Identification of Ca2+-binding subunit of myosin light chain kinase from skeletal muscle with modulator protein.

The protein present in various vertebrate tissues as smooth muscle, adrenal medulla, brain and platelets, previously identified with troponin C, has been found [ 1,2] to be identical with modulator protein of cyclic nucleotide phosphodiesterase. This protein was also found in skeletal and cardiac muscles, i.e., in the tissues which contain troponin C. Most of modulator protein was present in the cytosol of all tissues studied but part of it was bound to the structural insoluble proteins [l] . The 18 000 dalton skeletal myosin light chain (P-light chain) is phosphorylated by a specific Ca*dependent kinase [3 f . When this work was in progress, this kinase was reported [5] to consist of two subunits, one of which is a 20 000 dalton Ca”binding protein. We present here evidence that this Cap-binding protein is identical with the modulator protein of cyclic nucleotide phosphodiesterase and that this protein is necessary for Cap-dependent skeletal myosin light chain kinase activity. tion and dissolved in 0.5 M KCl. KCl concentration was lowered to 0.3 M and the solution was ultracentrifuged at 75 000 X g for 1 h. Supernatant was used for further studies. The activity of myosin light chain kinase was determined by the appearance of the phosphorylated form of myosin P-light chain detected by means of ureapolyacrylamide gel electrophoresis [3] . Myosin was incubated in 50 mM Tris-HCl (pH 7.5), 0.12 M KCl, 10 mM MgCl,, 2 mM ATP and either 0.1 mM CaC12 or 1 mM EGTA at 37°C. After 15 min the reaction was stopped by addition of urea to 6 M and the samples analysed by polyacrylamide gel electrophoresis on 8.0% gel, essentially according to [7] , with Tris-glycine buffer, pH 8.6, in the presence of 5 M urea. Polyacrylamide gel electrophoresis with SDS was performed according to [8]. Modulator protein and cyclic nucleotide phosphodiesterase were isolated from brain as in [9] , and phosphodiesterase activity was measured as in [lo] . Troponin C was prepared asin [ll].