Purification and characterization of choline kinase from rat liver

Thomas John Porter, Purdue University

Abstract

Choline kinase, the first enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis, was purified 26,000-fold from rat liver to a specific activity of 143,000 nmol$\cdot$min$\sp{-1}\cdot$mg$\sp{-1}$ protein. The subunit molecular weight was 47 kDa by SDS-polyacrylamide gel electrophoresis, while the apparent native molecular weight was 160 kDa by size exclusion chromatography, suggesting a tetrameric structure. Two peaks of choline kinase activity were obtained by chromatofocusing. These isoforms eluted at pH 4.7 (CKI) and 4.5 (CKII). CKII appeared to be homogeneous by SDS-polyacrylamide gel electrophoresis. Peptide mapping of the two isoforms indicated a high degree of similarity, although there were peptides not common to both. Ethanolamine kinase activity co-purified with both isoforms. The ratio of choline to ethanolamine kinase activity was 3.7 $\pm$ 0.7 throughout the purification procedure. Choline and ethanolamine were mutually competitive inhibitors. The respective $K\sb{m}$ values, 0.013 and 1.2 mM, were similar to the $K\sb{i}$ values of 0.014 and 2.2 mM. An antibody raised against CKII immunoprecipitated both choline and ethanolamine kinase activities from liver cytosol at the same titer. These data suggest that both activities reside on the same protein and occur at the same active site. Similarly, both activities were immunoprecipitated from brain, lung, and kidney cytosols. Western blot analysis showed both purified liver isoforms, as well as brain, lung and kidney enzymes, to have a molecular weight of 47 kDa.

Degree

Ph.D.

Advisors

Kent, Purdue University.

Subject Area

Biochemistry

COinS