Choline kinase and ethanolamine kinase are separate, soluble enzymes in rat liver

Choline kinase and ethanolamine kinase are located in the cytosol from rat liver and have been copurified more than 500-fold by affinity chromatography [P. J. Brophy and D. E. Vance (1976) FEBS Lett. 62, 123- 1251. Kinetic properties of the two activities were determined. Choline kinase had a K, for choline of 0.033 mM and ethanolamine was a competitive inhibitor (Ki = 6.2 mM). Ethanolamine kinase had a K, for ethanolamine of 7.7 mM and choline was a ‘mixed’ type of inhibitor with a Ki of 0.037 mM. Both enzyme activities responded in a similar fashion to the adenylate energy charge. Betaine and choline phosphate partially inhibited both kinases with a 93 % inhibition of the ethanolamine kinase by 5 mM choline phosphate. CTP and ethanolaminephosphate partially inhibited the ethanolamine kinase, but not the choline kinase. Other metabolites tested had negligible effects on both kinases. The affinity-column-purified enzyme was analyzed by disc gel electrophoresis which resolved the two activities. Hence, although many of the properties of the two activities are similar, choline kinase and ethanolamine kinase must be separate enzymes. Analysis of rat liver cytosol by disc gel electrophoresis indicated four isoenzymes for choline kinase and ethanolamine kinase. The phosphorylation of choline and ethanolamine is the first step in the synthesis de now of their respective phosphatides [l]. The ratio of choline kinase and the major ethanolamine kinase activities from rat liver remains constant when purified by DEAEcellulose and Sephadex G-200 chromatography and both activities have similar stability [2]. Recently we reported copurification of choline kinase and ethanolamine kinase more than 500-fold with a cholineSepharose 6B affinity column [3]. These data suggested that both kinase activities might be associated with the same protein. In contrast, a recent report stated that the activities from rat liver are associated with discrete proteins and choline kinase is predominantly a microsomal enzyme whereas ethanolamine kinase is localized in the cytosolic fraction [4]. In the light of these conflicting reports, we have studied the properties of highly purified choline kinase and ethanolamine kinase from rat liver. Our results unambiguously demonstrate that choline kinase and ethanolamine kinase are separate, soluble enzymes in rat liver.