Phosphatidylinositol metabolism is important to the growth of eukaryotic organisms. The agonist-stimulated hydrolysis of membrane polyphosphoinositides in animals result in the stimulation of various cellular responses. The enzyme that catalyzes the initial step in the synthesis of the polyphosphoinositides is phosphatidylinositol kinase. This application will focus on the regulation of phosphatidylinositol kinase in the simple eukaryote, Saccharomyces cerevisiae. Phosphatidylinositol kinase will be purified to homogeneity and will be characterized with respect to enzymological, chemical, and physical properties. The purified enzyme will be reconstituted into well defined unilamellar phospholipid vesicles to study the regulation of activity. Regulation will be studied by altering the composition and environment of the vesicles and by possible phosphorylation and dephosphorylation of the enzyme. Antibodies specific for phosphatidylinositol kinase will be prepared and used as a probe to study the regulation of phosphatidylinositol kinase formation in cells grown under various conditions. The proposed studies should provide insight into the regulation of polyphosphoinositide synthesis. The results found with S. cerevisiae should be relevant to higher eukaryotic organisms.
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