The sarcoplasmic reticulum ATPase concentrates cytosolic Ca2+ into intracellular compartments, thereby permitting relaxation and subsequent tension development of cardiac and skeletal muscle. We will use ATPase proteins and cDNA clones to study catalytic, transport and inhibitory mechanisms, as well as ATPase expression following gene transfer, as follows: 1-Characterization of the mechanism of Ca2+ binding, ATP utilization, intermediate enzyme phosphorylation and Ca2+ transport, as they relate to ATPase topology and structure. Following previous information obtained by indirect measurement, we will extend our mutational analysis by expressing mutated ATPase in large quantities for direct measurements of stoichiometry, equilibrium and kinetic constants, and the effects of specific amino acid mutations of the partial reactions of the catalytic and transport cycle. ATPase mutants will be also used for spectroscopic studies to relate functional steps to structural features, an explain intermolecular linkages of separate function domains. 2-Topology of the specific ATPase inhibition by thapsigargin. We will synthesize high affinity thapsigargin analogs, including a radioactive azido derivative for (covalent) photo labeling of the native ATPase, proteolytic digestion, proteolytic digestion, proteolytic digestion, sequencing and determination of labeled amino acid(s) as the specific binding site. Furthermore guided by previous findings on the effects of large chimerical exchanges, we will test the effects of specific amino acid mutations on the sensitivity of the Ca2+ ATPase to thapsigargin.
The specific aim i s to define the topology of the thapsigargin inhibitory site and characterize the inhibitory mechanism. 3-Gene transfer and ATPase expression in foreign cells and cardiac muscle. A variety of cultured cells including cardiac myocytes will be transfected with SERCA1 or SERCA2A cDNA using various methods to obtain maximal efficiency and expression of Ca2+ ATPase. A variety of DNA constructs will be used, including dual gene constructs for expression under selective pressure, high yield promoters for overexpression, and tissue specific promoters for cardiac cells. Expression and targeting will be monitored by immunofluorescence in permeabilized cells and in isolated subcellular fractions.
The specific aims are to: (a) recover large amounts of expressed ATPase for mutational studies of transport and catalytic function by the isolated enzyme, and (b) used gene transfer to established functional intracellular organelles and to influence cytosolic Ca2+ homeostasis.
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