Factors affecting the stability of detergent-solubilized cholinephosphotransferase and ethanolaminephosphotransferase.
Cholinephosphotransferase (CPT) and ethanolaminephosphotransferase (EPT) are the enzymes catalyzing the last step of the de novo pathway for phosphatidylcholine and phosphatidylethanolamine synthesis, respectively. A major limitation for the complete characterization of the reactions catalyzed by the two enzymes derives from their poor stability in detergent-containing buffers. CPT is heavily inactivated, when native membranes are solubilized using a series of detergents, whereas EPT activity is better preserved during solubilization. An investigation of the factors which could play a role in preserving both enzymes from inactivation was carried out. The dramatic loss of enzymatic activities occurring upon dilution of solubilized membranes with detergent-containing buffers can be reduced by supplementing the dilution medium with phospholipids. The addition of Mn2+ ions to the dispersion buffer increases the stability of both enzymes. The procedure previously described for solubilizing EPT from rat brain microsomes has been modified on the basis of this evidence. Microsomes were solubilized in buffered detergent solutions containing Mn2+ ions and both CPT and EPT were partially purified in their active form by anion-exchange chromatography.
Mancini
A
A
Institute of Biochemistry, Medical School, University of Perugia, Italy.
Roberti
R
R
Binaglia
L
L
el Missiri
A
A
Freysz
L
L
D000818:Animals; D001921:Brain / Q000201:enzymology; D002852:Chromatography, Ion Exchange; D003902:Detergents; D002798:Diacylglycerol Cholinephosphotransferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004982:Ethanolaminephosphotransferase / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats; D012995:Solubility
D003902:Detergents; D011092:Polyethylene Glycols; D017830:Octoxynol; D004982:Ethanolaminephosphotransferase; D002798:Diacylglycerol Cholinephosphotransferase