Oh P, Schnitzer J E
Department of Pathology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA.
J Biol Chem. 1999 Aug 13;274(33):23144-54. doi: 10.1074/jbc.274.33.23144.
Defining the molecular composition of caveolae is essential in establishing their molecular architecture and functions. Here, we identify a high affinity monoclonal antibody that is specific for caveolin-1alpha and rapidly binds caveolin oligomerized around intact caveolae. We use this antibody (i) to develop a new simplified method for rapidly isolating caveolae from cell and tissue homogenates without using the silica-coating technology and (ii) to analyze various caveolae isolation techniques to understand how they work and why they yield different compositions. Caveolae are immunoisolated from rat lung plasma membrane fractions subjected to mechanical disruption. Sonication of plasma membranes, isolated with or without silica coating, releases caveolae along with other similarly buoyant microdomains and, therefore, requires immunoisolations to purify caveolae. Shearing of silica-coated plasma membranes provides a homogeneous population of caveolae whose constituents (i) remain unchanged after immunoisolation, (ii) all fractionate bound to the immunobeads, and (iii) appear equivalent to caveolae immunoisolated after sonication. The caveolae immunoisolated from different low density fractions are quite similar in molecular composition. They contain a subset of key signaling molecules (i.e. G protein and endothelial nitric oxide synthase) and are markedly depleted in glycosylphosphatidylinositol-anchored proteins, beta-actin, and angiotensin-converting enzyme. All caveolae isolated from the cell surface of lung microvascular endothelium in vivo appear to be coated with caveolin-1alpha. Caveolin-1beta and -2 can also exist in these same caveolae. The isolation and analytical procedures as well as the time-dependent dissociation of signaling molecules from caveolae contribute to key compositional differences reported in the literature for caveolae. This new, rapid, magnetic immunoisolation procedure provides a consistent preparation for use in the molecular analysis of caveolae.
确定小窝的分子组成对于构建其分子结构和功能至关重要。在此，我们鉴定出一种对小窝蛋白-1α具有特异性的高亲和力单克隆抗体，它能迅速结合在完整小窝周围寡聚化的小窝蛋白。我们利用这种抗体：（i）开发一种新的简化方法，无需使用二氧化硅包被技术即可从细胞和组织匀浆中快速分离小窝；（ii）分析各种小窝分离技术，以了解它们的工作原理以及为何会产生不同的组成成分。通过对大鼠肺质膜组分进行机械破碎来免疫分离小窝。对有或没有二氧化硅包被的质膜进行超声处理，会释放出小窝以及其他浮力相似的微区，因此需要进行免疫分离来纯化小窝。对二氧化硅包被的质膜进行剪切可得到均匀的小窝群体，其成分：（i）在免疫分离后保持不变；（ii）全部与免疫磁珠结合进行分级分离；（iii）与超声处理后免疫分离得到的小窝相当。从不同低密度组分免疫分离得到的小窝在分子组成上非常相似。它们包含一组关键信号分子（即G蛋白和内皮型一氧化氮合酶），并且糖基磷脂酰肌醇锚定蛋白、β-肌动蛋白和血管紧张素转换酶明显减少。体内从肺微血管内皮细胞表面分离的所有小窝似乎都被小窝蛋白-1α包被。小窝蛋白-1β和-2也可存在于这些相同的小窝中。分离和分析程序以及信号分子从小窝中的时间依赖性解离导致了文献中报道的小窝关键组成差异。这种新的、快速的磁性免疫分离程序为小窝的分子分析提供了一致的制备方法。
