Abstract
BackgroundCholine kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (CKα1, α2, and β). Specific inhibition of CKα has been reported to selectively kill tumoral cells. Monoclonal and polyclonal antibodies against CKα used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the α1 or the α2 isoform.Methodology/Principal FindingsIn this study, an antiserum against CKα was produced by immunizing rabbits with denatured, purified recombinant CKα2 full-length protein. This antiserum was highly specific for CKα when tested with extracts from different cell lines, and there was no cross reactivity with purified CKβ and other related proteins like human ethanolamine kinases (EK) and yeast choline or ethanolamine kinases. The antiserum simultaneously detected both CKα1 and α2 isoforms in MCF-7 and HepG2 cell extracts, but not in HeLa, HCT-116, and mouse embryonic stem cell extracts. Subsequent protein dot blot assay of total CKα in a human normal/tumor protein array of 30 tissue samples by using the antiserum showed that CKα was not overexpressed in all tumor tissues when compared to their normal counterparts. Most striking differences between tumor and normal CKα expression levels were observed in kidney (11-fold higher in tumor) and liver (15-fold lower in tumor) samples.Conclusion/SignificanceApart from its high sensitivity and specificity, the antiserum produced in this work, which does not require further purification, has the advantage of co-detecting both α1 and α2 isoforms in cell extracts for direct comparison of their expression levels.
Highlights
Choline kinase (CK) (EC 2.7.1.32) catalyzes the phosphorylation of choline by ATP in the presence of Mg2+, yielding phosphocholine and ADP [1]
Previous antibodies against human choline kinase were generated by immunizations with either glutathione S-transferase (GST)-purified full-length CKa protein [17,18] or synthetic peptide [10]
The first antibody raised against choline kinase was a polyclonal antibody against rat CKa [7]
Summary
Choline kinase (CK) (EC 2.7.1.32) catalyzes the phosphorylation of choline by ATP in the presence of Mg2+, yielding phosphocholine and ADP [1]. CK commits choline to the socalled Kennedy pathway for the biosynthesis of phosphatidylcholine (PtdCho) [2]. PtdCho is the predominant membrane lipid in eukaryotes amounting to almost 50% of the total phospholipid content [3]. CK exists as at least three isoforms, encoded by two separate genes named ck-a and ck-b. Choline kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. Choline kinase exists as three isoforms (CKa1, a2, and b). Monoclonal and polyclonal antibodies against CKa used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the a1 or the a2 isoform
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