Complete co-purification of choline kinase and ethanolamine kinase from rat kidney and immunological evidence for both kinase activities residing on the same enzyme protein(s) in rat tissues.
Choline kinase and ethanolamine kinase were completely co-purified from rat kidney cytosol through acid treatment, ammonium sulfate fractionation, DEAE-cellulose column chromatography, Sephadex G-150 gel filtration followed by choline-Sepharose affinity chromatography. The final preparation appeared to be highly homogeneous with respect to both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Ishidate, K., Nakagomi, K. and Nakazawa, Y. (1984) J. Biol. Chem. 259, 14706-14710). Throughout the purification steps, the ratio of ethanolamine kinase activity to choline kinase activity was almost constant in a range of 0.3-0.4, which strongly indicated that, in rat kidney, both activities could reside on a single enzyme protein. The rabbit polyclonal antibody raised against highly purified rat kidney choline (ethanolamine) kinase protein inhibited both choline and ethanolamine kinase activities in a parallel manner in crude enzyme preparations not only from rat kidney, but also from rat liver, lung and intestinal cytosols. The results, together with our previous findings, suggested strongly that, in rat tissues, at least large portions of both kinase activities are present on the same enzyme protein(s). The kinetic properties of both kinase reactions with the highly purified kidney enzyme were compared in some detail and the overall result suggested that choline kinase and ethanolamine kinase activities may not have a common active site on a single enzyme protein.
Ishidate
K
K
Furusawa
K
K
Nakazawa
Y
Y
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002797:Choline Kinase / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D010770:Phosphotransferases / Q000302:isolation & purification*; D017853:Phosphotransferases (Alcohol Group Acceptor); D011817:Rabbits; D051381:Rats; D014018:Tissue Distribution
D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); D002797:Choline Kinase; C020125:ethanolamine kinase; D008274:Magnesium