Purification and characterization of inositolphosphoryl ceramide synthase from the yeast Saccharomyces cerevisiae

Yongsheng Liu, University of Rhode Island

Abstract

Sphingolipid synthesis is vital for growth and viability of the yeast Saccharomyces cerevisiae. A key enzyme in the biosynthesis of the yeast sphingolipids is inositol-phosphoryl ceramide (IPC) synthase, which catalyzes the transfer of phosphoinositol from phosphatidylinositol (PI) to ceramide to yield IPC. This enzyme is present in yeast as well as other fungi but absent in humans making it a unique target for antifungal drugs. In this study, IPC synthase was purified 3,431 fold from the yeast Saccharomyces cerevisiae. The purification procedure included: Trition X-100 solubilization of yeast microsomal membranes followed by chromatography on DEAE DE 52, Q-Sepharose, Octyl-Sepharose, Mono Q, and Resourse Phenyl. Examination of the purified protein by SDS gel electrophoresis has revealed four major protein bands suggesting the enzyme has been highly purified but not to homogeneity. The four major protein bands have minimum subunit molecular weights of 58 kDa, 56 kDa, 52 kDa and 45 kDa. Attempts to purify the enzyme further resulted in total loss of enzyme activity. Maximal IPC synthase activity was measured at 30$\sp\circ$C and pH 6.5 in the presence of 5 mM Triton X-100, 1 mM manganese, and 5 mM magnesium ions. IPC synthase activity was dependent upon the surface concentration of PI and ceramide in a Triton X-100 mixed micellar assay system. PI activated IPC synthase activity in a cooperative manner with a Hill constant of 3. The apparent K$\sb{\rm m}$ values were 5 mol% for the surface concentration of PI and 0.69 mol% for the surface concentration of ceramide in the Triton X-100 mixed micellar assay system. Sphingoid bases, nucleotides, cardiolipin, CDP-diacylglycerol and Aureobasidin A inhibited IPC synthase activity. Phosphatidylglycerol stimulated IPC synthase activity. The cooperative activation of IPC synthase by PI suggests a role for this lipid in stimulating or limiting the synthesis of the yeast inositol-containing sphingolipids via regulation of IPC synthase activity. ^

Subject Area

Biology, Molecular|Agriculture, Food Science and Technology|Biology, Cell|Chemistry, Biochemistry

Recommended Citation

Yongsheng Liu, "Purification and characterization of inositolphosphoryl ceramide synthase from the yeast Saccharomyces cerevisiae" (1998). Dissertations and Master's Theses (Campus Access). Paper AAI9908229.
http://digitalcommons.uri.edu/dissertations/AAI9908229

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