Hilfiker H, Guerini D, Carafoli E
Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Zürich.
J Biol Chem. 1994 Oct 21;269(42):26178-83.
Full-length cDNAs for the three human plasma membrane Ca2+ pump isoforms 2 (PMCA2) differently spliced at the A site were constructed and transferred to baculovirus. The corresponding proteins were expressed after infection in Sf9 insect cells. The proteins were expressed at high levels and retained the canonical properties of the plasma membrane Ca2+ pump. The alternative splicing process failed to produce functional differences detectable with the methods used. The Ca(2+)-dependent ATPase activity of the PMCA2 pumps had a 5-10-fold higher affinity for calmodulin than the PMCA4 pump expressed in the same system. Experiments on the formation of the phosphoenzyme intermediate from ATP revealed that the PMCA2 pumps had higher affinity for ATP than did the PMCA4 counterpart. The response of the two pump types to activating acidic phospholipids was the same.
构建了在A位点存在不同剪接的三种人质膜Ca2+泵同工型2（PMCA2）的全长cDNA，并将其转移到杆状病毒中。感染Sf9昆虫细胞后表达出相应的蛋白质。这些蛋白质高水平表达，并保留了质膜Ca2+泵的典型特性。替代剪接过程未能产生用所用方法可检测到的功能差异。与在同一系统中表达的PMCA4泵相比，PMCA2泵的Ca(2+)依赖性ATP酶活性对钙调蛋白的亲和力高5至10倍。由ATP形成磷酸化酶中间体的实验表明，PMCA2泵对ATP的亲和力高于PMCA4对应物。两种泵类型对激活酸性磷脂的反应相同。
