In the course of determining the primary structure of rabbit skeletal muscle myosin light chain kinase (MLCK; ATP:protein phosphotransferase, EC 2.7.1.37) a peptide fragment was obtained that appears to represent the calmodulin-binding domain of this enzyme. Low concentrations of the peptide inhibited calmodulin activation of MLCK (Ki 1 nM). The peptide was not associated with a catalytically active, calmodulin-independent form of MLCK that was obtained by limited proteolysis. The peptide is 27 residues in length and represents the carboxyl terminus of MLCK. The sequence of the peptide shows no significant homology with any known protein sequence. The peptide contains one tryptophanyl residue and a high percentage of basic and hydrophobic residues, but no acidic or prolyl residues. Much of the sequence has a high probability of forming a helix. A chemically synthesized peptide has been prepared to study the interactions of the peptide and calmodulin in more detail. The intrinsic tryptophan fluorescence of the synthetic peptide shows a significant enhancement (w45%) in the presence of Ca2' and calmodulin; fluorescence enhancement is maximal at a peptide:calmodulin stoichiometry of 1:1. Calmodulin-Sepharose affinity chromatography in the presence of 2 M urea indicates that the interaction of peptide and calmodulin is Ca2+-dependent. The results of these studies indicate that the catalytic and calmodulin-binding domains of MLCK represent distinct and separable regions of the protein. In addition, the results provide a basis for future studies of the molecular and evolutionary details of calmodulin-dependent enzyme regulation.Calmodulin is a Ca2+-binding protein that is ubiquitously distributed and highly conserved throughout eukaryotic evolution. Although it is known to regulate many Ca2+-dependent enzymes (recently reviewed in ref. 1), little is known about the interactions of calmodulin with these proteins at the molecular level. Myosin light chain kinase (MLCK; ATP:protein phosphotransferase, EC 2.7.1.37) is one of the better characterized calmodulin-regulated enzymes (reviewed in refs. 2 and 3) and is found in both muscle and nonmuscle tissues. The enzyme occurs in tissue-specific forms, which vary widely in molecular weight, antigenic determinants, and enzymatic properties (3, 4). It catalyzes the phosphorylation of a specific class of myosin light chain subunit, termed the P-light chain (5). In smooth muscle this phosphorylation is required for initiation of contraction (reviewed in refs. 2 and 6), whereas in skeletal muscle the phosphorylation appears to modulate the degree of tension produced during isometric contraction (3, 7).A determination of the amino acid sequence of rabbit skeletal muscle MLCK was undertaken by these several laboratories as part of a long-range study of protein kinase and cal- Ca2' and 5 gM calmodulin. This calmodulin-independent form of MLCK was denoted C35. Details of the preparation and properties of these proteolyzed forms of MLCK will be described elsewhere. CNBr digesti...

These results are consistent with the hypothesis that adventitial fibroblasts are transformed into myofibroblasts and begin to proliferate within hours after graft placement. Migration of these cells towards the vessel lumen with subsequent proliferation appears to be a major contributor to NH formation. The pivotal role of the adventitial fibroblasts in the pathogenesis of NH provides a compelling rationale for therapies that target the transformation, proliferation and migration of these cells to prevent arteriovenous graft stenosis.

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