The proposed project deals with two major problems related to the structure and function of sarcoplasmic reticulum in skeletal muscle. 1. The interaction of Ca2+ transport ATPase molecules in the membrane: The structural and functional implications of the interactions between Ca2+ ATPase molecules will be analyzed by fluorescence energey transfer, by the excimer fluorescence of pyrenemaleimide covalently bound to the Ca2+ ATPase, and by reconstruction of the structure of Ca2+ ATPase molecules from 2D and 3D crystals. Information obtained by these techniques will be correlated with steady state and rapid kinetic data on the mechanism of ATP hydrolysis and Ca transport. The ultimate aim of the project is to define the functional role of Ca2+ ATPase oligomers and to provide a 3-dimensional structure of the Ca ATPase polypeptide in the membrane at the beginning and at the end of the Ca transport cycle; this may allow conclusions about the physical basis of Ca2+ translocation. 2. The regulation of the biosynthesis and degradation of sarcoplasmic reticulum. The feedback regulation by Ca2+ of the synthesis of Ca2+ ATPase and other muscle proteins will be studied by correlating changes in cytoplasmic Ca2+ with the cellular concentration of mRNA for a 100 KD and an 80 KD proteinthat are induced in cultured muscle cells by Ca ionophores. Pure mRNA will be isolated fror the Ca-ATPase and for the 80 KD protein and used for the synthesis of cDNA probes. The concentration of the relevant mRNAs during development and in response to changes in cytoplasmic [Ca2+], induced by drugs, muscle exercise or muscle diseases, will be measured by cDNA and cloned DNA probes. The possible role of Ca2+ binding chromosomal proteins in the Ca2+ regulation of gene expression will be explored. Studies will be initiated on the mechanism of intracellular degradation of Ca2+ ATPase that appears to be an important element in the control of Ca2+ ATPase concentration in muscle cells. Although the proposed studies are aimed at the solution of basic biological problems related to sarcoplasmic reticulum, they have general implications to ion transport mechanisms, the structure of membrane proteins and to the mechanisms, of adaptation of sarcoplasmic reticulum content and composition to physiological and pathological requirements (hypertrophy, muscle diseases).
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