BINDING OF PHOSPHOLIPASE A2 TO BILAYERS

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Action of phospholipase A2 on phospholipid bilayers and biomembranes depends upon their organization. The experimental aim of this study is to characterize the action of phospholipase A2 on vesicles of anionic and zwitterionic lipids in order to understand the role of anionic surface charge, the role of substrate binding to the active site, the role of interface recognition site, and the role of calcium binding sites. From these experiments we also hope to develop an understanding of the interrelationships between the various binding sites and kinetic parameters. Thus, for example, intravesicle scooting of the enzyme leads to higher initial rate of hydrolysis, whereas faster intervesicle exchange of the bound enzyme would lower apparent rate of hydrolysis. We also plan to characterize the microenvironment of the bound enzyme by probing the state of the enzyme in the bilayer, and by monitoring the phase state of the substrate bilayer in the presence and in the absence of the enzyme. The phase state and local purturbation of the bilayer will be characterized by monitoring transbilayer movement, marker release, lateral phase separation, and fusion of vesicles. These experiments are designed with the paradigm that defects in the substrate interface regulate the binding and the catalytic turnover of the enzyme. Such experiments are expected to provide information about binding and catalytic specificity for the substrate structure and the phase state of their bilayer, about rates of transvesicle exchange of the bound enzyme, about the effect of the phase state on the kinetic and binding parameters. This information is central to understanding the mechanism of interfacial activation of phospholipase A2. Activation and specificity of phospholipase A2 is thought to be the first and the rate limiting step in the biosynthesis of prostaglandins in vivo, since the phospholipase A2 releases or regulates the release of arachidonic acid from membrane phospholipids. All these studies will be conducted on phospholipase A2 from pig pancreas, and could be later extended to the enzyme from other sources.