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.
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.
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鸡砂囊肌球蛋白轻链激酶的纯化及其钙、锶敏感性与超沉淀和肌动球蛋白ATP酶活性的比较。
DOI：
10.1093/oxfordjournals.jbchem.a133685
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发表时间：
1982
影响因子：
2.7
通讯作者：
Shizuo Watanabe
中科院分区：
文献类型：
--
作者：
Hideyo Uchiwa;Tomoyasu Kato;Hirofumi Onishi;Toshiaki Isobe;Tsuneo Okuyama;Shizuo Watanabe
1.从鸡的砂囊中分离纯化了肌球蛋白轻链激酶（MLCK），它由130，000（130 K）-道尔顿亚基和17，000（17 K）-道尔顿亚基组成。在氨基酸组成中，130 K和17 K亚基分别与Dabrowska等人（1977和1978）的105 K和17 K亚基相同。在盘状凝胶电泳中，MLCK制备物的17 K亚基以与牛钙调蛋白相同的方式对Ca 2+离子作出反应，而与骨骼肌钙蛋白C不同。17 K亚基和钙调素在C-末端区域的一级结构中似乎有一个微小的差异。2.测定了三种活性（ATP酶和超沉淀活性以及MLCK活性）所需的Ca 2+和Sr 2+浓度。使用了两种类型的“重构”肌球蛋白B;一种含有肌胃MLCK的17 K亚基，另一种含有牛脑钙调蛋白。两种类型的“重构”肌球蛋白B在上述三种活性的Ca 2+和Sr 2+需求方面与“天然”肌球蛋白B几乎相同。3.测定了超沉淀的程度和超沉淀的活性，以及ATP酶的有限活性和稳定活性。MLCK活性的估计在两种方式;尿素凝胶电泳和通过测量32 P掺入从[γ-32 P]ATP肌球蛋白。由此获得的结果有利于肌砂囊收缩的钙调节的激酶-磷酸酶机制。
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.