Core--Metal Ion Regulation /Transport

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The purpose of this core is to provide the instrumentation and technical support to fulfill the goals of this application. The core will provide atomic absorption analysis of metals; equipment and expertise to perform two-dimensional electrophoresis analysis of proteins, an IAsys apparatus for the analysis of protein protein interactions; a mass spectrometry facility for the analysis of proteins and peptides; and a confocal microscope facility for the immunolocalization of proteins in cells and tissues. The atomic absorption instrument will allow sensitive quantitative anlysis of copper, iron, and selenium ions necessary to study the cellular mechanisms regulating their transport and function. The two-dimensional electrophoresis equipment will be used to separate complex mixtures of cellular proteins to identify components of signal transduction pathways that regulate ion transport, and to elucidate mechanisms of metal ion toxicity. The IAsys apparatus will provide a direct way to measure the strength and specificity that ion transport proteins interact with other components in the cell that regulate their trafficking and activity. The mass spectrometer will be used to identify phosphorylation sites in ion transport proteins, identify new proteins regulating ion transport, determine how proteins, such as transferrin, interact to form protein complexes, and confirm the efficiency of incorporation of selenocysteine into proteins prepared for small angle x-ray diffraction experiments. The confocal microscope will allow direct visualization of the subcellular localization of ion transport proteins so that the mechanisms controlling their trafficking in normal and diseased tissue can be understood.