The long term goals of the proposed research are to gain a better understanding of the role of transverse tubules in the process of excitation/contraction coupling in muscle and to understand how that process is regulated by pharmacologically important and clinically significant ions and drugs. The transverse tubules play a pivotal role in excitation/contraction coupling and abnormalities of the transverse tubules may be important in muscular dystrophy. These membranes have very interesting pharmacological and biochemical characteristics, including a preponderance of Ca2+ antagonist drug binding sites, a high level of cholesterol, and a highly active, unique Mg2+-ATPase which may play a key role in excitation/contraction coupling.
The specific aims are to identify, purify, characterize in terms of possible physiological function, and affinity label the Mg2+-ATPase. Polyclonal antibodies will be raised to the subunits of the Mg2+ -Atpase. N-terminal sequencing of the purified subunits as well as enzymatic and chemical fragmentation followed by purified of peptides for sequencing will be performed. From the protein sequence obtained, oligonucleotide probes will be made to allow the identification of the cDNA coding for the Mg2+ -ATPase and the entire amino acid sequence deduced from the cDNA sequence. A membrane spanning model will be proposed based on hydropathy plots and labeling of sites on the cytoplasmic face of the protein. This ATPase will be compared structurally and functionally to other ATPases. The Mg2+ -ATPase may be a member of a previously unknown class of ATPases, and structural comparisons will be invaluable in the determination of essential and therefore conserved, domains. The techniques utilized in this work are centrifugation for native gel electrophoresis and sucrose density centrifugation for isolation of the enzyme, trypsin and cyanogen bromide fragmentation, reversed phase HPLC, and amino acid analysis and gas phase sequencing for the determination of the amino terminal sequence of the protein and generated peptide fragments. Techniques of cDNA cloning and sequencing will be used to determine the entire sequence of the gene coding for the Mg2+ -ATPase. This research will supply important information about a transverse tubule component involved in excitation/contraction coupling which will lead to a better understanding of this physiologically crucial process.
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