Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells.
Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells.
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DOI：
10.1083/jcb.106.4.1141
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发表时间：
1988-04
期刊：
影响因子：
--
通讯作者：
Mooseker MS
中科院分区：
文献类型：
--
作者：
Anderson JM;Stevenson BR;Jesaitis LA;Goodenough DA;Mooseker MS
ZO-1最初通过mAb技术鉴定，是第一个显示与紧密连接特异性相关的蛋白质。在这里，我们描述和比较的物理特性ZO-1从小鼠肝脏和Madin-Darby犬肾（MDCK）上皮细胞系。通过SDS-PAGE/免疫印迹分析测定，ZO-1多肽在小鼠组织中的表观大小为225 kD，在犬来源的MDCK细胞中的表观大小为210 kD。ZO-1从这两个来源是最佳的溶解从分离的质膜通过6 M尿素或高pH值条件;部分溶解发生与0.3 M KCl。非离子去污剂Triton X-100和辛基-β-D-吡喃葡萄糖苷不增溶ZO-1。这些溶解性表明ZO-1是一种外周相关的膜蛋白。通过凝胶过滤和免疫亲和层析的组合，从[35S]蛋氨酸代谢标记的MDCK细胞中纯化ZO-1至电泳均一。纯化的ZO-1具有5.3的s20，w和8.6nm的斯托克斯半径。这些值表明纯化的ZO-1是不对称单体分子。相应的值为小鼠肝脏ZO-1，其特征在于在不纯的蛋白质提取物，分别为6 s20，w和9 nm。ZO-1被证明是一个磷蛋白在MDCK细胞代谢标记的[32 P]正磷酸盐，从纯化的ZO-1磷酸氨基酸分析显示只有磷酸丝氨酸。ZO-1表位数通过Scatchard分析两种不同的125I-mAb与来自固定在硝酸纤维素上的肝和MDCK细胞的SDS溶解的蛋白质的竞争性和饱和结合来确定。饱和结合发生在26 ng mAb/mg肝脏和63 ng/mg MDCK细胞蛋白。假设单个表位/ZO-1分子，这相当于每个MDCK细胞30，000个ZO-1分子。
ZO-1, originally identified by mAb techniques, is the first protein shown to be specifically associated with the tight junction. Here we describe and compare the physical characteristics of ZO-1 from mouse liver and the Madin-Darby canine kidney (MDCK) epithelial cell line. The ZO-1 polypeptide has an apparent size of 225 kD in mouse tissues and 210 kD in canine-derived MDCK cells as determined by SDS- PAGE/immunoblot analysis. ZO-1 from both sources is optimally solubilized from isolated plasma membranes by either 6 M urea or high pH conditions; partial solubilization occurs with 0.3 M KCl. The nonionic detergents, Triton X-100 and octyl-beta-D-glucopyranoside, do not solubilize ZO-1. These solubility properties indicate that ZO-1 is a peripherally associated membrane protein. ZO-1 was purified to electrophoretic homogeneity from [35S]methionine metabolically labeled MDCK cells by a combination of gel filtration and immunoaffinity chromatography. Purified ZO-1 has an s20,w of 5.3 and Stokes radius of 8.6 nm. These values suggest that purified ZO-1 is an asymmetric monomeric molecule. Corresponding values for mouse liver ZO-1, characterized in impure protein extracts, were 6 s20,w and 9 nm. ZO-1 was shown to be a phosphoprotein in MDCK cells metabolically labeled with [32P]orthophosphate; analysis of phosphoamino acids from purified ZO-1 revealed only phosphoserine. ZO-1 epitope number was determined by Scatchard analysis of competitive and saturable binding of two different 125I-mAbs to SDS-solubilized proteins from liver and MDCK cells immobilized on nitrocellulose. Saturation binding occurs at 26 ng mAb/mg liver and 63 ng/mg of MDCK cell protein. This is equivalent to 30,000 ZO-1 molecules per MDCK cell assuming a single epitope/ZO-1 molecule.
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影响因子：
64.8
作者：
HORWITZ, A;DUGGAN, K;BURRIDGE, K
通讯作者：
BURRIDGE, K
影响因子：
--
作者：
Ackers, G K
通讯作者：
Ackers, G K
影响因子：
11.4
作者：
VAN MEER, G;SIMONS, K
通讯作者：
SIMONS, K
DOI：
10.1083/jcb.101.2.506
发表时间：
1985-08
期刊：
The Journal of cell biology
影响因子：
--
作者：
Jones JC;Goldman RD
通讯作者：
Goldman RD
DOI：
10.1083/jcb.104.4.905
发表时间：
1987-04
期刊：
The Journal of cell biology
影响因子：
--
作者：
Vega-Salas DE;Salas PJ;Gundersen D;Rodriguez-Boulan E
通讯作者：
Rodriguez-Boulan E