Chou Y H, Rebhun L I
J Biol Chem. 1986 Apr 25;261(12):5389-95.
The crude actomyosin precipitate from sea urchin (Arbacia punctulata) egg extracts contains Ca2+-sensitive myosin light chain kinase activity. Activity can be further increased by exogenous calmodulin (CaM). Egg myosin light chain kinase activity is purified from total egg extract by fractionating on three different chromatographic columns: DEAE ion exchange, gel filtration on Sephacryl-300, and Affi-Gel-CaM affinity. The purified egg kinase depends totally on Ca2+ and CaM for activity. Unphosphorylated egg myosin has very little actin-activated ATPase. After phosphorylation of the phosphorylable light chain by either egg kinase or gizzard myosin light chain kinase, the actin-activated ATPase of egg myosin is enhanced several fold. However, the egg kinase bears some unique characteristics which are very different from conventional myosin light chain kinases of differentiated tissues. The purified egg kinase has a native molecular mass of 405 kDa, while on sodium dodecyl sulfate-polyacrylamide electrophoresis it shows a single subunit of 56 kDa. The affinity of egg kinase for CaM (Ka = 0.4 microM) is relatively weaker than that of the gizzard myosin light chain kinase. The egg kinase autophosphorylates in the presence of Ca2+ and CaM and has a rather broad substrate specificity. The possible relationship between this egg Ca2+-CaM-dependent kinase and the Ca2+-CaM-dependent kinases from brain and liver is discussed.
从海胆（刺冠海胆）卵提取物中得到的粗制肌动球蛋白沉淀含有对Ca2+敏感的肌球蛋白轻链激酶活性。外源性钙调蛋白（CaM）可进一步增强该活性。通过在三种不同的色谱柱上进行分级分离，即DEAE离子交换柱、Sephacryl - 300凝胶过滤柱和Affi - Gel - CaM亲和柱，从全卵提取物中纯化出海胆卵肌球蛋白轻链激酶活性。纯化后的卵激酶的活性完全依赖于Ca2+和CaM。未磷酸化的海胆卵肌球蛋白的肌动蛋白激活ATP酶活性很低。用海胆卵激酶或鸡胗肌球蛋白轻链激酶对可磷酸化轻链进行磷酸化后，海胆卵肌球蛋白的肌动蛋白激活ATP酶活性增强了几倍。然而，海胆卵激酶具有一些与分化组织中的传统肌球蛋白轻链激酶非常不同的独特特性。纯化后的海胆卵激酶的天然分子量为405 kDa，而在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上显示为一个56 kDa的单一亚基。海胆卵激酶对CaM的亲和力（Ka = 0.4 microM）相对弱于鸡胗肌球蛋白轻链激酶。海胆卵激酶在Ca2+和CaM存在下会发生自身磷酸化，并且具有相当广泛的底物特异性。本文还讨论了这种海胆卵Ca2+ - CaM依赖性激酶与来自脑和肝脏的Ca2+ - CaM依赖性激酶之间可能的关系。
