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) Protein phosphorylation in the mitochondrial matrix is beginning to be appreciated as a wide spread mechanism of regulating mitochondrial energy metabolism. A 32P screen of the mitochondrial matrix phospho-protein turnover has been expanded to evaluate the role of different experimental conditions on the rate of 32P incorporation into the matrix proteins. Using this approach we have found that independent of the enzyme concentrations, the rate of matrix protein phosphorylation is much different in different tissue types. These results imply that protein phosphorylation may be regulating the """"""""tuning"""""""" of mitochondrial function in different tissues along with the types of protein deposited. 2) Using minimal detergent gels, so called Native gels, it was shown that we could maintain kinase activity in isolated protein complexes from oxidative phosphorylation, in vitro. These studies demonstrate that the kinase activities are associated within these protein complexes and provide a novel, in vitro, tool for isolating and studying mitochondrial matrix kinases. 3) Using the same gel system, the activity of Complex 1(initial oxidation of NADH) and Complex 5 (actual formation of ATP) have been directly within the gel. These assay systems revealed that the binding of NADH in complex 1 uniquely enhances NADH fluorescence and likely dominates the NADH fluorescent signal in most differentiated cells. The Complex 5 assay has been used to confirm the permissive action of calcium in the formation of ATP potentially a major site of ATP synthetic regulation. Using both of these assays we have also demonstrated rather striking differences in the activity of these two complexes on a per molecular basis. Thus, despite essentially identical protein sequences, the activity of each of these complexes is likely tuned to a tissues function by the aforementioned post-translational modifications and not protein content alone. 4) A hypothesis has been developed that tissues with large swings in metabolic activity (i.e. heart and muscle) hold mitochondrial electron transport activity in reserve via post translational modifications while more constant activity tissues (i.e. liver) have most of their enzymatic activity active and available. The active tissues inhibit electron transport activity during rest to prevent the formation of reactive oxygen species under these low flux conditions. 5) Succinyl-Coa Synthetase (SCS) has been identified as a significant phosphate binding protein in the matrix. The association of phosphate with SCS may represent a marker of matrix energization. Function significance of the binding is still under investigation.
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