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Myosin II is the main molecular motor in the actomyosin-dependent motility in cells. Phosphorylation of the myosin regulatory light chain (RLC) at Ser19 is a prerequisite for smooth muscle/non-muscle myosin II activation and serves as a biochemical equivalent of myosin II activity. Simultaneous phosphorylation at Thr18 further promotes the myosin II ATPase activity. A number of methods have been developed to measure myosin RLC phosphorylation at Ser19 or di-phosphorylation at Thr18/Ser19. While these methods are straightforward and robust in myosin-rich muscle tissues, they demonstrate limited applicability in non-muscle cells that have low myosin II content and are usually available in lesser amounts than muscle tissue. Because of this, dynamic analysis of RLC phosphorylation in multiple samples of non-muscle cells is difficult and requires large number of cells. The use of phospho-specific antibodies increases detection sensitivity but allows estimation of only relative levels of RLC phosphorylation at specific residues, which makes it difficult to estimate the physiologic relevancy of the observed changes in RLC phosphorylation. To measure RLC phosphorylation in small amounts of non-muscle cells, we used external calibration standards of non-phosphorylated and in vitro phosphorylated RLC in standard SDS-PAGE and Western blot procedures with phospho-specific RLC antibodies. Here, we describe the method in detail and demonstrate its application for quantitative measurement of myosin RLC phosphorylation in endothelial cells in response to natural agonists (thrombin or insulin) and intact human platelets. We discuss the advantages and limitations of the proposed method vs other approaches for measuring myosin RLC phosphorylation in non-muscle cells.
This article was published in the following journal.
Name: Biochemistry. Biokhimiia
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A method that is used to detect DNA-protein interactions. Proteins are separated by electrophoresis and blotted onto a nitrocellulose membrane similar to Western blotting (BLOTTING, WESTERN) but the proteins are identified when they bind labeled DNA PROBES (as with Southern blotting (BLOTTING, SOUTHERN)) instead of antibodies.
A method that is derived from western blotting (BLOTTING, WESTERN) and is used to detect protein-protein interactions. The blotted proteins are probed with a non-antibody protein which can then be tagged with a labeled antibody.
The smaller subunits of MYOSINS that bind near the head groups of MYOSIN HEAVY CHAINS. The myosin light chains have a molecular weight of about 20 KDa and there are usually one essential and one regulatory pair of light chains associated with each heavy chain. Many myosin light chains that bind calcium are considered "calmodulin-like" proteins.
An enzyme that phosphorylates myosin light chains in the presence of ATP to yield myosin-light chain phosphate and ADP, and requires calcium and CALMODULIN. The 20-kDa light chain is phosphorylated more rapidly than any other acceptor, but light chains from other myosins and myosin itself can act as acceptors. The enzyme plays a central role in the regulation of smooth muscle contraction.
A phosphoprotein phosphatase that is specific for MYOSIN LIGHT CHAINS. It is composed of three subunits, which include a catalytic subunit, a myosin binding subunit, and a third subunit of unknown function.
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