Purification and characterization of myosin light-chain kinase from porcine myometrium and its phosphorylation and modulation by cyclic amp-dependent protein kinase

Myosin light-chain kinase was purified from porcine myometrium to apparent homogeneity at about 262-fold with an M_r of 130 000 as determined by SDS-polyacrylamide gel electrophoresis and a sedimentation coefficient of 4.5 S. The approximate content of the soluble myosin light-chain kinasee was estimated to be about 0.85 μ M. The purified enzyme exhibited strict substrate specificity only for 20-kDa myosin light chain and _a values of 0.6 nM and 0.3 μM for calmodulin and Ca²⁺, respectively. The enzyme was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, which resulted in a decrease in the affinity for calmodulin of 4-7-fold without effect on the V_max. The maximal amount of phosphate incorporated into the enzyme was 0.5-0.8 and 1.0-1.4 mol per mol of the enzme in the presence and absence of Ca²⁺ and calmodulin, respectively. In the presence of a subsaturating concentration of calmodulin, the enzyme showed a lower sensitivity for Ca²⁺ by phosphorylation.