It is our hypothesis that membrane structural fluctuations play an important role in the modulation of biological function. Assuming that the site of anesthetic action is the lipid matrix of the biological membrane, we propose that the initial event in anesthesia is alteration of such fluctuations. To test this hypothesis we plan to use the gel-liquid crystalline transition of single bilayer vesicles as a model for such fluctuations and investigate the affect of various anesthetics on the melting temperature, and the average cluster size which is related to the half-width of the transition. Additional studies will include the effect of anesthetics on the kinetics of the transition using a novel volume perturbation, dynamic calorimeter and their effect on lipid-related protein fluorescence noise. A most important aspect of this study will be the investigation of the effect of anesthetics on the activation of pancreatic phospholipase A2 for which we have proposed a model which quantitatively describes data for the lipase's action on phosphatydilcholine vesicle substrates. This model has three key ingredients: the enzyme binds preferentially to the lipid in the gel state; the activation process involves dimer formation on the membrane surface; and the rate of activation is directly proportional to the magnitude of structural fluctuations within the membrane. This latter aspect of the model is particularly important in developing an understanding of mechanisms of enzyme regulation since all membrane constituents can potentially alter membrane fluctuations. The proposed series of experiments will provide a library of data of several thermodynamic and kinetic parameters related to the lipid-enzyme system. This library of information will allow construction of specific quantitative statements describing anesthetic action in terms of its influence on membrane structure and membrane-mediated protein function. Reaction microcalorimetry, differential scanning calorimetry and pH stat technique will be used in this investigation. This laboratory possesses unusual expertise in these techniques which will be complemented by other procedures such as fluorescence measurements, electron microscopy and nuclear magnetic resonance. The experimental worked will be supplemented by computer simulation (e.g. Monte Carlo Calculations) of models describing the composite of the experimental results.
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