Quantitation of energy coupling between Ca2+, calmodulin, skeletal muscle myosin light chain kinase, and kinase substrates.
Interactions between Ca2+, calmodulin, rabbit skeletal muscle myosin light chain kinase, and kinase substrates were investigated by microequilibrium dialysis using 45CaCl2 and by fluorescence anisotropy using fluorescent labeled calmodulin. We have determined the free energy coupling for the interaction of Ca2+ and skeletal muscle myosin light chain kinase with calmodulin (delta GCM), and the free energy coupling for the interaction of calmodulin and enzyme substrates with myosin light chain kinase (delta GSCaM). The mean dissociation constants for Ca2+ interaction with calmodulin in the presence and absence of enzyme were 0.40 and 14 microM, respectively, yielding a minimal estimate for delta GCM of -2.11 kcal/mol of Ca2+ (-8.44 kcal/4 mol of Ca2+). Dissociation constants for the interaction of fluorescent 5-iodo-amino-ethyl-amino-naphthalene-1-sulfonic acid-labeled calmodulin and native calmodulin with skeletal muscle myosin light 100 and 15 nM, respectively. In the presence of substrates (phosphorylatable skeletal muscle myosin light chain and App(NH)p) these dissociation constants were 30 and 3 nM, respectively. The free energy coupling for binding of calmodulin and substrates to myosin light chain kinase was -0.95 kcal/mol of calmodulin. These data confirm that interaction of Ca2+ with calmodulin is highly cooperative in the presence of myosin light chain kinase with the degree of cooperativity dependent upon the concentrations of Ca2+, enzyme, and substrates.
D000818:Animals; D001921:Brain / Q000378:metabolism; D002122:Calcium Chloride / Q000378:metabolism*; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D009210:Myofibrils / Q000201:enzymology; D009219:Myosin-Light-Chain Kinase; D009282:Naphthalenesulfonates; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits
C032162:AEDANS-calmodulin; D002147:Calmodulin; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase; D002122:Calcium Chloride