Although hyperthermia is a promising modality in cancer therapy, the underlying molecular mechanisms of heat-induced cell killing and thermotolerance are still speculative. This proposal aims at studying the molecular mechanisms of thermal-inactivation of Ca-transport functions of intracellular Ca-pumping ATPase and ligand-regulated Ca-channel proteins. The intracellular Ca- pumping ATPase and Ca-channel proteins will be isolated from the fractionated sarcoplasmic reticulum membranes of rabbit muscles. Using the membrane protein reconstitution and the lipid-exchange protein-mediated lipid modification techniques, the above membrane Ca-transport proteins in native, lipid- modified, and reconstituted membranes will be studied. The thermal-inactivation kinetics of the Ca-transport functions for the above membranes at different lipid compositions will be measured in the presence or absence of different Ca-regulating factors (Ca2+, pH, calmodulin, glutathione, and IP3) as well as exogenous hyperthermic sensitizing and protective chemicals (sugars, polyols, local anesthetics, alcohols, and antibiotics). The thermal-induced structural alteration of the specific functional site and the overall conformational change of the protein will be determined by studying the fluorescence intensity of the covalently attached fluorescent probes and the native protein fluorescence, respectively. These structural alterations will then be related to the corresponding functional-inactivation of the protein. The physical state of lipids in modulating the structural and functional impairment of the Ca-transport proteins will also be examined using lipophilic probe molecules. This study, which employs a well-defined model system, will advance our present knowledge of the heat-induced inactivation of cell membrane Ca- transport proteins, and provide insight toward understanding the mechanisms of the alteration of Ca-homeostasis within the cell which will lead to cell death or thermotolerance.
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