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Journal of Undergraduate Research

Keywords

secretory phospholipase A2, sPLA2, inflammatory responses, diseases

College

Life Sciences

Department

Physiology and Developmental Biology

Abstract

Secretory phospholipase A2 (sPLA2) binds to and hydrolyzes cell membranes. It is important in inflammatory responses and diseases including septic shock, atherosclerosis, and cancer. Normally, cells resist the enzyme’s action, but they become susceptible early during the process of either biochemically-programmed or of traumatic cell death. A recent discovery demonstrated that the activity of different isozymes of sPLA2 depends on the type of cell death involved. Understanding the relationship between this specificity and physiological roles of these isozymes requires elucidation of the relevant molecular mechanisms. This project will test the hypothesis that the relative levels of four membrane biophysical parameters determine the activity of each isozyme to hydrolyze dying cells: 1) binding of merocyanine 540, 2) membrane solvation, 3) permeability of propidium iodide, and 4) phosphatidylserine exposure on the outer membrane face. The hypothesis will be examined by using a variety of agents to induce a spectrum of cell death modes ranging from apoptosis to necrotic death caused by oxidative stress. The activity of various sPLA2 isozymes to hydrolyze the cell membrane and the levels of the four parameters will be assayed for each type of cell death. A quantitative global model that combines the information from these assays to predict each isozyme’s specificity toward the mode of cell death will then be generated and evaluated. The primary method employed to asses membrane physical properties will be fluorescence spectroscopy. Subtle changes in membrane permeability to propidium iodide will be quantified by flow cytometry. Membrane hydrolysis will be assayed with a fluorescent fatty acid binding protein. In summary, this project will help guide a novel direction in sPLA2 research relating the biophysics of cell membranes to enzyme activity in the physiological and pathological setting of cell death.

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