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
Source: PubMed
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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.
    Analytical Biochemistry 11/1997; 252(1):96-105. DOI:10.1006/abio.1997.2319 · 2.22 Impact Factor