Highly specific antibodies for co-detection of human choline kinase ?1 and ?2 isoforms.

ABSTRACT: BACKGROUND: Choline 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 (CK?1, ?2, and ?). Specific inhibition of CK? has been reported to selectively kill tumoral cells. Monoclonal and polyclonal antibodies against CK? used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the ?1 or the ?2 isoform. METHODOLOGY/PRINCIPAL FINDINGS: In this study, an antiserum against CK? was produced by immunizing rabbits with denatured, purified recombinant CK?2 full-length protein. This antiserum was highly specific for CK? when tested with extracts from different cell lines, and there was no cross reactivity with purified CK? and other related proteins like human ethanolamine kinases (EK) and yeast choline or ethanolamine kinases. The antiserum simultaneously detected both CK?1 and ?2 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 CK? in a human normal/tumor protein array of 30 tissue samples by using the antiserum showed that CK? was not overexpressed in all tumor tissues when compared to their normal counterparts. Most striking differences between tumor and normal CK? expression levels were observed in kidney (11-fold higher in tumor) and liver (15-fold lower in tumor) samples. CONCLUSION/SIGNIFICANCE: Apart 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 ?1 and ?2 isoforms in cell extracts for direct comparison of their expression levels.

SUBMITTER: Too WC 

PROVIDER: S-EPMC2946341 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature