Purification and characterization of choline/ethanolamine kinase from rat liver.
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.min-1.mg-1 protein. The subunit molecular mass was 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the apparent native molecular mass 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 sodium dodecyl sulfate gel electrophoresis. Peptide mapping of two isoforms indicated a high degree of similarity, although there were peptides not common to both. Ethanolamine kinase activity copurified with both isoforms. The ratio of choline to ethanolamine kinase activity was 3.7 +/- 0.7 throughout the purification procedure. Choline and ethanolamine were mutually competitive inhibitors. The respective Km values, 0.013 and 1.2 mM, were similar to the Ki 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 mass of 47 kDa.
Porter
T J
TJ
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Kent
C
C
D000085:Acetates; D019342:Acetic Acid; D000645:Ammonium Sulfate; D000818:Animals; D001667:Binding, Competitive; D005591:Chemical Fractionation; D002794:Choline / Q000494:pharmacology; D002797:Choline Kinase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography; D003600:Cytosol / Q000201:enzymology; D019856:Ethanolamine; D004983:Ethanolamines / Q000494:pharmacology; D005260:Female; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010449:Peptide Mapping; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D051381:Rats; D011919:Rats, Inbred Strains
D000085:Acetates; D004983:Ethanolamines; D019856:Ethanolamine; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D002797:Choline Kinase; C020125:ethanolamine kinase; D002794:Choline; D019342:Acetic Acid; D000645:Ammonium Sulfate