Biosynthesis of phosphatidylinositol in Crithidia fasciculata

Microsomal preparations from the protozoan Crithidia fasciculata were shown to incorporate myo-[2-³H]inositol into phosphatidylinositol by both the CDPdiacylglycerol: myo-inositol phosphatidyltransferase reaction and by a myo-inositol exchange reaction. Non-ionic detergent and Mg²⁺ were necessary for the measurement of transferase activity. Untreated preparations could not be saturated with Mg²⁺, even at very high concentrations (50-75 mM). However, low concentrations of EGTA (75 μM) both stimulated the activity 3-fold and reduced the Mg²⁺ required for saturation to 15-20 mM. EGTA also increased the apparent K_m for CDPdiacylglycerol while increasing the sensitivity to substrate inhibition above 1 mM. The transferase activity was inhibited by relatively low concentrations of Ca²⁺ (50 μM). This and the EGTA effect suggest a possible role for Ca²⁺ in the modulation of phosphatidylinositol synthesis. The myo-inositol exchange activity required Mn²⁺, was insensitive to Ca²⁺ inhibition and was only slightly stimulated by detergents and EGTA. This activity was preferentially inactivated by heating at 50°C in the presence of Triton X-100. In a detergent solubilized preparation the exchange activity but not the transferase exhibited a non-specific requirement for phospholipid. The differences in properties of the two activities suggest the presence of a separate exchange enzyme.