Our objective is to elucidate the molecular basis for structural breakdown in energy deprived cells. Our previous research showed that cell associated glycine is a major factor that maintains cell integrity during depletion of adenosine triphosphate (ATP). ATP depleted cells lose glycine because of compromised transport function. Under these conditions, provision of glycine protects cells from lethal injury. Moreover, glycine, by preserving structure, promotes cell survival. We showed that ATP depleted cells develop porous defects in plasma membranes accompanied by marked aggregation of intramembranous (protein) particles and other structural lesions similar to those induced by complement. These findings suggested that membrane proteins were being redistributed. Plasma membrane lesions were accompanied by specific patterns of cytoplasmic protein aggregation. Glycine prevented not only the functional defects and structural lesions in plasma membranes, but also the cytoplasmic protein alterations. When proteins on the extrinsic surfaces of plasma membranes were selectively cross-linked, the full cytoprotective spectrum of effects mediated by glycine was reproduced, including the promotion of survival. Based on these findings, we postulate that in ATP depleted cells, a single glycine preventable event is initiated at the plasma membrane, triggering not only the formation of porous lesions in membranes, but also a degenerative biochemical cascade that aggregates specific, important proteins and disrupts the cytoplasm. Because equilibrium between phosphorylation and dephosphorylation shifts during ATP depletion, phosphatase activity is likely to be involved in the pathogenesis of these alterations. In view of our findings, we propose the following studies to test our hypotheses regarding the pathogenesis of membrane and cytoplasmic structural damage that is specifically preventable by glycine. 1. Characterization of the molecular basis for cytoplasmic protein aggregation with emphasis on protein dephosphorylation. 2. Definition of the structural basis for increased plasma membrane permeability using freeze-fracture and freeze-etch methods. 3. Comparison of the biochemical and structural bases for cytoprotection conferred by glycine and membrane impermeant cross- linkers. 4. Investigation of membrane protein phosphorylation during ATP depletion. In all these studies, we will focus on aspects of structural and biochemical damage that critically determine loss of viability. Glycine is the most effective agent yet discovered that confers hypoxia tolerance to mammalian cells. Because its actions on cell damage at multiple levels of cytoplasmic organization are so powerful, and since it suppresses biochemical events associated with ATP depletion that are relevant to injury, we are confident that our studies will yield significant new information on hypoxic cell death and its prevention.
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