SYNTHESIS OF PHOSPHATIDYLINOSITOL IN RAT-LIVER MICROSOMES IS ACCOMPANIED BY THE RAPID FORMATION OF LYSOPHOSPHATIDYLINOSITOL

In mammalian cells, newly synthesized phosphatidylinositol (PI) has a fatty acid composition similar to its precursors, phosphatidic acid and CDP-diacylglycerol (DAG). It is then remodelled by deacylation/reacylation cycles to the predominant form, 1-stearoyl, 2-arachidonoyl PI. Incubation of dipalmitoyl CDP-DAG, [H-3]inositol and Mg2+ with rat liver microsomes results in the rapid synthesis of PI, along with the simultaneous formation of multiple species of lysoPI. Analysis of the kinetics of formation of PI and lysoPI reveals no lag in the formation of lysoPI from PI. Moreover, evaluation of the concentration dependencies indicate nearly identical apparent K(m) values for PI synthesis compared with lysoPI synthesis for the substrates inositol (180-mu-M) and CDP-DAG (100-mu-M). The dependence on pH and the requirement for Mg2+ or Mn2+ are nearly identical for PI and lysoPI formation and the labelling of both lipids is similarly inhibited by submicromolar concentrations of calcium and by NEM. These results suggest that the formation of lysoPI is dependent on the initial, rate-limiting synthesis of PI. Pulse-chase analysis of the labelling of these lipids indicates that PI and lysoPI rapidly equilibrate after the initial slow synthesis of PI. In addition, it appears that only newly synthesized PI is involved in lysoPI formation. The extent of lysoPI formation depends upon the fatty acid composition of the added CDP-DAG. A number of experimental approaches demonstrate that lysoPI is not formed when pre-existing microsomal PI is labelled by head group exchange, perhaps because this PI has already undergone remodelling to polyenoic forms. These data suggest that the rapid deacylation of newly synthesized PI may represent the first step in PI remodeling.