Association of calmodulin and smooth muscle myosin light chain kinase: application of a label selection technique with trace acetylated calmodulin.
A method is described for rapidly surveying the effects of modifying individual amino acid residues of a protein on its ability to interact specifically with another macromolecule. The procedure has been used to examine the individual roles of the seven lysyl residues of calmodulin in its ability to bind to smooth muscle myosin light chain kinase; previous studies by Jackson et al. (J. Biol. Chem. 261:1226-12232, 1986) have suggested that certain lysines may be located close to the interaction site. Trace [3H]-acetylated calmodulin, consisting predominantly of molecules acetylated at single sites together with unmodified protein, was incubated in excess (five- to 20-fold) with smooth muscle MLC kinase to allow the modified and unmodified molecules to compete for binding to the enzyme. Subsequently, the calmodulin-enzyme complex was separated from unbound calmodulin, and the level of acetylation of each of the seven lysines of the bound fraction of calmodulin was determined and compared to that of each corresponding group of the starting preparation. Significant changes were found at only two of the lysines, 21 and 75, where the extent of acetylation in the bound fraction was three- and fivefold lower, respectively, than that in the original preparation. These results were reproducible in three separate selection experiments employing both chicken and turkey gizzard MLC kinase. It is concluded that acetylation of calmodulin at either lysine 21 or 75 markedly reduces its affinity for MLC kinase, but acetylation at any of the other lysines (13, 30, 77, 94, or 148) has only minor effects.(ABSTRACT TRUNCATED AT 250 WORDS)
D000107:Acetylation; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002147:Calmodulin / Q000378:metabolism*; D066298:In Vitro Techniques; D008239:Lysine; D009130:Muscle, Smooth / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*
D002147:Calmodulin; D009219:Myosin-Light-Chain Kinase; D008239:Lysine