Vectorial phosphorylation of filamentous smooth muscle myosin by calmodulin and myosin light chain kinase complex.
Vertebrate smooth muscle myosin extracted from myofibrils and isolated via filament assembly was co-purified with calmodulin (CaM) and myosin light chain kinase (MLCK) which are tightly associated with the filament architecture and, therefore, it may be considered as a native-like preparation. These endogenous contaminates also co-precipitated with a native-like actomyosin, for both cases, at levels sufficient to fully phosphorylate myosin within 10-20 s after addition of ATP and calcium, although their molar ratio to myosin was only about 1 to 100. Phosphorylation progress curves obtained from mixtures of the native-like, and CaM- and MLCK-free filaments indicated that the CaM/MLCK complex preferentially phosphorylated its parent filaments and, as result, the whole myosin present was not maximally phosphorylated. Solubilization of the filaments' mixtures at high ionic strength resulted in slower phosphorylation rates but with maximal phosphorylation levels being attainable. Similar observations were made on the filamentous myosin system reconstituted from the kinase- and CaM-free myosin with added purified MLCK and CaM as well as on the native-like myosin from which only one of these endogenous contaminates was removed by affinity chromatography. These data indicated that not only the MLCK but also CaM was necessary for the observed preferential phosphorylation kinetics. Thus, the native-like filamentous myosin appeared to be phosphorylated by some kind of vectorial mechanism. Similar experiments were carried out on the native-like actomyosin where these vectorial effects were even more pronounced.
Sobieszek
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Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg. asobieszek@imb.oeaw.ac.at
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D010766:Phosphorylation; D014422:Turkeys
D002147:Calmodulin; D009219:Myosin-Light-Chain Kinase; D009218:Myosins