Kissmehl R, Huber S, Kottwitz B, Hauser K, Plattner H
Department of Biology, University of Konstanz, Germany.
Cell Calcium. 1998 Sep;24(3):193-203. doi: 10.1016/s0143-4160(98)90128-2.
Considering increasing interest in calcium stores in protozoa, including parasitic forms, and specifically in subplasmalemmal stores in higher eukaryotes, we have isolated subplasmalemmal calcium stores (alveolar sacs) from the ciliated protozoan, Paramecium tetraurelia. Using antibodies against established sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCAs) we detected in Western blots of subcellular fractions a band of approximately 106 kDa size selectively in alveolar sacs--but not, for example, in plasma membranes--and concomitant restriction of immunofluorecence labelling to the cell cortex of permeabilised cells. These results are the same as with ABs against a peptide derived from a cloned SERCA-like gene from Paramecium [Hauser K., Pavlovic N., Kissmehl R., Plattner H. Molecular characterization of a sarco(endo)plasmic reticulum Ca(2+)-ATPase gene from Paramecium tetraurelia and localisation of its gene product to subplasmalemmal calcium stores. Biochem J 1998; 334: 31-38]. When such isolated alveolar sacs were now tested for phosphoenzyme intermediate (EP) formation, a phosphoprotein of the same apparent molecular mass (approximately 106 kDa) as in blots could be identified in gel autoradiograms. This EP corresponds to that formed in the reaction cycle of different SERCA-types, with dependency on Ca2+ and Mg2+, sensitivity to La3+ or insensitivity towards calmodulin, calmodulin antagonists and vanadate. However, EP formation in alveolar sacs is not inhibited by established SERCA inhibitors (e.g. thapsigargi[ci]n tested up to 100 microM). Surprisingly, caffeine, which is frequently used to mobilise Ca2+ from intracellular stores, strongly inhibits EP formation. In parallel experiments, we did not find any similar effect with sarcoplasmic reticulum isolated from skeletal muscle. We conclude that the approximately 106 kDa protein of alveolar sacs in Paramecium may represent a SERCA-like Ca(2+)-ATPase with some unorthodox features, which might be relevant also for some other protozoan systems. In this case, the established Ca(2+)-mobilizing effect of caffeine may be amplified by inhibiting store refilling.
鉴于对原生动物（包括寄生形式）钙储存的兴趣日益增加，特别是对高等真核生物中质膜下储存的关注，我们从纤毛原生动物四膜虫中分离出了质膜下钙储存（肺泡囊）。使用针对已确定的肌质（内质）网Ca(2+)-ATP酶（SERCAs）的抗体，我们在亚细胞组分的蛋白质印迹中检测到一条约106 kDa大小的条带，该条带仅在肺泡囊中选择性出现——例如，不在质膜中出现——并且免疫荧光标记仅限于通透细胞的细胞皮质。这些结果与用针对来自四膜虫的克隆的类SERCA基因衍生的肽的抗体得到的结果相同[豪泽K.，帕夫洛维奇N.，基斯梅尔R.，普拉特纳H.四膜虫肌质（内质）网Ca(2+)-ATP酶基因的分子特征及其基因产物在质膜下钙储存中的定位。生物化学杂志1998；334：31 - 38]。当现在对这种分离的肺泡囊进行磷酸酶中间体（EP）形成测试时，在凝胶放射自显影片中可以鉴定出一种与印迹中表观分子量相同（约106 kDa）的磷蛋白。这种EP与不同SERCA类型反应循环中形成的EP相对应，依赖于Ca2+和Mg2+，对La3+敏感或对钙调蛋白、钙调蛋白拮抗剂和钒酸盐不敏感。然而，肺泡囊中EP的形成不受已确定的SERCA抑制剂（例如高达100 microM的毒胡萝卜素）的抑制。令人惊讶的是，常用于从细胞内储存中动员Ca2+的咖啡因强烈抑制EP的形成。在平行实验中，我们在从骨骼肌分离的肌质网中未发现任何类似的作用。我们得出结论，四膜虫肺泡囊中约106 kDa的蛋白质可能代表一种具有一些非正统特征的类SERCA Ca(2+)-ATP酶，这可能对其他一些原生动物系统也有意义。在这种情况下，咖啡因已确定的Ca(2+)动员作用可能通过抑制储存再填充而被放大。
