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J. Biochem, 1982, Vol. 91, No. 1 273-282
© 1982 Japanese Biochemical Society

research-article

Purification of Chicken Gizzard Myosin Light-Chain Kinase, and Its Calcium and Strontium Sensitivities as Compared with Those of Superprecipitation and ATPase Activities of Actomyosin1

Hideyo UCHIWA*, Tomoyasu KATO*, Hirofumi ONISHI*, Toshiaki ISOBE*, Tsuneo OKUYAMA** and Shizuo WATANABE*,2

*Department of Chemistry, Tokyo Institute of Technology, Faculty of Science Meguro-ku, Tokyo 152
**Tokyo Metropolitan University, Faculty of Science Setagaya-ku, Tokyo 158

  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 130K and 17K subunits were identical with the 105K and 17K subunits of Dabrowska et al. (1977 and 1978), respectively. In disc gel electrophoresis, the 17K 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 struc ture 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 initial and steady activities of ATPase were measured. The MLCK activity was estimated in two ways; by urea gel electrophoresis and by measuring 32P incorporation from [{gamma}–32]ATP into myosin. The results thus obtained favor the kinase-phosphatase mechanism of calcium regulation of gizzard muscle contraction.

1 Supported by research grants to the Cardiovascular Research Group, to the Regulatory Protein Research Group and to S. Watanabe from the Ministry of Education, Science and Culture of Japan. Presented by H. Uchiwa at the annual meeting of the Japanese Biochemical Society, held in Tokyo on Oct. 13, 1980.

2 Present address: Department of Chemistry, Gumma University Faculty of Technology, Kiryu, Gumma 376.


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