Effects of Ca2+ and Mg2+ on the actomyosin adenosine-5'-triphosphatase of stably phosphorylated gizzard myosin.
Effects of Ca2+ and Mg2+ on the actomyosin adenosine-5'-triphosphatase of stably phosphorylated gizzard myosin.
复制标题
Ca2 和 Mg2 对稳定磷酸化砂囊肌球蛋白的肌动球蛋白腺苷-5-三磷酸酶的影响。
DOI：
10.1021/bi00332a020
复制
发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Chacko,S
中科院分区：
文献类型：
--
作者：
Heaslip,RJ;Chacko,S
宾夕法尼亚大学兽医学院病理生物学系，费城，宾夕法尼亚州19104接收于1984年5月29日;修订后的Mandarin pt接收于1984年10月5日摘要：一旦轻链被钙钙调蛋白依赖性激酶完全磷酸化，关于Ca 2+对肌球蛋白腺苷三磷酸酶（ATP酶）肌动蛋白激活的影响有相互矛盾的报道。使用硫代磷酸化肌球蛋白，Sherry et al. [雪莉，J.M. F.、Gorecka，A.，Aksoy，M. O.，Dabrowska河，&哈茨霍恩，D. J.（1978）Biochemistry 17，4417-4418]观察到ATP酶的肌动蛋白活化不受Ca 2+去除的抑制。因此，我们认为钙离子对肌球蛋白ATP酶活性的调节只通过磷酸化作用来实现。在本研究中，磷酸化和硫代磷酸化的肌球蛋白制备的激酶和磷酸酶活性的自由，因此，theATP酶活性可以在不同浓度的Ca 2+和Mg 2+测量，而不影响磷酸化的水平。肌球蛋白的ATP酶活性被激活的骨骼肌或肌胃肌动蛋白在不同浓度的Mg 2+，无论是在pCa 5或pCa 8。激活是敏感的Ca 2+在低Mg 2+浓度与肌动蛋白。原肌球蛋白增强肌动蛋白激活的ATP酶活性在所有的Mg 2+和Ca 2+浓度。磷酸化和硫代磷酸化肌球蛋白与肌动蛋白和原肌球蛋白重建的钙敏感性是最明显的游离Mg 2+浓度约为3 mM。125 I-原肌球蛋白的结合肌动蛋白表明，在低Mg 2+浓度下观察到的ATP酶的钙敏感性是不是由于钙介导的结合原肌球蛋白的F-肌动蛋白。当Mg 2+浓度增加到5 mM以上时，两种肌球蛋白的肌动蛋白激活对Ca 2+不敏感。数据表明，在低Mg 2+浓度下，Ca 2+不仅通过影响钙钙调蛋白依赖性激酶对轻链的磷酸化，而且通过直接影响肌动蛋白激活的ATP水解来调节肌动球蛋白ATP酶。平滑肌肌球蛋白的20000-道尔顿（Da）1轻链的磷酸化与其肌动蛋白激活的ATP酶活性的几倍增加有关（Gorecka等，1976; Sobieszek，1977; Chacko等人，1977; Ikebe等人，1978; Rees & Frederiksen，1981）。轻链的磷酸化由称为肌球蛋白轻链激酶的钙钙调蛋白依赖性激酶催化（Frearson &佩里，1975; Dabrowska等人，1978; Adelstein等人，1978年）。因此，钙通过影响肌球蛋白轻链的磷酸化激活肌动球蛋白ATP酶活性。使用分离的肌球蛋白
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania19104 Received May 29, 1984; Revised Manuscript Received October 5, 1984 abstract: There are conflicting reports on the effect of Ca2+ on actin activation of myosin adenosinetriphosphatase (ATPase) once the light chain is fully phosphorylated by a calcium calmodulin dependent kinase. Using thiophosphorylated gizzard myosin, Sherry et al.[Sherry, J. M. F., Gorecka, A., Aksoy, M. O., Dabrowska, R., & Hartshorne, D. J.(1978) Biochemistry 17, 4417-4418] observed that the actin activation of ATPase was not inhibited by the removal of Ca2+. Hence, it was suggested that the regulation of actomyosin ATPase activity of gizzardmyosin by calcium occurs only via phosphorylation. In the present study, phosphorylated and thiophosphorylated myosins were prepared free of kinase and phosphatase activity; hence, theATPase activity could be measured at various concentrations of Ca2+ and Mg2+ without affecting the level of phosphorylation. The ATPase activity of myosin was activated either by skeletal muscle or by gizzard actin at various concentrations of Mg2+ and either at pCa 5 or at pCa 8. The activation was sensitive to Ca2+ at low Mg2+ concentrations with both actins. Tropomyosin potentiated the actin-activated ATPase activity at all Mg2+ and Ca2+ concentrations. The calcium sensitivity of phosphorylated and thiophosphorylated myosin reconstituted with actin and tropomyosin was most pronounced at a free Mg2+ concentration of about 3 mM. The binding of 125I-tropomyosin to actin showed that the calcium sensitivity of ATPase observed at low Mg2+ concentration is not due to a calcium-mediated binding of tropomyosin to F-actin. The actin activation of both myosins was insensitive to Ca2+ when the Mg2+ concentration was increased above 5 mM. The data indicate that, at low Mg2+ concentrations, Ca2+ regulates the actomyosin ATPase not only by its effect on the phosphorylation of light chain by calcium calmodulin dependent kinase but also by a direct effect on the actin-activated ATP hydrolysis.^^ osphorylation of the 20000-dalton (Da) 1 light chain of smooth muscle myosin is associated with severalfold increase in its actin-activated ATPase activity (Gorecka et al., 1976; Sobieszek, 1977; Chacko et al., 1977; Ikebe et al., 1978; Rees & Frederiksen, 1981). Phosphorylation of the light chain is catalyzed by a calcium calmodulin dependent kinase called myosin light chain kinase (Frearson & Perry, 1975; Dabrowska et al., 1978; Adelstein et al., 1978). Hence, calcium activates the actomyosin ATPase activity via its effect on the phos-phorylation of the myosin light chain. Using myosin isolated