Partial purification and properties of ethanolamine kinase from spinach leaf.

Ethanolamine kinase was purified 60-fold by fractionation with ammonium sulfate, freeze-thawing, and gel filtration from a 100,000g supernatant from spinach leaf. The 100,00g supernatant preparation was stable for weeks, but the partially purified preparation lost half of the ethanolamine kinase activity in 10–14 days at 0–4 °C or ?20 °C. A molecular weight of 110,000 was estimated by gel filtration on Sephadex G-200. The reaction required ethanolamine (K_m, 42 μm), MgATP (K_m, 63 μm), and free magnesium ions. The enzyme was inhibited by MgATP, with an apparent K_i of 6.7 mm. Ethanolamine kinase was inhibited by calcium (in the presence of magnesium) and o-phenanthroline. EDTA above 0.9 mm inhibited the formation of phosphorylethanolamine and EGTA stimulated at low concentrations (0.4-0.9 mm) and inhibited at 1.8 mm. Ethanolamine kinase was inhibited by monomethylethanolamine and dimethylethanolamine, but not by choline (5 mm). The ethanolamine kinase and choline kinase activities of the 100,000g supernatant preparation could be separated by gel electrophoresis