Purification of modulator-deficient myosin light-chain kinase by modulator protein-Sepharose affinity chromatography.
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 two substrates, ATP and g2 light-chain, Km values for ATP and g2 light chain were evaluated as 0.28 mM and 0.024 mM, respectively. Vm/e was 5.7 s-1.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002846:Chromatography, Affinity; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009124:Muscle Proteins / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology; D009218:Myosins; D010446:Peptide Fragments; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits
D002147:Calmodulin; D009124:Muscle Proteins; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D000251:Adenosine Triphosphatases; D009218:Myosins; D008274:Magnesium; D002118:Calcium