Hauser K, Pavlovic N, Kissmehl R, Plattner H
Faculty of Biology, University of Konstanz, P.O. Box 5560, D-78434 Konstanz, Germany.
Biochem J. 1998 Aug 15;334 ( Pt 1)(Pt 1):31-8. doi: 10.1042/bj3340031.
A cDNA encoding the gene for a sarco(endo)plasmic reticulum-type Ca2+-ATPase (SERCA) was isolated from a cDNA library of Paramecium tetraurelia by using degenerated primers according to conserved domains of SERCA-type ATPases. The identified nucleotide sequence (PtSERCA) is 3114 nucleotides in length with an open reading frame of 1037 amino acids. An intron of only 22 nucleotides occurs. Homology searches for the deduced amino acid sequence revealed 38-49% similarity to SERCA-type ATPases from organisms ranging from protozoans to mammals, with no more similarity to some parasitic protozoa of the same phylum. The calculated molecular mass of the encoded protein is 114.7 kDa. It contains the typical 10 transmembrane domains of SERCA-type ATPases and other conserved domains, such as the phosphorylation site and the ATP binding site. However, there are no binding sites for phospholamban and thapsigargin present in the PtSERCA. Antibodies raised against a cytoplasmic loop peptide between the phosphorylation site and the ATP binding site recognize on Western blots a protein of 106 kDa, exclusively in the fraction of sub-plasmalemmal calcium stores ('alveolar sacs'). In immunofluorescence studies the antibodies show labelling exclusively in the cell cortex of permeabilized cells in a pattern characteristic of the arrangement of alveolar sacs. When alveolar sacs where tested for phosphoenzyme-intermediate formation a phosphoprotein of the same molecular mass (106 kDa) could be identified.
根据肌浆网型Ca2+ -ATP酶（SERCA）的保守结构域，使用简并引物从四膜虫的cDNA文库中分离出编码该基因的cDNA。鉴定出的核苷酸序列（PtSERCA）长度为3114个核苷酸，开放阅读框为1037个氨基酸。仅存在一个22个核苷酸的内含子。对推导的氨基酸序列进行同源性搜索发现，与从原生动物到哺乳动物的生物的SERCA型ATP酶有38%-49%的相似性，与同一门的一些寄生原生动物没有更多相似性。计算得出的编码蛋白分子量为114.7 kDa。它包含SERCA型ATP酶典型的10个跨膜结构域以及其他保守结构域，如磷酸化位点和ATP结合位点。然而，PtSERCA中不存在受磷蛋白调节和毒胡萝卜素的结合位点。针对磷酸化位点和ATP结合位点之间的胞质环肽产生的抗体在蛋白质印迹上识别出一种106 kDa的蛋白质，仅在质膜下钙储存（“肺泡囊”）部分出现。在免疫荧光研究中，抗体仅在通透细胞的细胞皮质中显示出标记，其模式具有肺泡囊排列的特征。当检测肺泡囊的磷酸酶中间体形成时，可以鉴定出相同分子量（106 kDa）的磷蛋白。
