ELECTRIC FIELD SENSITIVITY OF YEAST H+

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The proposed research is targeted towards investigating the interactions between low frequency AC electric fields and membrane proteins. The basic approach will involve measuring the rate of reactions catalyzed by proton ATPase as a function of the frequency and amplitude of the field. We will use a very broad range of frequencies and amplitudes in order to establish a connection between effects seen at moderate fields (1-10 V/cm) and weak fields (1-100 mV/cm). Since our experiments will be carried out on vesicles rather than much larger mammalian cells, the corresponding fields anticipated to cause similar effects in human tissue are much smaller, so this work is directly relevant to understanding possible effects of powerline or household appliance fields (10(-6)-10(-3) V/m). The experiments will be carried out at many different conditions in order to test postulated mechanisms for the field-protein interactions. Some of these include electro-conformational coupling, effects of electric polarization of the double layer, and field inducted changes in ionic concentration in the double layer. We have derived a general expression for the field and amplitude dependence of the response to a weak oscillating field. This can be described as a sum of Lorenzian curves, where the amplitudes depend on the square of the applied field strength. The dependence of these amplitudes on the experimental conditions is different for different mechanisms. Thus, by measuring these amplitudes at different conditions we can distinguish between different mechanisms. The enzyme used in our study will be the H+ATPase from yeast because it is readily available in purified form from our collaborator, and because much evidence suggests a strong membrane potential dependence for this enzyme. Thus, there is a very good likelihood of experimental success. In initial experiments we will use enzyme in plasma membranes from fractions with very high specific activity. Later, we will use purified enzyme reconstituted in phospholipid bilayer vesicles.