Purification and Biochemical Characterization of Ethanolamine Kinase from Spinach
| Autor: | Mercer, Shelly |
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| Jazyk: | angličtina |
| Rok vydání: | 1994 |
| Předmět: | |
| Druh dokumentu: | Diplomová práce |
| Popis: | Ethanolamine kinase (EC 2.7.1.82) catalyses the reaction of ethanolamine and Mg^2+-ATP to produce phosphoethanolamine and Mg^2+ -ADP. For spinach (Spinacia oleracea) the activity of ethanolamine kinase is increased in leaf extracts of salinized plants. A comparison of ethanolamine kinase activity between extracts from control and salinized plants after native polyacrylamide gel electrophoresis shows that ethanolamine kinase activity migrates to the same position on a gel. This observation suggests that salinization does not induce the activity of a novel ethanolamine kinase isozyme. Ethanolamine kinase has been purified 6,537 fold to apparent homogeneity from spinach leaves by ammonium sulphate fractionation and sequential fractionation by both open-bed and HPLC chromatography, using ion-exchange and hydrophobic interaction matrices. The enzyme has an estimated molecular weight of 80,000 D by gel filtration chromatography and a subunit size of 38,000 D by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Ethanolamine kinase has a broad pH optimum between pH 7 and 9 and the optimal ratio of Mg^2+:ATP for the reaction is 1: 1 at 5 mM. The apparent K^m value for the substrate ethanolamine is 16 μM and the V^max is 438 nmol • min^-1 •mg^-l protein. Monomethylethanolamine and dimethylethanolamine serve as substrates for. ethanolamine kinase but not trimethylethanolamine (ie choline). Enzyme activity is slightly stimulated by NaCl and KCl and inhibited to varying degrees by phosphate, ammonium, phosphoethanolamine and phosphodimethylethanolamine. Not surprisingly enzyme activity is also inhibited by ADP and to varying degrees by the divalent cations Mn^2+, Ca^2+, Co^2+, Ba^2+ and Ni^2+. This work is the first purification and biochemical characterization of ethanolamine kinase in spinach and is the first step towards understanding the contribution ethanolamine kinase makes towards the synthesis of choline. Thesis Master of Science (MSc) |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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