Solubilization and Characterization of Microsomal-Associated Phosphatidylinositol: Ceramide Phosphoinositol Transferase From Saccharomyces Cerevisiae

The membrane associated enzyme phosphatidylinositol: ceramide phosphoinositol transferase (IPC synthase) catalyzes an essential step in the biosynthesis of yeast inositol-containing sphingolipids. A variety of solubilization agents were examined for their ability to release IPC synthase activity from yeast membranes. The most effective solubilization agent was Triton X-100 which released over 90% of the IPC synthase activity. The basic enzymological properties of the solubilized enzyme were determined using a Triton X-100/phosphatidylinositol/ceramide/enzyme mixed micellar assay system. IPC synthase activity was dependent upon the surface concentrations of phosphatidylinositol and ceramide in a phosphatidylinositol/ceramide/Triton X-100 mixed micelle. Maximal enzyme activity was measured at 30C and pH 7.0 in the presence of 5 mM Triton X-100, 1 mM manganese and 5 mM magnesium ions.