The development of a new computer-based system for obtaining realtime measurements of delta psi, and delta pH, delta mu H and H+-fluxes has been completed. This system monitors [TPP+], makes corrections for TPP+-binding, computes delta psi, measures fluorescence of internally trapped pyranine and converts this to internal pH, measures external pH, reports delta pH, measures [02), and computes proton fluxes. This system was used to compare the energy transducing abilities of 13-subunit mammalian cytochrome aa3 with that of the 2-subunit enzyme from Paracoccus denitrificans, using proteoliposomes. The bacterial enzyme was able to form a delta psi of 217 mV compared to 184 mV for the mammalian enzyme. Both systems form a delta pH of about 0.15 units, or 9 mV. The small delta pH has a greater role, however, in respiratory control than the delta psi. The H+/O pumping stoichiometry is quite comparable for the 2 systems. Therefore, only 2 peptide subunits are necessary for energy transduction by cytochrome aa3. It was determined that because of the very low internal volume of cholate-dialysis-prepared liposomes and the high buffer capacity of the liposomal membrane, this system cannot be used for proton flux measurements between the outer and inner aqueous compartments. Preliminary experiments to measure ATP synthesis using liposomes also uncovered insurmountable problems that prevent the use of ATPase liposomes to study delta pH and delta psi-driven ATP synthesis. For these reasons, attention is being shifted from liposomes to submitochondrial particles for a continuation of these studies.
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