Chacko S, Conti M A, Adelstein R S
Proc Natl Acad Sci U S A. 1977 Jan;74(1):129-33. doi: 10.1073/pnas.74.1.129.
A 35--70% ammonium sulfate fraction of smooth muscle actomyosin was prepared from guinea pig vas deferens. This fraction also contains a smooth muscle myosin kinase and a phosphatase that phosphorylates and dephosphorylates, respectively, the 20,000-dalton light chain of smooth muscle myosin. Phosphorylated and dephosphorylated 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 have identical stained patterns after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. They also have similar ATPase activities measured in 0.5 M KCl in the presence of K+-EDTA and Ca2+. However, the actin-activated myosin ATPase activity is markedly increased after phosphorylation. Moreover, the actin-activated ATPase activity of phosphorylated myosin is inhibited by the removal of Ca2+ in the absence of any added regulatory proteins. Dephosphorylation of myosin results 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+.
从豚鼠输精管制备了平滑肌肌动球蛋白的35%-70%硫酸铵级分。该级分还含有一种平滑肌肌球蛋白激酶和一种磷酸酶，它们分别使平滑肌肌球蛋白的20,000道尔顿轻链磷酸化和去磷酸化。通过凝胶过滤从该硫酸铵级分中纯化了磷酸化和去磷酸化的平滑肌肌球蛋白，凝胶过滤还将激酶和磷酸酶与肌球蛋白分离。纯化的磷酸化和去磷酸化肌球蛋白在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳后具有相同的染色模式。在存在K⁺-EDTA和Ca²⁺的0.5 M KCl中测量时，它们也具有相似的ATP酶活性。然而，磷酸化后肌动蛋白激活的肌球蛋白ATP酶活性显著增加。此外，在没有添加任何调节蛋白的情况下，去除Ca²⁺会抑制磷酸化肌球蛋白的肌动蛋白激活的ATP酶活性。肌球蛋白的去磷酸化导致肌动蛋白激活的ATP酶活性降低。在有Ca²⁺存在但无Ca²⁺不存在时，骨骼肌原肌球蛋白显著增加了磷酸化但未去磷酸化的肌球蛋白的肌动蛋白激活的ATP酶活性。
