Isolation of an immunoreactive analogue of brain fodrin that is associated with the cell cortex of Dictyostelium amoebae.
Isolation of an immunoreactive analogue of brain fodrin that is associated with the cell cortex of Dictyostelium amoebae.
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分离与阿米巴盘基网柄菌细胞皮层相关的脑胞因子的免疫反应类似物。
DOI：
10.1002/cm.970110408
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发表时间：
1988
影响因子：
--
通讯作者：
Condeelis,J
中科院分区：
文献类型：
--
作者：
Bennett,H;Condeelis,J
我们用一种多克隆亲和纯化的抗体(包括240和235kD两个亚基)作为探针，以确定盘基网眼菌的阿米巴中是否存在类口感蛋白。在整个细胞和分离的细胞皮质的Western blotts中，由Fodrin抗体识别的220和70kD的多肽。间接免疫荧光定位表明，免疫活性多肽为皮质多肽。免疫反应类似物COPATE和COCAP与刀豆蛋白A结合。在电子显微镜分辨率水平上，用抗角蛋白和胶体金进行免疫细胞化学证实，免疫反应类似物是与细胞膜一侧的微丝相关的皮质蛋白。我们已经分离和鉴定了220kD的蛋白，以确定它是否与Fodrin相似，并研究它与70kD多肽的关系。通过凝胶过滤和蔗糖密度梯度沉淀法，可以在不使用洗涤剂的情况下从皮层中提取220kD的蛋白质。220kD为杆状蛋白，全长118±17.8 nm(N=37)。其沉淀系数S为9.3，斯托克斯半径为13 nm，以约500,000道尔顿(MR)的二聚体形式存在。用旋转阴影法检测分离的220kD蛋白在体外与肌动蛋白细丝结合。形态特征区分了Dictyosteliummyosin II重链(215kD)和240kD的丝氨酸样蛋白。70kD的多肽似乎是220kD的切割片段，因为它是在长时间储存后发现的，而以前只有220kD。此外，当TPCK胰酶处理时，220和70kD多肽显示出相似的一维肽图。根据其物理和免疫反应特性以及在细胞中的定位，220kD可能是一种类口感蛋白。
We have used a polyclonal affinity‐purified antibody made against chicken brain fodrin (both 240 and 235 Kd subunits) as a probe to determine if a fodrinlike protein exists in amoebae ofDictyostelium discoideum. In Western blots of whole cells and the isolated cell cortex, polypeptides measuring 220 and 70 Kd are recognized by the fodrin antibodies. In situ localization by indirect immunofluorescence with antifodrin indicates that the immunoreactive polypeptides are cortical. The immunoreactive analogues copatch and cocap with concanavalin A. At the level of resolution of the electron microscope, immunocytochemistry with antifodrin and colloidal gold confirms that the immunoreactive analogues are cortical proteins associated with microfilaments on the cytoplasmic side of the plasma membrane. We have isolated and characterized the 220 Kd protein to determine if it is similar to fodrin and to investigate its relationship to the 70 Kd polypeptide. The 220 Kd protein can be extracted from the cortex in the absence of detergent and isolated by gel filtration and sucrose density gradient sedimentation. The 220 Kd is a rod‐shaped protein 118 ± 17.8 nm (N = 37) in length. It has a sedimentation coefficient of 9.3 S and Stokes' radius of 13 nm and exists as a dimer of approximately 500,000 daltons (Mr). Isolated 220 Kd binds to actin filaments in vitro when assayed by rotary shadowing. Morphological criteria distinguish 220 Kd fromDictyosteliummyosin II heavy chain (215 Kd) and the filaminlike protein at 240 Kd. The 70 Kd polypeptide appears to be a cleavage fragment of the 220 Kd, since it is found after prolonged storage when formerly only the 220 Kd was present. Furthermore, the 220 and 70 Kd polypeptides exhibit similar one‐dimensional peptide maps when treated with TPCK trypsin. On the basis of its physical and immunoreactive characteristics, and location in the cell, the 220 Kd may be a fodrinlike protein.