Subcellular localization of myosin light chain kinase in skeletal, cardiac, and smooth muscles.

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Abstract
Antibodies were elicited against turkey gizzard myosin L chain kinase (MLCK), purified by affinity chromatography on the enzyme bound to Sepharose, and used to localize myosin kinase, in rabbit fast skeletal, slow skeletal, cardiac and smooth muscles, by indirect immunofluorescence. When studied on nitrocellulose replicas of sodium dodecyl sulfate/polyacrylamide gel electrophoretograms, antibodies were specific for the MW 140,000 MLCK of gizzard smooth muscle. By using the same technique, they recognized the MW 140,000 MLCK and a MW 75,000 polypeptide, presumably derived from the former by proteolysis, in rat arterial and stomach smooth muscle as well as in rat thyroid cells. The same antibodies reacted only with a MW .apprxeq. 75,000 protein from rat cardiac and skeletal muscle. Antibodies inhibited the activity of smooth and skeletal myosin kinases in an in vitro assay with .apprxeq. 11 mol of antibody needed for 50% inhibition of 1 mol of gizzard enzyme. The antibodies stain vascular and gizzard smooth muscle cells with no apparent segregation of the enzyme in a specific part of the cell. Sarcomeric muscles exhibit a striated staining pattern, superimposable to the staining by antiactin antibodies. Antibodies are not species- or tissue-specific; they recognize kinases that differ in their MW and ability to be phosphorylated, probably at the level of their common catalytic and calmodulin-binding domains. Sarcomeric muscle kinases are at least in part bound to the contractile apparatus and their distribution is restricted to a specific part of the sarcomere. Myosin phosphorylation may possibly be controlled not only by the Ca2+ concentration but also by actin-myosin interaction.