Abstract
Modulator-deficient myosin light-chain kinase from rabbit skeletal muscle was purified by modulator protein-Sepharose 4B affinity chromatography. The purified protein showed a single band (MW 80, 000) on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and it exists as a monomer in the native state as determined by gel filtration. The modulatordeficient myosin light-chain kinase (MW 80, 000), modulator protein (MW 16, 500) and Ca2+ were essential for the kinase activity. The half-maximal activity of the kinase in the presence of excess modulator protein with 10mM MgCl2 was at pCa 5.1, where full activity of actomyosin-ATPase is observed in the presence of the troponin-tropomyosin system. Assuming a rapid equilibrium between myosin light-chain kinase and the two substrates, ATP and g2 light-chain, Km values for ATP and g2 light chain were evaluated as 0.28mM and 0.024mM, respectively. Vm/e was 5.7s-1.
