Alpha-actinin and vinculin from nonmuscle cells: calcium-sensitive interactions with actin.
Alpha-actinin and vinculin from nonmuscle cells: calcium-sensitive interactions with actin.
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来自非肌肉细胞的α-肌动蛋白和纽蛋白：与肌动蛋白的钙敏感相互作用。
DOI：
10.1101/sqb.1982.046.01.055
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
Feramisco,JR
中科院分区：
文献类型：
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作者：
Burridge,K;Feramisco,JR
材料和方法纯化蛋白质。按照我们以前使用的程序（Feramisco和Burridge 1980）纯化鸡砂囊平滑肌α-辅肌动蛋白。对于某些制剂，使用羟基磷灰石色谱法的额外步骤来去除杂质。将α-辅肌动蛋白加载到缓冲液B（Feramisco和Burridge 1980）中的柱上。然后用50 mM磷酸钾（pH 7.0）洗涤，并用该缓冲液至300 mM磷酸钾（pH 7.0）的梯度洗脱。用基本上相同的方法纯化来自HeLa细胞的α-辅肌动蛋白，但如别处所述进行了修改（JR Feramisco和K. Burridge，in prep.）。简而言之，将约500 g（湿重）培养的HeLa细胞用于单次制备。Th-celis是。在缓冲液A（Feramisco：and，Burridge 1980）中在37 ℃下提取，并将提取物上清液用（NH 4）2SO 4分级。The-fraeIio ~：在27.5%和37.5%饱和度之间的沉淀用于在DEAE-纤维素上的层析，随后在Sepharose CL-6 B上凝胶过滤。馏分含有-
MATERIALS AND METHODSPurification of proteins. The purification of chicken gizzard smooth-muscle a-actinin followed our previously used procedures (Feramisco and Burridge 1980). For some preparations an additional step of chromatography on hydroxyapatite was used to remove impurities. The a-actinin was loaded onto the column in Buffer B (Feramisco and Burridge 1980). This was then washed with 50 mM potassium phosphate (pH 7.0) and eluted with a gradient of this buffer to 300 mM potassium phosphate (pH 7.0). a-Actinin from HeLa cells was purified by essentially the same procedure, but modified as will be described elsewhere (JR Feramisco and K. Burridge, in prep.). In brief;~ approxi, mately 50 g (wet weight) of culture~ HeLa cells was used for a single preparation,. Th~ ae;-celis were. extracted at 37~ in Buffer A (Feramisco: and, Burridge 1980), and the extract supematam, was,: subje~ ted to fractionation with (NH4) 2SO4. The-fraeIio~: pmopitating between 27.5% and 37.5% saturation was used for chromatography on DEAE-cellulose, followed by gel filtration on Sepharose CL-6B. The fractions contain-