Hartwig J H, Stossel T P
J Biol Chem. 1975 Jul 25;250(14):5696-705.
Actin, myosin, and a high molecular weight actin-binding protein were extracted from rabbit alveolar macrophages with low ionic strength sucrose solutions containing ATP, EDTA, and dithiothreitol, pH 7.0. Addition of KCl, 75 to 100 mM, to sucrose extracts of macrophages stirred at 25 degrees caused actin to polymerize and bind to a protein of high molecualr weight. The complex precipitated and sedimented at low centrifugal forces. Macrophage actin was dissociated from the binding protein with 0.6 M KCl, and purified by repetitive depolymerization and polymerization. Purified macrophage actin migrated as a polypeptide of molecular weight 45,000 on polyacrylamide gels with dodecyl sulfate, formed extended filaments in 0.1 M KCl, bound rabbit skeletal muscle myosin in the absence of Mg-2+ATP and activated its Mg-2+ATPase activity. Macrophage myosin was bound to actin remaining in the macrophage extracts after removal of the actin precipitated with the high molecular weight protein by KCl. The myosin-actin complex and other proteins were collected by ultracentrifugation. Macrophage myosin was purified from this complex or from a 20 to 50% saturated ammonium sulfate fraction of macrophage extracts by gel filtration on agarose columns in 0.6 M Kl and 0.6 M Kl solutions. Purified macrophage myosin had high specific K-+- and EDTA- and K-+- and Ca-2+ATPase activities and low specific Mg-2+ATPase activity. It had subunits of 200,000, 20,000, and 15,000 molecular weight, and formed bipolar filaments in 0.1 M KCl, both in the presence and absence of divalent cations. The high molecular weight protein that precipitated with actin in the sucrose extracts of macrophages was purified by gel filtration in 0.6 M Kl-0.6 M KCl solutions. It was designated a macrophage actin-binding protein, because of its association with actin at physiological pH and ionic strength. On polyacrylamide gels in dodecyl sulfate, the purified high molecular weight protein contained one band which co-migrated with the lighter polypeptide (molecular weight 220,000) of the doublet comprising purified rabbit erythrocyte spectrin. The macrophage protein, like rabbit erythrocyte spectrin, was soluble in 2 mM EDTA and 80% ethanol as well as in 0.6 M KCl solutions, and precipitated in 2 mM CaCl2 or 0.075 to 0.1 M KCl solutions. The macrophage actin-binding protein and rabbit erythrocyte spectrin eluted from agarose columns with a KAV of 0.24 and in the excluded volumes. The protein did not form filaments in 0.1 M KCl and had no detectable ATPase activity under the conditions tested.
用含有ATP、EDTA和二硫苏糖醇（pH 7.0）的低离子强度蔗糖溶液从兔肺泡巨噬细胞中提取肌动蛋白、肌球蛋白和一种高分子量肌动蛋白结合蛋白。向在25℃搅拌的巨噬细胞蔗糖提取物中加入75至100 mM的KCl，可使肌动蛋白聚合并与一种高分子量蛋白质结合。该复合物沉淀并在低离心力下沉降。巨噬细胞肌动蛋白用0.6 M KCl从结合蛋白上解离下来，并通过重复解聚和聚合进行纯化。纯化的巨噬细胞肌动蛋白在含十二烷基硫酸钠的聚丙烯酰胺凝胶上迁移时表现为分子量45,000的多肽，在0.1 M KCl中形成伸展的细丝，在没有Mg-2+ATP的情况下与兔骨骼肌肌球蛋白结合并激活其Mg-2+ATP酶活性。巨噬细胞肌球蛋白与用KCl沉淀高分子量蛋白质后残留在巨噬细胞提取物中的肌动蛋白结合。肌球蛋白-肌动蛋白复合物和其他蛋白质通过超速离心收集。巨噬细胞肌球蛋白通过在0.6 M Kl和0.6 M Kl溶液中在琼脂糖柱上进行凝胶过滤从该复合物或巨噬细胞提取物的20%至50%饱和硫酸铵部分中纯化得到。纯化的巨噬细胞肌球蛋白具有高特异性的K+-、EDTA-、K+-和Ca-2+ATP酶活性以及低特异性的Mg-2+ATP酶活性。它具有分子量为200,000、20,000和15,000的亚基，并且在0.1 M KCl中，无论有无二价阳离子都形成双极细丝。在巨噬细胞蔗糖提取物中与肌动蛋白一起沉淀的高分子量蛋白质通过在0.6 M Kl-0.6 M KCl溶液中进行凝胶过滤进行纯化。由于其在生理pH和离子强度下与肌动蛋白的结合，它被命名为巨噬细胞肌动蛋白结合蛋白。在含十二烷基硫酸钠的聚丙烯酰胺凝胶上，纯化的高分子量蛋白质包含一条带，该带与纯化的兔红细胞血影蛋白双峰中较轻的多肽（分子量220,000）共迁移。巨噬细胞蛋白与兔红细胞血影蛋白一样，可溶于2 mM EDTA、80%乙醇以及0.6 M KCl溶液中，并在2 mM CaCl2或0.075至0.1 M KCl溶液中沉淀。巨噬细胞肌动蛋白结合蛋白和兔红细胞血影蛋白从琼脂糖柱上洗脱时的KAV为0.24且处于排阻体积。该蛋白质在0.1 M KCl中不形成细丝，并且在测试条件下没有可检测到的ATP酶活性。
