Stelly N, Mauger J P, Claret M, Adoutte A
Laboratoire de Biologie Cellulaire 4, Unité de Recherche Associée 1134 du Centre National de la Recherche Scientifique, Université Paris-Sud, Orsay, France.
J Cell Biol. 1991 Apr;113(1):103-12. doi: 10.1083/jcb.113.1.103.
The plasma membrane of Paramecium is underlain by a continuous layer of membrane vesicles known as cortical alveoli, whose function was unknown but whose organization had suggested some resemblance with muscle sarcoplasmic reticulum. The occurrence of antimonate precipitates within the alveoli first indicated to us that they may indeed correspond to a vast calcium storage site. To analyze the possible involvement of this compartment in calcium sequestration more directly, we have developed a new fractionation method, involving a Percoll gradient, that allows rapid purification of the surface layer (cortex) of Paramecium in good yield and purity and in which the alveoli retain their in vivo topological orientation. This fraction pumped calcium very actively in a closed membrane compartment, with strict dependence on ATP and Mg2+. The pumping activity was affected by anti-calmodulin drugs but no Triton-soluble calmodulin binding protein could be identified, using gel overlay procedures. The high affinity of the pump for calcium (Km = 0.5 microM) suggests that it plays an important role in the normal physiological environment of the cytosol. This may be related to at least three calcium-regulated processes that take place in the immediate vicinity of alveoli: trichocyst exocytosis, ciliary beating and cytoskeletal elements dynamics during division.
草履虫的质膜下方是一层连续的膜泡，称为皮质泡，其功能尚不清楚，但其结构显示出与肌肉肌质网有一些相似之处。泡内出现锑酸盐沉淀首先向我们表明，它们可能确实对应一个巨大的钙储存位点。为了更直接地分析这个区室在钙螯合中的可能作用，我们开发了一种新的分级分离方法，涉及Percoll梯度，该方法能够以良好的产量和纯度快速纯化草履虫的表层（皮质），并且泡在其中保持其体内拓扑方向。该级分在封闭的膜区室中非常活跃地泵送钙，严格依赖于ATP和Mg2+。泵送活性受抗钙调蛋白药物影响，但使用凝胶覆盖程序未鉴定出任何可溶于Triton的钙调蛋白结合蛋白。该泵对钙的高亲和力（Km = 0.5 microM）表明它在细胞质的正常生理环境中起重要作用。这可能与至少三个在泡附近发生的钙调节过程有关：刺丝泡胞吐作用、纤毛摆动以及分裂过程中的细胞骨架元件动态变化。
