EFFECT OF PHOSPHORYLATION OF SMOOTH-MUSCLE MYOSIN ON ACTIN ACTIVATION AND CA2+ REGULATION
EFFECT OF PHOSPHORYLATION OF SMOOTH-MUSCLE MYOSIN ON ACTIN ACTIVATION AND CA2+ REGULATION
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DOI：
10.1073/pnas.74.1.129
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发表时间：
1977-01-01
影响因子：
11.1
通讯作者：
ADELSTEIN, RS
中科院分区：
文献类型：
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作者：
CHACKO, S;CONTI, MA;ADELSTEIN, RS
以豚鼠输精管为原料制备了35 ~ 70%硫酸铵的平滑肌肌动球蛋白组分。该部分还含有一个平滑肌肌球蛋白激酶和一个磷酸酶，分别磷酸化和去磷酸化平滑肌肌球蛋白的20,000道尔顿L链。通过凝胶过滤从硫酸铵部分中分离出磷酸化和去磷酸化的平滑肌肌球蛋白，并从肌球蛋白中分离出激酶和磷酸酶。经十二烷基硫酸钠/聚丙烯酰胺凝胶电泳，纯化磷酸化和去磷酸化的肌球蛋白具有相同的染色模式。在0.5 M KCl中，在K+-EDTA和Ca2+的存在下，它们也具有相似的atp酶活性。而肌动蛋白激活的肌球蛋白atp酶活性在磷酸化后明显升高。在没有添加任何调节蛋白的情况下，肌球蛋白磷酸化的肌动蛋白激活的atp酶活性被Ca2+去除所抑制。肌球蛋白的去磷酸化导致肌动蛋白激活的atp酶活性降低。骨骼肌原肌球蛋白显著增加肌动蛋白激活的atp酶活性，而不是去磷酸化肌动蛋白，在Ca2+存在的情况下，而不是在没有Ca2+的情况下。
A 35-70% ammonium sulfate fraction of smooth muscle actomyosin was prepared from guinea pig vas deferens. This fraction also contained a smooth muscle myosin kinase and a phosphatase that phosphorylates and dephosphorylates, respectively, the 20,000-dalton L chain of smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin were purified from this ammonium sulfate fraction by gel filtration, which also separated the kinase and the phosphatase from the myosin. Purified phosphorylated and dephosphorylated myosin had identical staining patterns after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. They also had similar ATPase activities measured in 0.5 M KCl in the presence of K+-EDTA and Ca2+. However, the actin-activated myosin ATPase activity was markedly increased after phosphorylation. The actin-activiated ATPase activity of phosphorylated myosin was inhibited by the removal of Ca2+ in the absence of any added regulatory proteins. Dephosphorylation of myosin resulted in a decrease in the actin-activated ATPase activity. Skeletal muscle tropomyosin markedly increased the actin-activated ATPase activity of phosphorylated but not dephosphorylated myosin in the presence, but not in the absence, of Ca2+.