Regulation of Smooth Muscle Actomyosin Function
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106.Advances in Experimental Medicine and Biology (Impact Factor: 1.96). 02/1991; 304:25-36. DOI: 10.1007/978-1-4684-6003-2_4
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ABSTRACT: The expression of fast and slow myosin isoforms in individual cells is associated with differences in shortening velocities and power output in fully differentiated vertebrate striated muscle. This paradigm in which shortening velocity is determined by the myosin isoform (and load) is inappropriate for smooth muscle. Smooth muscle tissues express multiple myosin heavy and light chain isoforms, and it is not currently possible to separate and identify chemically distinct native myosin hexamers (i.e., isoforms). It is not known if different isoforms are localized in subpopulations of cells or in specific cellular domains nor whether they combine preferentially to form a small number of native myosin hexamer isoforms. Potentially, thick filaments are aggregates of many different combinations of heavy and light chain isoforms that may or may not exhibit different kinetics. Shortening velocities in smooth muscle are regulated by Ca(2+)-dependent crossbridge phosphorylation of the myosin regulatory light chains. Much of the observed diversity in power output in smooth muscle may be attributed to regulatory mechanisms modulating crossbridge cycling rates rather than contractile protein isoform expression.
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ABSTRACT: An epitope-tagged calmodulin (CaM), capable of interacting with CaM-binding proteins in cellular extracts, would be a valuable tool for identifying proteins in signal transduction pathways involving calcium. A bacterial overexpression vector for epitope-tagged CaM has been constructed by inserting the coding sequence for a nine amino acid portion of the influenza virus hemagglutinin (HA) protein into the initiation site of an overexpression vector for chicken CaM. The HA-CaM fusion produced in bacteria was compared to native CaM for its ability to activate smooth muscle myosin light chain kinase (MLCK), one of the best understood CaM-dependent enzymes. MLCK activity was tested in both a purified system and a CaM-depleted "native actomyosin" preparation maintaining many of the regulatory properties of the intact smooth muscle. HA-CaM behaves identically to unmodified CaM in both systems, indicating that the HA epitope does not adversely affect CaM function. The recombinant HA-CaM was used to sensitively detect CaM interactions with smooth muscle proteins in a modified gel overlay assay, using a monoclonal antibody against the HA epitope as the secondary reagent. Enzymatically active complexes of HA-CaM and MLCK could be immunoprecipitated from actomyosin preparations using the same monoclonal antibody and protein G-Sepharose beads.
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