Partial purification and properties of ethanolamine kinase from spinach leaf

Abstract

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 μ m), MgATP ( K m , 63 μ m), and free magnesium ions. The enzyme was inhibited by MgATP, with an apparent K i of 6.7 m m. Ethanolamine kinase was inhibited by calcium (in the presence of magnesium) and o-phenanthroline. EDTA above 0.9 m m inhibited the formation of phosphorylethanolamine and EGTA stimulated at low concentrations (0.4-0.9 m m) and inhibited at 1.8 m m. Ethanolamine kinase was inhibited by monomethylethanolamine and dimethylethanolamine, but not by choline (5 m m). The ethanolamine kinase and choline kinase activities of the 100,000g supernatant preparation could be separated by gel electrophoresis