Irene Litosch - US grants

The proposed studies are designed to investigate the regulation of phosphoinositide breakdown by hormones in a cell-free system from the blowfly salivary gland. Phosphoinositide breakdown has been linked to a receptor mechanism, distinct from the cyclic AMP system, involved in the elevation of cytosolic Ca2+. The system to be used in these studies, the salivary gland of the blowfly, Calliphora erythrocephala, selectively incorporates (3H)ubisutik and (32P)Pi into the pool of phosphoinositides involved in hormone action. Using a membrane fraction, obtained from salivary glands prelabeled with (3H)inositol, it is possible to demonstrate 5-hydroxytryptamine-stimulated production of inositol phosphates derived from phosphoinositide breakdown. Hormone-stimulated breakdown in the cell-free system is rapid and is mediated through a phospholipase active at physiological pH. The relationship between phosphatidylinositol and the polyphosphoinositides in the cell-free system has not been defined. The isolated membrane fraction will be used for the biochemical characterization of the phospholipases involved in hormone-stimulated phosphoinositide breakdown, the mechanism by which hormone stimulates phosphoinositide breakdown, the nature of the active pool of phosphoinositides involved in hormone action and the metabolic interrelationship between phosphatidylinositol and the polyphosphoinositides.

A major mechanism of transmembrane signal transduction utilizes regulatory GTP-binding proteins to mediate events from receptor activation to the effector. Adenylyl cyclase, cyclic GMP phosphodiesterase and a number of ion channels are regulated by heterotrimeric G proteins. Increasing evidence indicates that another class of important enzymes, phospholipases, are also regulated through a receptor-G protein dependent mechanism. The identity and properties of the GTP-binding proteins which regulate phospholipase activity has eluded identification suggesting that regulation of this class of enzymes occurs through a novel G protein or novel G protein regulated mechanism. The phosphoinositide specific phospholipase C is linked to receptor mechanisms involved in elevation of cytosolic Ca2+ levels and increase in protein kinase C activity. The identity of phospholipase C and its regulatory GTP-binding protein (Gp) however has yet to be established. Guanine nucleotide-dependent inhibition of phospholipase C activity has been demonstrated in membranes, suggesting that phospholipase C activity may be regulated by both stimulatory and inhibitory G proteins. A major goal of the proposed studies is to purify the Gp-regulated PLC and to characterize its regulation by Gp. To accomplish these goals, conditions which solubilize a Gp-regulated phospholipase C activity from bovine brain membranes have been established. Purification of the solubilized activity results in the resolution of two distinct activities, a Gp-insensitive and Gp-sensitive phospholipase C. GTP-gamma-S promotes a marked increase in the Ca2+ sensitivity of the Gp regulated activity. This suggests that the Gp regulated phospholipase C is associated with its regulatory component and constitutes a source of the regulated phospholipase C. The properties of Gp will be characterized. The nature of the interaction between Gp and phospholipase C will be ascertained by hydrodynamic studies. The Gp regulated PLC will be used to purify or identify Gp. The interaction between these components will be studied in a reconstituted system. These studies will extend our knowledge concerning the mechanism of regulation of phospholipase C by GTP-binding proteins and provide insight into the regulation of this important class of enzymes.