An improved procedure for the purification of ethanolaminephosphotransferase. Reconstitution of the purified enzyme with lipids.
Ethanolaminephosphotransferase (CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) has been purified in active form from rat brain microsomes by a two-step chromatographic procedure. Enzyme preparations characterized by high specific activity and stability were obtained supplementing the solubilization and elution buffers, containing 1% Triton X-100, with 0.01% 2,6-di-tert-butyl-4-methylphenol. The specific activity of the purified enzyme was about 1200-times higher than that of the crude solubilized enzyme. The lipid dependence of ethanolaminephosphotransferase was studied both in the presence of Triton X-100 and in detergent-free enzyme preparations. The activity of the detergent-solubilized ethanolaminephosphotransferase was strongly modified by phospholipids. The kinetic behaviour of the enzyme was also dependent on the lipids contained in the aggregates obtained by removal of the detergent from detergent/lipid/protein suspensions. A regulatory role of phospholipids on the activity of the membrane-bound ethanolaminephosphotransferase is discussed.
Roberti
R
R
Department of Biochemistry, Faculty of Medicine, University of Perugia, Italy.
Vecchini
A
A
Freysz
L
L
Masoom
M
M
Binaglia
L
L
D000818:Animals; D001921:Brain / Q000201:enzymology; D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004982:Ethanolaminephosphotransferase / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008563:Membrane Lipids / Q000502:physiology*; D008861:Microsomes / Q000201:enzymology; D017830:Octoxynol; D010770:Phosphotransferases / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D051381:Rats; D012995:Solubility
D008563:Membrane Lipids; D011092:Polyethylene Glycols; D017830:Octoxynol; D010770:Phosphotransferases; D004982:Ethanolaminephosphotransferase