Calmodulin activates bovine-cardiac myosin light-chain kinase by increasing the affinity for myosin light-chain 2

Abstract

The basic mechanism by which calmodulin activates bovine-cardiac muscle myosin light-chain kinase was investigated using highly purified preparations of mixed bovine-cardiac myosin light chains or isolated myosin light chain 2. The apparent contamination of these substrate proteins by calmodulin, as detected by activation of calmodulin-sensitive phosphodiesterase, was < 4 PPM and was undetectable by antibodies against calmodulin. The apparent KA for calmodulin was 2 nM and 20 nM in the presence of isolated myosin light-chain 2 and mixed myosin light chains, respectively. Purified bovine cardiac troponin C activated myosin light-chain kinase by about 10% at a concentration of 2 .mu.M. Mixed myosin light chains were phosphorylated in the absence and presence of calmodulin and the presence of Ca with a V of 11.1 and 11.0 .mu.mol phosphate transferred min-1 (mg enzyme)-1, respectively. The apparent Km values for mixed myosin light chains were 8.0 and 0.35 mg/ml in the absence and presence of calmodulin, respectively. Similarly, calmodulin lowered the Km value for isolated myosin light-chain 2 over 20-fold and increased the V value only about 1.5-fold. Activity observed in the absence of calmodulin was dependent on the presence of Ca and was suppressed by chelating free Ca either before or during a phosphorylation reaction. The apparent KA for Ca was 1.2 .mu.M and 0.4 .mu.M in the absence and presence of calmodulin. Activity in the absence of calmodulin was inhibited at very high concentrations of the specific calmodulin antagonists W-7 [N-(6-aminohexyl-5-chloro-1-naphthalinesulfonamide)], trifluoperazine and R24571 1-[bis(P-chlorophenyl)methyl]-3-[2,4-dichloro-.beta.-(2,4-dichlorobenzyloxy)phenethyl] midazolium chloride with apparent IC50 values of 0.3 mM, 0.2 mM and 0.02 mM. Antibodies raised against calmodulin suppressed completely the kinase activity in the presence of calmodulin but had no effect on the activity measured in its absence. Evidently, calmodulin stimulates the activity of bovine-cardiac myosin light-chain kinase by increasing over 20-fold the affinty for its substrate myosin light-chain 2.