Changes in contractile proteins during differentiation of myeloid leukemia cells. II. Purification and characterization of actin.
Changes in contractile proteins during differentiation of myeloid leukemia cells. II. Purification and characterization of actin.
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DOI：
10.1083/jcb.93.2.470
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发表时间：
1982-05
影响因子：
7.8
通讯作者：
Ichikawa, Y
中科院分区：
文献类型：
--
作者：
Nagata, K;Sagara, J;Ichikawa, Y
骨髓性白血病细胞系M1在与来自成纤维细胞培养物的条件培养基（CM）和细菌内毒素孵育时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-Sephadex A- 50，聚合/解聚和Sephadex G-150层析。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差异;发生了与骨骼肌肌动蛋白相同的剂量反应。但经CM处理后，肌动蛋白对HMMM Mg（2+）-ATP酶的激活比活性是未处理的M1肌动蛋白的2倍，是骨骼肌肌动蛋白的1/3。对照组和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，K（应用程序）值为控制和CM处理的M1细胞肌动蛋白的3倍和1.5倍的值为腹肌肌动蛋白。这意味着CM对M1肌动蛋白的处理产生了对腹主肌肌球蛋白的Mg（2+）-ATP酶的两倍亲和力。比较了不同盐浓度和温度下聚合反应的临界浓度。尽管在2 mM MgCl（2）的存在下没有发现显着差异，但在5 ℃下用0.1 M KCl代替MgCl（2）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。虽然D（-）肌动蛋白的临界浓度非常高，但在临界浓度以上，这种肌动蛋白显示正常聚合。这与我们以前的论文中提出的数据一起，即粗提物中的D（-）肌动蛋白没有溶解，表明肌动蛋白聚合的抑制剂存在于亚系中。在0.1 M KCl和25 ℃下的动力学实验显示，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。这种延迟的聚合是由于成核阶段期间的延迟，而不是在伸长阶段期间。通过等电聚焦，β-肌动蛋白与γ-肌动蛋白的比值显示出明显的差异，这取决于细胞的状态：对照M1约为4.9，CM处理的M1约为2.8，D（-）-亚细胞肌动蛋白约为7.6。胰蛋白酶肽图谱也揭示了不同肽的存在。因此，肌动蛋白在分化前后的功能差异伴随着肌动蛋白分子的一些化学变化。
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 β- to γ-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.