Phospholipase A/2s (PLA/2) are a diverse group of enzymes that are ubiquitous in nature and are found in every cell where they have been sought. This enzyme catalyzes the hydrolysis of the sn-2-acyl ester bond of phospholipids with or without calcium (depending on the source), and provides precursors for eicosanoid generation. PLA/2 plays a central role in phospholipid metabolism and digestion, signal transduction and host defense. This enzyme has been implicated in various diseases including asthma, arthritis, pulmonary disorders, gastrointestinal disorders, heart disease, septic shock and a host of other metabolic disorders. PLA/2 activity has been most recently correlated with glucose metabolism, and data suggest that this phospholipase is associated with diabetes. The mammalian phospholipases have been found from a variety of sources, and are widely diverse in nature. The long-term goal of this research project is to: (1.) Study on a molecular level how the human placental phospholipase A/2s are affected by its natural protein inhibitor, the human placental lipocortin; (2.) To elucidate the catalytic mechanism of the calcium-dependent and the calcium-independent PLA/2s from human placenta; (3.) To relate these findings to what is known about the abnormal cellular systems; and (4.) Define an experimental model for this inhibitory process. In order to properly characterize this inhibitory process important baseline information has to be established. The baseline data needed are: (1.) The determination of the optimal physical state of the substrate phospholipid for the measurement of the human placental phospholipase A/2 activity at the lipid/water interface, (2.) The determination of the backbone structure of the human placental enzymes, and (3.) The determination of the effects of calcium and phospholipid binding on the backbone structure of the human placental phospholipase A/2s. The experiments in this proposed research project are designed to answer the fundamental questions: What is the backbone structure of the human placental phospholipase A/2s? What is the substrate specificity of these human placental phospholipase A/2s? What effect does calcium and phospholipid binding have on the backbone structure of these phospholipases? How does the interaction of lipocortin with phospholipase A2 effect the secondary structure of the phospholipases. What is the secondary structure of the human placental lipocortin? Three types of human placental phospholipase A/2s: high molecular weight calcium-dependent (70 KDa), high molecular weight calcium-independent (70 KDa) and low molecular weight calcium-dependent (15 KDa) enzymes will be analyzed in this study. The kinetic and structural characterization of these proteins will be done by: Fluorescence spectroscopy, Fourier Transform Infrared (FTIR) Spectroscopy and lipid chemistry. These methods will be employed to study protein structure, protein-lipid, protein-calcium interactions and substrate specificity of the human placental phospholipases.
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