The purpose of these studies is to establish a better understanding of the energy metabolism of biological tissues. Towards this goal, the laboratory concentrates on the use of screening approaches in proteomics and post-translational modifications. The following major findings were made over the last year: 1) We have continued to develop a non-destructive optical spectroscopy method using a center mounted integrating sphere and a rapid scanning spectroscopy system to monitor the redox sensitive chromophores of mitochondrial oxidative phosphorylation minimizing light scattering effects. Using this approach we have established characterized all of the redox chromophores in the mitochondria and begun to establish the regulation of reducing equivalent distribution within the network. We have described that both the activation, with calcium, and deactivation, ischemia reperfusion in intact heart, that all of the Complexes of oxidative phosphorylation are modified in concert. These data imply that the activity of these Complexes is orchestrated together in the intact mitochondria. The mechanisms responsible for the coordination of the Complex activities are still under investigation. Our working hypothesis is based on the fact that the mitochondria evolved from early symbiotic bacteria retaining many of bacteria protein synthesis processes and even DNA. We speculate that the signaling mechanisms within the mitochondria may also be closer to bacterial signaling systems than the more familiar eukaryotic systems. 2) To test the bacterial signaling hypothesis stated above, we have initiated studies on isolated bacteria believed to be closest to the mitochondrial origins, paracoccus denitrificans. In these studies we have established that paracoccus oxidative phosphorylation system has very similar optical properties as the mammalian mitochondria and alterations in ATP production can be induced by simply modifying the bathing potassium concentration. Proteomic screening tools, established in mitochondria, have also been successfully modified to evaluate the protein programming of these bacteria. It is hoped that this simplified system will provide insights into the regulation of mitochondrial oxidative phosphorylation, in the coming year . 3) Our previous work on the regulation of oxidative phosphorylation has concentrated on isolated mitochondria that we have extrapolated to in vivo conditions. We are now moving our non-invasive optical studies of the chromophores of oxidative phosphorylation into the study of the isolated perfused heart. Our initial studies revealed that we could quantitate all of the redox sensitive chromophores of the perfused heart by simply adding myoglobin to our fitting array. However, applying this technology to the isolated perfused rabbit heart revealed that myoglobin oxygenation was highly labile in the saline perfused heart unlike the blood perfused heart in vivo. These data suggested that the heart was partially hypoxic under these conditions and may not reflect in vivo dynamics. We could not use a blood perfused system since the optical interference from hemoglobin dominated the optical properties. Thus, we devised methods to construct a colorless artificial blood substitute using lipid vesicles containing perfluorocarbon oxygen carrying polymers to improve oxygen delivery to the perfused heart. We have demonstrated that this artificial oxygen delivery system works in the perfused heart increasing oxygen delivery by more than 5 fold. It is hoped that that this system will permit us to extrapolate our isolated mitochondria observations to a working intact tissue. 4) One of the proposed mechanisms of signaling oxidative phosphorylation is via cell signaling through the protein kinase A system modulated by cAMP. We have evaluated this process in porcine heart isolated mitochondria and crude cytosol homogenates using lipid permeable cAMP analogs and numerous inhibitors of this signaling network. No significant effects of the permeable cAMP analogs or network inhibitors could be found. This data suggest that the cAMP regulatory network is not a significant acute modulator of cardiac oxidative phosphorylation. 5) Protein phosphorylation at conventional and non-conventional sites has been proposed as an important modulator of oxidative phosphorylation. One of the broad inhibitors of protein phosphorylation is fluoride. We have found that potassium fluoride (KF) is an effective inhibitor of oxidative phosphorylation. We have shown that KF inhibits several of the oxidative phosphorylation Complexes in intact mitochondria consistent with the phosphorylation hypothesis. In addition, we have shown that with regards to Complex V, the effect is persistent through the isolation of the purified Complex V for in depth proteomic analysis. We are current now attempting to characterize the post-translational modifications responsible for the persistent KF effect on Complex V. We are not assuming the KF effect is due to protein phosphorylation as we are screening for all the post-translational modification, including protein-protein interactions, we can detect using mass spectroscopy techniques. The reversible effects of KF together with the ability of these effects to persist through a careful isolation of Complex V suggest that these studies might reveal a post-translational network modifying the function of this critical Complex of oxidative phosphorylation.
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