Plasmalemmal caveolae and intracellular calcium regulation

1)发明了一种简便、快速的观察单层培养细胞质膜的方法。金属夹心法简单，不需要任何昂贵的设备，并为后续的组织化学操作提供了较大的质膜破裂面。2)将胆固醇结合毒素Rheta毒素(Perringolysin O)部分蛋白分解和生物素化(BChita)，作为细胞化学探针。由于质膜在与BChita结合后仍保持完整，该探针不仅可用于固定细胞的细胞化学标记，还可用于追踪活细胞中交联的胆固醇分子的行为。通过使用这一新的探针，揭示了质膜中的交联胆固醇被隔离到小窝中。3)通过免疫电子显微镜，关键地评估了Gi2a是否结构性地集中在小窝中。首先，重新考察了小窝蛋白和Gi2a在密度平衡超速离心法中的行为。通过有效地收集组分，发现洞穴和Gi2a的分布不同。其次，通过新的免疫细胞化学方法发现，Gi2a在小窝中的标记密度是非小窝质膜的2.29倍。结果表明，小窝中Gi2a的浓度低于大多数生化研究的结果。

1) A simple quick freezing method to observe the plasma membrane from monolayr cultured cells was invented. The metal sandwich method is simple, does not require any expensive equipment, and provides a large fracture plane of the plasma membrane for subsequent histochemical manipulation.2) rheta-Toxin (perfringolysin O), a cholesterol-binding toxin, was partially proteolysed and biotinylated (BCrheta) and used as a cytochemical probe. Because the plasma membrane keeps integrity even after the binding of BCrheta, the probe can be used not only for cytochemical labeling of fixed cells, but for pursuing the behavior of cross-linked cholesterol molecules in living cells. By using this new probe, it was revealed that cross-linked cholesterol in the plasma membrane are sequestered to caveolae.3) By immunoelectron microscopy, whether Gi2a is constitutively concentrated in caveolae was critically assessed. First, behavior of caveolin and Gi2a in density-equilibrium ultracentrifugation was reexamined. By collecting fractions efficiently, caveolin and Gi2a were found to distribute differently. Secondly, by novel immunocytochemical methods it was found that the labeling density of Gi2a was 2.29 times more in caveolae than in the non-caveolar plasma membrane. The results indicate that concentration of Gi2a in caveolae is less than deduced from most biochemical studies.