FACTORS AFFECTING THE STABILITY OF DETERGENT-SOLUBILIZED CHOLINEPHOSPHOTRANSFERASE AND ETHANOLAMINEPHOSPHOTRANSFERASE

Cholinephosphotransferase (CPT) and ethanolaminephosphotransferase (EP T) are the enzymes catalyzing the last step of the de novo pathway for phosphatidylcholine and phosphatidylethanolamine synthesis, respectiv ely. A major limitation for the complete characterization of the react ions 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, were as EPT a ctivity is better preserved during solubilization. An investigation of the factors which could play a role in preserving both enzymes from i nactivation was carried out. The dramatic loss of enzymatic activities occurring upon dilution of solubilized membranes with detergent-conta ining buffers can be reduced by supplementing the dilution medium with phospholipids. The addition of Mn2+ ions to the dispersion buffer inc reases the stability of both enzymes. The procedure previously describ ed for solubilizing EPT from rat brain microsomes has been modified on the basis of this evidence. Microsomes were solubilized in buffered d etergent solutions containing Mn2+ ions and both CPT and EPT were part ially purified in their active form by anion-exchange chromatography.