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| TITLE: | Study of the Electric Field Sensitivity of Yeast H+ ATPase | ||
| Principal Investigator |
R. Dean Astumian, Ph.D. | University of Chicago | |
| Health Relevance |
Other: Biophysics of Cell Field Interactions | ||
| Research Categories |
Cellular Function | Biophysics Cell/Field Interactions | Cellular Processes |
| FY95 Funds | R29ES06620 $108,267 | Start Date 09/28/94 | End Date 08/31/98 |
| Rationale and Summary |
The focus of this research project is to study ion transport proteins in order to determine
possible mechanisms of interaction between electric fields and biological systems. Many
experiments have focused on what we refer to as "downstream" effects, effects that may
be caused by electric field exposure but require several intermediate processes before a
biological response is seen. Thus, any condition of the cell that influences any of the
intermediary states can change the overall response to a field in sometimes difficult to
understand ways. Our approach is to study a reaction where the process that is the primary
site of interaction can be directly assayed and studied as a function of field parameters
such as frequency and amplitude. While ultimately, we are interested in the effects of 60
Hz fields at amplitudes below a few mV/cm, it is essential to investigate the mechanism of
interaction over a wide range of frequencies and amplitudes to establish a baseline
understanding of how electric fields and cells may interact.
Our specific hypothesis is that the proton ATPase that we are studying will have similar field dependence to the related protein Sodium-Potassium ATPase (NaKATPase) for which both frequency and amplitude optima have been observed. The proton ATPase is a simpler system, and so a more exhaustive study than is possible with NaKATPase can be accomplished. Specifically, we want to understand the biophysics behind the frequency and amplitude peaks. Many theories have been advanced which can only be tested be studying the behavior under many different conditions and comparing the results between these experiments. Ultimately, we anticipate that our research will provide insight that will help us to predict what proteins are likely to be very sensitive to electric fields, and under what conditions. |
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| Experimental Design and Exposure Conditions |
Our experimental design employs an electric field applied to a suspension of yeast proteoliposomes that contain the proton ATPase using plane parallel electrodes. As a precaution against artefacts resulting from electrolysis products, we separate the electrode from the sample solution by using a salt bridge, although preliminary experiments suggest that this cumbersome precaution is not necessary and we may dispense with the salt bridge, particularly for the very low field experiments. When the proteoliposome are made by breaking open whole yeast cells, most of the vesicles turn inside out. In this topology, the ATPase site faces outside, and the ATP mediated proton pumping is directed from out to in. This makes the assay quite convenient, since the level of ATP can be changed from the outside, and the proton uptake into the vesicle can be followed by acridine orange (a weak base) localization into the vesicle as an optical signal. All of these facts combine to make it possible to carry out experiments at many different field strengths and amplitudes ranging from .01 mV/cm to 1 V/cm, and 1 Hz to 100,000 Hz. The measured quantities are the steady state rates of proton uptake and ATP hydrolysis so transients are not expected to play a role. | ||
| Quality Assurance Measures |
Many of the types of problems that plague very week field experiments using inductive coupling, such as stray fields, etc. are not relevant for the type of experiments we are performing. A particular advantage is the experiments will always include zero field (no exposure) which serves as a negative control, and high field (1 V/cm) where an effect is always observed, serving as a positive control. | ||
| Results and Discussion |
Most of this last year has been devoted to setting up our laboratory and to validating the
established assays for ATP hydrolysis and proton uptake in our own labs. The assays were
developed in the laboratory of our collaborator, David Perlin at the Public Health Research
Institute in New York. We have also worked on theoretical models of electric field driven
transport which will be used in interpreting our data. These include "Brownian Ratchet"
models for transport which offer an explanation of how an oscillating field, with an
average of zero, can lead to a net "DC" effect on the transport or catalytic rate of an
enzyme.
By next year, we expect to have the first results for the frequency dependence of ATP hydrolysis and proton transport under identical conditions. In order to have an easily measurable effect, we will probably start with a relatively high field amplitude. Once this benchmark is established we will systematically decrease the amplitude of the field to establish detection limits. Since it is possible to vary the size of the vesicles, we anticipate that it will be possible to directly test theories for the effects of signal averaging and signal pooling on the "noise" governed detection limits. |
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| Recent Publications |
R.D. Astumian and M. Bier. Fluctuation Driven Ratchets - Molecular Motors, Phys. Rev.
Letters 72:1766-1769 (1994).
R. Dean Astumian, Electroconformational coupling of membrane proteins, Annals. N.Y. Acad. Sci. 720: 136-141 (1994). R. Dean Astumian, J. C. Weaver, and R. K. Adair, Rectification and Stochastic Resonance for signal Averaging of Weak Electric Fields in Biological Systems, Proc. Natl. Acad. Sci. USA 92: 3740-3743 (1995). R. D. Astumian, Physical Models of Molecular Motors and Pumps, invited refereed review for Science, in press (1995) M. Bier and R. Dean Astumian, Biased Brownian Motion as the Operating Principle for Microscopic Engines, in press, Bioelectrochemistry and Bioenergetics, (1995). C.K. Bagdassarian and R. Dean Astumian, Conformational Fluctuations and Protein Function, submitted , Phys. Rev. Letters. C.K. Bagdassarian and R. Dean Astumian, Conformational Fluctuations and Protein Function: The Thermodynamics of a Brownian Motor, submitted, J. Chem. Physic. |
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