The major contractile protein of muscle, myosin, has recently been shown to exist in a number of different molecular forms (isomyosins) in both the heart and skeletal muscle. As many as 15 different genes for various types of isomyosin may exist in a single organism whose expression appears to be regulated by a variety of developmental and physiological conditions. While current evidence suggests that these isomyosins differ in biochemical properties (ATPase activity) and physiological function (contraction speed), little else is known about the physiological significance of different isomyosins. This application proposes to examine 2 fundamental questions regarding myosin polymorphism. First, to better understand the relation between the structural and functional properties of myosin, the molecular substructure will be examined to determine which regions are most closely associated with a specific functional type of isomyosin, e.g, cardiac actrial and ventricular types V1 and V3, and fast and slow skeletal myosins. Secondly, factors controlling the expression of different isomyosin heavy chains will be examined in both cardiac and skeletal muscle cells maintained under a number of specific conditions in tissue culture. Analysis of myosin substructure will be conducted on purified proteins using a unique new technology combining partial proteolytic digestion """"""""peptide fingerprinting"""""""" and monoclonal antibodies as a probe for specific structural domains. This analysis, which has not previously been employed for examining the structure of isomyosins, is capable of locating and distinguishing highly conserved or divergent regions between different isomyosins. Correlating these structural domains with the physical properties of different isomyosins may provide evidence for the functional role of different regions of the myosin molecule. Secondly, by using monoclonal antibodies specific for different isomyosin types in immunofluorescence and in newly developed procedures for analysis of specific protein synthesis and turnover in culture, both the qualititative and quantitative characteristics of isomyosin expression in tissue culture will be evaluated. The results of these studies should provide new insight into the structural and functional relationships between different members of the isomyosin family, and thereby increase our understanding of the physiological adaptation provided by multiple isomyosins in the heart and skeletal muscle.
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