Calmodulin-dependent myosin light chain kinases from cardiac and smooth muscle: a comparative study

Authors:
  • MWMichael P. Walsh
    Michael P. Walsh
  • JCJean-Claude Cavadore
    Jean-Claude Cavadore
  • BVBernard Vallet
    Bernard Vallet
  • JDJacques G. Demaille
    Jacques G. Demaille
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Abstract
Various properties of cardiac and smooth muscle calmodulin-dependent myosin light chain kinases (MLCKs) have been compared. The enzymes exhibit the same isoelectric point (6.5) but differ markedly in molecular weight (Mr = 72 000 for both canine and bovine cardiac MLCK, and Mr = 130 000 for smooth muscle MLCK). Comparison of the tryptic peptide maps of bovine cardiac and turkey gizzard MLCKs indicates that the cardiac enzyme is a fragment of a protein homologous to the smooth muscle kinase. While the smooth muscle kinase can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, the same is not true for either bovine or canine cardiac MLCK. Controlled tryptic hydrolysis of phosphorylated smooth muscle MLCK, followed by affinity chromatography on a column of calmodulin–Sepharose, enables separation of a phosphopeptide (Mr = 22 000) from a mixture of peptides of Mr = 50 000 and 24 000 which are bound to the column in the presence of Ca2+ and eluted with ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid. The phosphorylation site, therefore, is distinct from the calmodulin-binding site. It appears that cardiac MLCK is proteolyzed during the isolation procedure. The purified cardiac enzyme represents a proteolytic fragment which retains Ca2+ and calmodulin dependence but only a fraction of the specific activity of the native enzyme, and has lost the site of phosphorylation by cAMP-dependent protein kinase. A protease is shown to exist in myocardium which is capable of digesting smooth muscle MLCK rapidly at low temperature, and which is resistant to classical antiproteases.

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