Phospholipases A2 (PLA2s) catalyze the hydrolysis of the sn-2 ester of membrane phospholipids to produce a free fatty acid and a lysophospholipid. There has been considerable interest in these enzymes because of their role in the liberation of arachidonic acid from mammalian cell membranes for the biosynthesis of the eicosanoid mediators of inflammation (prostaglandins, leukotrienes, and others). There is also considerable interest in these enzymes as a paradigm for understanding interfacial enzymology (the action of enzymes at the lipid-water interface). Many enzymes in cells operate on membranes. Mammalian cells contain two types of PLA2s that are involved in arachidonic acid production. The secreted PLA2s (sPLA2s) are 14-18 kDa, calcium-dependent enzymes that are secreted from a large number of mammalian cells following stimulation with pro-inflammatory mediators. Mammalian cells also contain an 87-kDA cytosolic PLA2 (cPLA2) that translocates to the membrane in response to a rise in cytosolic calcium. Recently we have cloned several new mouse and human sPLA2s. Thus, it is now clear that the sPLA2s constitute a superfamily of proteins in mammals. We have made significant progress toward the production of recombinant forms of the full set of mouse and human sPLA2s. We will complete this process and also study the interfacial kinetic and binding properties of these enzymes. We are now faced with the task of determining the expression profile of these sPLA2s in mammalian cells, and our studies will focus on cells from the airways of normal and asthmatic patients. It is well established that eicosanoids play a prominent role in causing many of the symptoms associated with asthma. It is important to determine which PLA2s are involved in releasing arachidonic acid. Such information will be useful in designing a new generation of therapeutics for treatment of airway inflammatory disorders. Once we have identified the PLA2s that are expressed in human airway cells, we will use PLA2-specific antibodies and small molecular weight inhibitors to probe whether these enzymes are involved in arachidonic acid release. The mechanisms by which PLA2s act in mammalian cells to liberate arachidonic acid are partially understood. Some sPLA2s act extracellularly on the plasma membrane of cells, while others are internalized into punctate intracellular membrane compartments. For the latter case, it is important to define this membrane compartment in more detail and to determine where the enzyme acts to release arachidonic acid. These studies will contribute to our fundamental understanding of how mammalian cells initiate the eicosanoid cascade.
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