Preianò B S, Guerini D, Carafoli E
Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Zürich, Switzerland.
Biochemistry. 1996 Jun 18;35(24):7946-53. doi: 10.1021/bi9527404.
PMCA isoforms 4CII (generated by splicing at the C-terminus) and 4BICI (a pump version lacking the 10th transmembrane domain) were expressed in Sf9 cells using the baculovirus system. The purified PMCA4CII had a 20-fold lower affinity for calmodulin than the PMCA4CI, the PMCA4 isoform of the erythrocytes' membranes, but had a higher activity in the absence of calmodulin. The amount of phosphoenzyme intermediate formed by PMCA4CII in the presence of Ca2+ alone was almost 3 times higher than in PMCA4CI and was increased by La3+ less than in the PMCA4CI. The isoform lacking the 10th transmembrane domain (PMCA4BICI) had no Ca2+-dependent ATPase activity, but was still able to form the phosphoenzyme intermediate starting from phosphate. When expressed in COS cells, this isoform was retained in the endoplasmic reticulum; changes in membrane architecture apparently occurred during its expression; the C-terminal portion of the isoform was located in the cytosol, indicating that the deletion of the 10th transmembrane domain resulted in the loss of at least another transmembrane domain.
使用杆状病毒系统在Sf9细胞中表达了PMCA亚型4CII（通过C末端剪接产生）和4BICI（一种缺少第10个跨膜结构域的泵变体）。纯化后的PMCA4CII对钙调蛋白的亲和力比红细胞膜的PMCA4亚型PMCA4CI低20倍，但在没有钙调蛋白的情况下具有更高的活性。仅在Ca2+存在下，PMCA4CII形成的磷酸酶中间体的量几乎是PMCA4CI的3倍，并且La3+对其的增加作用小于对PMCA4CI的作用。缺少第10个跨膜结构域的亚型（PMCA4BICI）没有Ca2+依赖性ATP酶活性，但仍能够从磷酸盐开始形成磷酸酶中间体。当在COS细胞中表达时，该亚型保留在内质网中；在其表达过程中显然发生了膜结构的变化；该亚型的C末端部分位于胞质溶胶中，这表明第10个跨膜结构域的缺失导致至少另一个跨膜结构域的丧失。
