Ravid S, Spudich J A
Department of Cell Biology, Stanford University School of Medicine, California 94305.
J Biol Chem. 1989 Sep 5;264(25):15144-50.
We purified to homogeneity the Dictyostelium discoideum myosin heavy chain kinase that is implicated in the heavy chain phosphorylation increases that occur during chemotaxis. The kinase is initially found in the insoluble fraction of developed cells. The major purification step was achieved by affinity chromatography using a tail fragment of Dictyostelium myosin (LMM58) expressed in Escherichia coli (De Lozanne, A., Berlot, C. H., Leinwand, L. A., and Spudich, J. A. (1988) J. Cell Biol. 105, 2990-3005). The kinase has an apparent molecular weight of 84,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent native molecular weight by gel filtration is 240,000. The kinase catalyzes phosphorylation of myosin heavy chain or LMM58 with similar kinetics, and the extent of phosphorylation for both is 4 mol of phosphate/mol. With both substrates the Vmax is about 18 mumol/min/mg and the Km is 15 microM. The myosin heavy chain kinase is specific to Dictyostelium myosin heavy chain, and the phosphorylated amino acid is threonine. The kinase undergoes autophosphorylation. Each mole of kinase subunit incorporates about 20 mol of phosphates. Phosphorylation of myosin by this kinase inhibits myosin thick filament formation, suggesting that the kinase plays a role in the regulation of myosin assembly.
我们将盘基网柄菌肌球蛋白重链激酶纯化至同质，该激酶与趋化作用过程中发生的重链磷酸化增加有关。该激酶最初在发育细胞的不溶性部分中被发现。主要的纯化步骤是通过亲和色谱法实现的，使用在大肠杆菌中表达的盘基网柄菌肌球蛋白的尾部片段（LMM58）（德洛赞，A.，贝洛特，C. H.，莱因万德，L. A.，和斯普迪奇，J. A.（1988年）《细胞生物学杂志》105卷，2990 - 3005页）。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳测定，该激酶的表观分子量为84,000。通过凝胶过滤法测定的表观天然分子量为240,000。该激酶以相似的动力学催化肌球蛋白重链或LMM58的磷酸化，两者的磷酸化程度均为4摩尔磷酸/摩尔。对于这两种底物，Vmax约为18微摩尔/分钟/毫克，Km为15微摩尔。肌球蛋白重链激酶对盘基网柄菌肌球蛋白重链具有特异性，磷酸化的氨基酸是苏氨酸。该激酶会进行自身磷酸化。每摩尔激酶亚基掺入约20摩尔磷酸盐。该激酶对肌球蛋白的磷酸化抑制了肌球蛋白粗丝的形成，这表明该激酶在肌球蛋白组装的调节中起作用。
