Secretory phospholipase A2 (sPLA2) binds to lipid bilayers and catalyzes hydrolysis of phospholipids. Normally, cells resist the enzyme's action, but they become susceptible during apoptosis or trauma. Hydrolysis at the membrane surface apparently requires two steps: enzyme adsorption to the membrane surface followed by migration of phospholipids from their normal bilayer position up into the enzyme's active site. Experiments using erythrocytes as a model suggest that when cells become susceptible to sPLA2, boundaries between domains of ordered and disordered lipids proliferate. Reduction of favorable interactions among neighboring phospholipids at those boundaries is hypothesized to enhance sPLA2 activity by facilitating migration of phospholipids into the enzyme active site. This proposal will extend these studies to nucleated cells and test the hypothesis during hormone-stimulated apoptosis. Four questions will be asked. 1) Do changes in membrane order occur during apoptosis and do they reduce phospholipids-neighbor interactions? 2) How does apoptosis increase susceptibility to sPLA2; does it promote enhanced adsorption of the enzyme to the membrane surface, migration of lipids into the active site of the adsorbed enzyme, or both? 3) Is the hypothesis theoretically feasible? 4) How do these mechanisms apply to the various types of mammalian sPLA2? To answer these questions, six general procedures will be used to study changes in lymphoma cells during apoptosis stimulated by dexamethasone. First, alterations to membrane physical properties will be examined by fluorescence spectroscopy and microscopy using the membrane probe laurdan. Second, the strength of phospholipid-neighbor interactions will be assessed by the fluorescence of merocyanine 540 and by measuring the rate at which albumin extracts fluorescent phospholipids from the cell membrane. Third, the kinetics of membrane hydrolysis will be assayed at various enzyme concentrations and mathematically analyzed in the context of the two-step model described above. These experiments will be repeated using various forms of mammalian sPLA2. Fourth, the hypothesis will be evaluated theoretically by computer simulations. Fifth, the binding of sPLA2 to the surface of the cell membranes will be measured. Sixth, the ability of phospholipids to migrate to the active site of bound enzyme will be assessed by measuring the rate of extraction of phospholipids by sPLA2.
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