Energetic and Stoichiometric Relationships Involving Respiration, H+, and Atp

Hendler, R

Abstract

With the use of a fiber-optic probe we have been able to measure light scattering changes which reflect conformational changes as mitochondria are energized and de-energized following respiration and uncoupling. The light scattering has been quantitatively linked to internal volumes of the mitochondria using 3H20 and 14C- sucrose to measure the latter. Pure cytochrome oxidase has been incorporated into azolectin proteoliposomes. The enzyme in the vesicles displays as high respiratory control ratios as the best of mitochondrial preparations. The vesicles develop a delta phi as measured with a TPP+ electrode during respiration using cytochrome c. Corrections for TPP+ binding to the vesicle membrane can be made based on a mathematical model and direct measurements of KD and concentration of binding sites. A salicylate electrode can not be used to monitor deltapH as the vesicles interfere with the electrode. Instead, we have trapped a pH-sensitive fluorescent dye, pyranine, inside. Using our fiber optic probe and suitable interference filters for excitation and emission, we have calibrated fluorescence in terms of pH. Knowing the internal pH and monitoring the external pH with an electrode, delta pH is now followed. Computer software development has proceeded, allowing us to convert all of the incoming signals to delta phi, delta pH, and delta muH+, in real time. Work has started on the study of a group bacteriocidal peptides produced by the skin of the frog, Xenopus laevis. We have tested a group of these peptides on coupled respiration and the formation of delta phi in mitochondria, bacteria and cytochrome oxidase liposomes. The results, so far indicate that the bacteriocidal action of these agents may be exerted at the level of uncoupling of oxidative phosphorylation.