The structural unit of the secretory Na+-K+-2Cl- cotransporter (NKCCl) is a homodimer
The structural unit of the secretory Na+-K+-2Cl- cotransporter (NKCCl) is a homodimer
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DOI：
10.1021/bi992301v
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发表时间：
2000-04-04
期刊：
影响因子：
2.9
通讯作者：
Turner, RJ
中科院分区：
文献类型：
--
作者：
Moore-Hoon, ML;Turner, RJ
采用可逆化学交联剂DTSSP[3,3'-二硫代比斯-（磺基琥珀酰丙酸）]研究了大鼠腮腺质膜分泌Na+- k +- 2cl（-）共转运体（NKCCl）的寡聚状态。NKCCl的单体表观分子质量约为170 kDa。然而，我们在这里发现，在DTSSP膜处理后，该蛋白以类似于355 kDa复合物的形式在SDS-PAGE凝胶上迁移，这表明NKCCl以低聚物的形式存在于质膜中。低浓度的非离子洗涤剂Triton X-100（0.3%）或温和的离子洗涤剂脱氧胆酸盐（20mm）对膜的增溶作用不会破坏这种低聚物的稳定性；然而，较高浓度的Triton X-100或变性洗涤剂SDS处理确实会导致NKCCl复合物的不稳定。在其他实验中，我们从生物素化膜中免疫沉淀355 kDa交联复合物，然后切割交联弯曲，并通过亲和素印迹分析得到的NKCCl低聚物成分。银染色和二维电泳。在这些研究中，我们无法在355 kDa寡聚物中检测到除了NKCCl本身之外的任何蛋白质的存在，这表明该复合物是NKCCl二聚体。通过对全长NKCCl和在HEK293细胞中表达的n端截断版NKCCl形成的寡聚物的分子大小的定量分析，为这一结论提供了有力的证据。综上所述，我们的数据提供了令人信服的证据，证明NKCCl在质膜中的主要结构单元是同型二聚体。
The oligomeric state of the secretory Na+-K+-2Cl(-) cotransporter (NKCCl) in rat parotid plasma membranes was studied using the reversible chemical cross-linker DTSSP [3,3'-dithiobis-(sulfosuccinimidyl propionate)]. The monomeric apparent molecular mass of NKCCl is similar to 170 kDa. However, we show here that this protein migrates as a similar to 355 kDa complex on SDS-PAGE gels after membrane treatment with DTSSP, indicating that NKCCl exists as an oligomer in the plasma membrane. The stability of this oligomer is such that it is not disrupted by solubilization of the membrane by Low concentrations of the nonionic detergent Triton X-100 (0.3%) or the mild ionic detergent deoxycholate (20 mM); however, higher concentrations of Triton X-100 or treatment with the denaturing detergent SDS do result in destabilization of the NKCCl complex. In additional experiments, we immunoprecipitated the 355 kDa cross-linked complex from biotinylated membranes, then cleaved the cross-linking bends and analyzed the resulting components of the NKCCl oligomer by avidin blotting? silver staining, and 2D electrophoresis. In these studies, we were unable to detect the presence of any proteins other than NKCCl itself in the 355 kDa oligomer, suggesting that this complex is tin NKCCl dimer. Strong evidence for this conclusion was provided by a quantitative analysis of the molecular sizes of oligomers formed by full-length NKCCl and an N-terminally truncated version of NKCCl expressed in HEK293 cells. Taken together, our data provide convincing evidence that the dominant structural unit of NKCCl in the plasma membrane is a homodimer.