Nagata K, Sagara J, Ichikawa Y
J Cell Biol. 1982 May;93(2):470-8. doi: 10.1083/jcb.93.2.470.
A myeloid leukemia cell line, M1, differentiates to macrophage and gains locomotive and phagocytic activity when incubated with conditioned medium (CM) from a fibroblast culture and bacterial endotoxin. To characterize the actin molecules before and after differentiation, the actin was purified through three sequential steps: DEAE-sephadex A- 50, polymerization/depolymerization, and sephadex G-150 chromatography. There were no essential differences between the inhibitory activity of actins from control M1 cells and CM-treated M1 cells on both DNase I and heavy meromyosin (HMMM) K(+)-EDTA-ATPase; the same dose response as with skeletal muscle actin took place. After the treatment with CM, however, the specific activity for the activation of HMMM Mg(2+)- ATPase by actin became two-fold that of untreated M1 actin, which was one third of the value for skeletal muscle actin. The V(max) for the control and the CM-treated M1 cell, as well as the skeletal muscle actins, proved to be the same. By contrast, the K(app) values for the control and CM-treated M1-cell actins were 3- and 1.5-fold the value for skeletal-muscle actin. This means that CM treatment of the M1 actin produced a twofold affinity for the Mg(2+)-ATPase of skeletal-muscle myosin. The critical concentrations for polymerization were compared under different salt concentrations and temperatures. Although no marked difference was found for the presence of 2 mM MgCl(2), 0.1 M KCl in place of MgCl(2) at 5 degrees C gave the following values: 0.1 mg/ml for skeletal-muscle actin, 0.7 mg/ml for control M1 actin, 0,5 mg/ml for CM- treated M1 actin, and 1.0 mg/ml for the D(-) subline that is insensitive to CM. Although the critical concentration of D(-) actin is extraordinarily high, this actin showed normal polymerization above the critical concentration. This together with the data presented in our previous paper, that the D(-) actin in the crude extract did not polymerize, suggests that an inhibitor for actin polymerization is present in the subline. The kinetics experiment at 0.1 M KCl and 25 degrees C revealed a slower polymerization of untreated M1- and D(-)-cell actins as compared with CM-treated M1 actin. This delayed polymerization was due to a delay during the nucleation stage, not during the elongation stage. By isoelectric focusing, the ratios of beta- to gamma-actin showed a marked difference depending on the states of cells: about 4.9 for control M1, 2.8 for CM-treated M1, and 7.6 for D(-)-subline actins. Tryptic peptide maps also revealed the presence of different peptides. Thus, the functional differences of actin before and after the differentiation was accompanied by some chemical changes in actin molecules.
一种髓样白血病细胞系M1，在与成纤维细胞培养的条件培养基（CM）和细菌内毒素一起孵育时，会分化为巨噬细胞并获得运动和吞噬活性。为了表征分化前后的肌动蛋白分子，通过三个连续步骤纯化肌动蛋白：DEAE-葡聚糖A-50、聚合/解聚以及葡聚糖G-150色谱法。对照M1细胞和CM处理的M1细胞的肌动蛋白对DNase I和重酶解肌球蛋白（HMMM）K(+)-EDTA-ATP酶的抑制活性之间没有本质区别；与骨骼肌肌动蛋白的剂量反应相同。然而，用CM处理后，肌动蛋白激活HMMM Mg(2+)-ATP酶的比活性变为未处理的M1肌动蛋白的两倍，而未处理的M1肌动蛋白的该值是骨骼肌肌动蛋白的三分之一。对照和CM处理的M1细胞以及骨骼肌肌动蛋白的V(max)被证明是相同的。相比之下，对照和CM处理的M1细胞肌动蛋白的K(app)值分别是骨骼肌肌动蛋白值的3倍和1.5倍。这意味着用CM处理M1肌动蛋白会使其对骨骼肌肌球蛋白的Mg(2+)-ATP酶的亲和力提高两倍。在不同盐浓度和温度下比较了聚合的临界浓度。尽管在存在2 mM MgCl(2)时未发现明显差异，但在5℃用0.1 M KCl代替MgCl(2)时得到以下值：骨骼肌肌动蛋白为0.1 mg/ml，对照M1肌动蛋白为0.7 mg/ml，CM处理的M1肌动蛋白为0.5 mg/ml，对CM不敏感的D(-)亚系为1.0 mg/ml。尽管D(-)肌动蛋白的临界浓度非常高，但该肌动蛋白在临界浓度以上显示出正常的聚合。这与我们之前论文中的数据一起，即粗提物中的D(-)肌动蛋白不聚合，表明该亚系中存在肌动蛋白聚合抑制剂。在0.1 M KCl和25℃下的动力学实验表明，与CM处理的M1肌动蛋白相比，未处理的M1-和D(-)-细胞肌动蛋白的聚合较慢。这种延迟聚合是由于成核阶段的延迟，而不是延伸阶段的延迟。通过等电聚焦，β-肌动蛋白与γ-肌动蛋白的比例根据细胞状态显示出明显差异：对照M1约为4.9，CM处理的M1为2.8，D(-)-亚系肌动蛋白为7.6。胰蛋白酶肽图谱也显示存在不同的肽。因此，分化前后肌动蛋白的功能差异伴随着肌动蛋白分子的一些化学变化。
