Copurification of actin and desmin from chicken smooth muscle and their copolymerization in vitro to intermediate filaments.
Copurification of actin and desmin from chicken smooth muscle and their copolymerization in vitro to intermediate filaments.
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DOI：
10.1083/jcb.80.1.166
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发表时间：
1979-01
影响因子：
7.8
通讯作者：
Lazarides, E
中科院分区：
文献类型：
--
作者：
Hubbard, B D;Lazarides, E
Desmin是一种50,000 mol wt的蛋白质，它与鸡砂囊中的100-A细丝一起富集，用1m KI提取。虽然1mki从砂囊中去除大部分肌动蛋白，但该蛋白的一小部分仍与去蛋白蛋白一起持续不溶。这种肌动蛋白的溶解度与聚乳酸蛋白相同：它们在高浓度盐中都不溶，但在低pH值下或被解离疏水键的试剂溶解。可通过1 M醋酸的反复增溶和随后的中和至pH 4的沉淀来纯化地丝蛋白。在这个过程中，肌动蛋白与蛋白的非化学计量比达到恒定。在0.5%的萨科齐NL-97存在下，在Ultrogel AcA34上进行凝胶过滤，发现肌动蛋白和蛋白的非单体组分通过色谱柱。在Bio-Gel P300上，在1 M醋酸的存在下进行凝胶过滤，发现在这些条件下，大多数乙酰氨基乙酯是单体的。一小部分去蛋白和所有的肌动蛋白以排除的体积洗脱。当通过透析将醋酸从肌动蛋白-desmin溶液中除去时，形成由直径为120-140 a的细丝组成的凝胶。这些纤维与抗肌动蛋白和抗desmin抗血清反应均匀。这些结果表明，肌动蛋白是肌肉100-A纤维的主要亚基，它可能与肌动蛋白形成非化学计量复合物。
Desmin is a 50,000-mol wt protein that is enriched along with 100-A filaments in chicken gizzard that has been extracted with 1 M KI. Although 1 M KI removes most of the actin from gizzard, a small fraction of this protein remains persistently insoluble, along with desmin. The solubility properties of this actin are the same as for desmin: they are both insoluble in high salt concentrations, but are solubilized at low pH or by agents that dissociate hydrophobic bonds. Desmin may be purified by repeated cycles of solubilization by 1 M acetic acid and subsequent precipitation by neutralization to pH 4. During this process, a constant nonstoichiometric ratio of actin to desmin is attained. Gel filtration on Ultrogel AcA34 in the presence of 0.5% Sarkosyl NL-97 reveals nonmonomeric fractions of actin and desmin that comigrate through the column. Gel filtration on Bio-Gel P300 in the presence of 1 M acetic acid reveals that the majority of desmin is monomeric under these conditions. A small fraction of desmin and all of the actin elute with the excluded volume. When the acetic acid is removed from actin-desmin solutions by dialysis, a gel forms that is composed of filaments with diameters of 120-140 A. These filaments react uniformly with both anti-actin and anti-desmin antiserum. These results suggest that desmin is the major subunit of the muscle 100-A filaments and that it may form nonstoichiometric complexes with actin.