Purification and characterization of IPC Synthase from saccharornyct'f- cf-revisiae

I lie membrane associated onzyme inositolphosphorylceramidem synthase (IPC' >ynth;iM\ phosphatidyUnositot:ceramide phosphoinositol transferase) catalyzes ,ii! essential step in the biosynthesis of yeast inositol-containing sphingolipids. IPC syntlid_o was purified 3,-liîl fold from the yoast Saccharomycts cerfrJMfl. The purification procedure included Triton X- !00 solubilization of yeast rnicrosornal membranes followed by chromatography on Q-Sepharose, Octyl St'pharose. Mono Q and Resource PHE. Fxamination of the purified protein by SOS -4\ elertrophoresis has jevealod four major protein bands suggesting the cn/yini: lias been highly purified but not to homogeneity. The four major protein hdnds have minimum subunit molecular weights of 97 kl)a. 66 kl)a. 50 kl>;i und 16 kl)a. Attempts to purify the en/yme further resulted in total loss of en/yme activity. Maximal IPC .synthase activity was measured at ,'IOT and pH 7,0 in the presence of 5 m M Triton X 100, l m M manganese and 5 m M magnesium mns, [['(' synthase activity was dependent upon the surface concentration of phosphaudylinositol (PI) and ceramide in the Triton X-100 mixed micellar assay syvi.-m. PI activated IPC synthase activity in a cooperative manner with .1 Hill construit of 3. The apparent Km values were 5 itiol % for the surface con ecu t r at im of PI and 1.35 mol 9e for the surface concentration of rerurnide in the Triton X-100 mixed micellar assay hVulom. Thioreactive reagents, sphingoid hases rind Aurcohasidin A inhibited IPC svnthase activity. (Supported by NUI Grant CM i'121 1 i.