Purification of chicken gizzard myosin light-chain kinase, and its calcium and strontium sensitivities as compared with those of superprecipitation and ATPase activities of actomyosin.
1. A purified preparation of myosin light-chain kinase (MLCK) was obtained from chicken gizzard, and it was shown to consist of two subunits; 130,000 (130 K)-dalton subunit and 17,000 (17 K)-dalton subunit. In amino acid composition the 130 K and 17 K subunits were identical with the 105 K and 17 K subunits of Dabrowska et al. (1977 and 1978), respectively. In disc gel electrophoresis, the 17 K subunit of our MLCK preparation responded to Ca2+ ions in the same way as bovine calmodulin, and differently from skeletal troponin C. There appeared to be one minor difference between 17 K subunit and calmodulin in the primary structure of the C-terminal region. 2. The Ca2+ and Sr2+ concentrations required for the three activities (ATPase and superprecipitation activities and MLCK activity) were measured. Two types of "reconstituted" myosin B were used; one contained 17 K subunit of gizzard MLCK and the other contained bovine brain calmodulin. The two types of "reconstituted" myosin B were practically identical with "natural" myosin B in the Ca2+ and Sr2+ requirements for the three activities measured above. 3. Both the extent and the activity of superprecipitation, and both the limited and steady activities of ATPase were measured. The MLCK activity was estimated in two ways; by urea gel electrophoresis and by measuring 32 P incorporation from [gamma-32P]ATP into myosin. The results thus obtained favor the kinase-phosphatase mechanism of calcium regulation of gizzard muscle contraction.
D000205:Actomyosin / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000032:analysis; D011232:Chemical Precipitation; D002645:Chickens; D005895:Gizzard, Avian / Q000201:enzymology; D007700:Kinetics; D009219:Myosin-Light-Chain Kinase; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D013324:Strontium / Q000494:pharmacology*
D000596:Amino Acids; D002147:Calmodulin; D000205:Actomyosin; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase; D000251:Adenosine Triphosphatases; D002118:Calcium; D013324:Strontium