Sobieszek A, Borkowski J, Babiychuk V S
Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstrasse 11, A-5020 Salzburg, Austria.
J Biol Chem. 1997 Mar 14;272(11):7034-41. doi: 10.1074/jbc.272.11.7034.
We show that a myofibrillar form of smooth muscle myosin light chain phosphatase (MLCPase) forms a multienzyme complex with myosin light chain kinase (MLCKase). The stability of the complex was indicated by the copurification of MLCKase and MLCPase activities through multiple steps that included myofibril preparation, gel filtration chromatography, cation (SP-Sepharose BB) and anion (Q-Sepharose FF) exchange chromatography, and affinity purification on calmodulin and on thiophosphorylated regulatory light chain columns. In addition, the purified complex eluted as a single peak from a final gel filtration column in the presence of calmodulin (CaM). Because a similar MLCPase is present in varying amounts in standard preparations of both MLCKase and myosin filaments, we have named it a kinase- and myosin-associated protein phosphatase (KAMPPase). The KAMPPase multienzyme complex was composed of a 37-kDa catalytic (PC) subunit, a 67-kDa targeting (PT) subunit, and MLCKase with or without CaM. The approximate molar ratio of the PC and PT subunits was 1:2 with a variable and usually higher molar content of MLCKase. The targeting role of the PT subunit was directly demonstrated in binding experiments in which the PT subunit bound to both the kinase and to CaM. Its binding to CaM was, however, Ca2+-independent. MLCKase and the PT subunit potentiated activity of the PC subunit when intact myosin was used as the substrate. These data indicated that there is a Ca2+-independent interaction among the MLCPase, MLCKase, and CaM that are involved in the regulation of phosphatase activity.
我们发现，平滑肌肌球蛋白轻链磷酸酶（MLCPase）的肌原纤维形式与肌球蛋白轻链激酶（MLCKase）形成多酶复合物。通过包括肌原纤维制备、凝胶过滤色谱、阳离子（SP-琼脂糖凝胶BB）和阴离子（Q-琼脂糖凝胶FF）交换色谱以及在钙调蛋白和硫代磷酸化调节轻链柱上进行亲和纯化等多个步骤，MLCKase和MLCPase活性的共纯化表明了该复合物的稳定性。此外，在钙调蛋白（CaM）存在的情况下，纯化后的复合物从最终的凝胶过滤柱上以单峰形式洗脱。由于在MLCKase和肌球蛋白丝的标准制剂中存在数量不等的类似MLCPase，我们将其命名为激酶和肌球蛋白相关蛋白磷酸酶（KAMPPase）。KAMPPase多酶复合物由一个37 kDa的催化（PC）亚基、一个67 kDa的靶向（PT）亚基以及带有或不带有CaM的MLCKase组成。PC和PT亚基的摩尔比约为1:2，MLCKase的摩尔含量可变且通常更高。PT亚基的靶向作用在结合实验中得到直接证明，其中PT亚基与激酶和CaM都结合。然而，它与CaM的结合不依赖于Ca2+。当完整的肌球蛋白作为底物时，MLCKase和PT亚基增强了PC亚基的活性。这些数据表明，参与磷酸酶活性调节的MLCPase、MLCKase和CaM之间存在不依赖于Ca2+的相互作用。
