not be a good antigen. However, Dr Harris Jamil was able to generate antibodies in the chicken. This antibody detected cytidylyltransferase by the Western-blotting technique and inhibited enzyme activity in a concentration-dependent manner, but was unable to immunoprecipitate the cytidylyltransferase in solution. Using this antibody, the cytidylyltransferase was found to occur in a variety of isoforms. Kinetic studies using the purified enzyme showed the K[sub m] for CTP and phosphocholine to be 0.31 mM and 0.15 mM respectively. Activation of cytidylyltransferase by commercial and microsomal lipids showed the enzyme to be activated by anionic phospholipids. The presence of oleate did not greatly enhance the activation of the enzyme by these lipids. The purified cytidylyltransferase was shown to be a substrate for cAMP-dependent protein kinase. Phosphorylation of the enzyme led to inactivation and increased recovery of the enzyme in the cytosol, while dephosphorylation by alkaline phosphatase led to activation and increased recovery of the enzyme in the microsomes. A serine residue(s) was phosphorylated on cytidylyltransferase. Twice as much phosphate was incorporated if cytidylyltransferase were dephosphorylated prior to phosphorylation. Incubation of hepatocytes with ³²Pi and then detection of cytidylyltransferase with antibody after 2-D-PAGE and Western-blotting, showed the cytidylyltransferase may be phosphorylated in vivo. Incubation of hepatocytes with Ca²⁺, Ca²⁺-mobilizing agents and phospholipase A₂ resulted in translocation of cytidylyltransferase from the cytosol to the microsomes. Incubation of hepatocytes with Ca²⁺ resulted in an increase in PC and LPC formation. Incubation of hepatocytes with phospholipase A₂ resulted in an increase in PC formation. Regulation of cytidylyltransferase activity by reversible phosphorylation and by Ca²⁺ may be important during short term regulation of enzyme activity by hormones.

Text

2010-10-18

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http://hdl.handle.net/2429/29280

Biochemistry and Molecular Biology

University of British Columbia

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