Purification of Modulator-Deficient Myosin Light-Chain Kinase by Modulator Protein-Sepharose Affinity Chromatography1

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 modulator-deficient 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 10 mM 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, K_m values for ATP and g₂ light chain were evaluated as 0.28 mM and 0.024 mM, respectively. V_m/e was 5.7 s⁻¹.