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The long range goals of the proposed research are: to determine what role translational regulation play in the control of cellular proliferation, differentiation and aging; and to determine how heavy metal ions, through their effects on protein synthesis, may adversely affect these processes. The initial objective is to elucidate the mechanism(s) by which heavy metal ions (i.e. Pb++ and Cd++) inhibit protein synthesis in eukaryotic cells, utilizing the rabbit reticulocyte lysate as a model system. The principal hypothesis to be tested is that heavy metals inhibit protein synthesis by affecting the redox potential of a cell which brings about the activation of an eIF-2 alpha kinase through a process involving thiol/disulfide exchange. The specific aims include: characterizing the effects of heavy metal ions on protein synthesis in the reticulocyte lysate; determining the effects of heavy metal ions on the redox state of the reticulocyte lysate and its relationship to the activation of an eIF-2 alpha kinase activity; determining the functional effects of eIF-2 alpha phosphorylation on protein synthesis in the reticulocyte lysate; and studying he effects of heavy metal ions on the growth and differentiation of neuroblastoma cells in culture, in relation to their effects on protein synthesis and eIF-2 alpha phosphorylation. This research is relevant not only for understanding the regulation of protein synthesis generally in eukaryotic cells, but also in determining the mechanism by which environmental exposure to heavy metals can bring about damage to various organ systems and cause developmental abnormalities. The methodology involve in situ and in vitro studies of the components involved in the initiation of protein synthesis and its regulation, and the major enzymes responsible for maintaining the redox state of the lysate (glucose 6-phosphate dehydrogenase, glutathione reductase and thioredoxin/thioredoxin reductase). The effect of heavy metal ions on: protein synthesis ((14C)Leu incorporation); eIF-2 alpha phosphorylation ((32P) phosphoprotein profiles by SDS/PAGE); reversing factor activity (catalysis of guanine nucleotide (eIF- 2.(3H)GDP) exchange); the composition of initiation complexes (analysis of polyribosome profiles using radiolabeled components of initiation and immunoblotting techniques); the GSH/GSSG and NADPH/NADP ratio (enzymatic cycling assays); and the sulfhydryl status of the heme-regulated eIF-2 alpha kinase (covalent modification), will be determined. Results will be related to effects which heavy metal ions have on protein synthesis protein phosphorylation and redox potential of neuroblastoma cells.
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