Isolation and characterization of circumferential microfilament bundles from retinal pigmented epithelial cells.
Isolation and characterization of circumferential microfilament bundles from retinal pigmented epithelial cells.
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DOI：
10.1083/jcb.95.1.310
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发表时间：
1982-10
期刊：
影响因子：
--
通讯作者：
Masuda H
中科院分区：
文献类型：
--
作者：
Owaribe K;Masuda H
鸡视网膜色素上皮细胞在粘附小带区域具有周向微丝束（CMB）。我们已经分离出这些CMB在完整的形式，其结构和生化特征。用甘油和Triton处理从11日龄鸡胚获得的色素上皮细胞。然后，将上皮细胞通过注射器针头进行均质化。相差显微镜观察发现，在匀浆中有许多孤立的CMB。它们形成多边形，大多数是五边形和六边形，或者多边形的碎片。多孔板充满了网状结构，即它们是多边形板。暴露于Mg-ATP后，分离的CMB显示出明显的和大的收缩。用N-乙基马来酰亚胺修饰的肌球蛋白亚片段-1处理可抑制收缩。经Percoll密度梯度离心纯化后，用SDS PAGE分析CMB。电泳图谱给出了200、55和42 kdalton的三种主要组分和几种次要组分。电子显微镜观察表明，多边形的肌动蛋白包含微丝的粗束，和网络主要由中间丝。
Chicken retinal pigmented epithelial cells have circumferential microfilament bundles (CMBs) at the zonula adherens region. We have isolated these CMBs in intact form and characterized them structurally and biochemically. Pigmented epithelia obtained from 11-d-old chick embryos were treated with glycerol and Triton. Then, the epithelia were homogenized by passing them through syringe needles. Many isolated CMBs were found in the homogenate by phase-contrast microscopy. They formed polygons, mostly pentagons and hexagons, or fragments of polygons. Polygons were filled with meshwork structures, i.e. they were polygonal plates. Upon exposure to Mg-ATP, isolated CMBs showed clear and large contraction. The contraction was inhibited by treatment with N- ethylmaleimide-modified myosin subfragment-1. After purification by centrifugation with the density gradient of Percoll, CMBs were analyzed by SDS PAGE. The electrophoretic pattern gave three major components of 200, 55, and 42 kdaltons and several minor components. Electron microscopy showed that the polygons were composed of thick bundles of actin-containing microfilaments, and the meshworks were composed primarily of intermediate filaments.