Nørregaard A, Vilsen B, Andersen J P
Danish Biomembrane Research Centre, Institute of Physiology, University of Aarhus.
J Biol Chem. 1994 Oct 28;269(43):26598-601.
Five different chimeric fusion proteins between the sarcoplasmic reticulum Ca(2+)-ATPase and the rat kidney alpha 1 isoform of the Na+,K(+)-ATPase were expressed in COS-1 cells and analyzed functionally. In two chimeras, the only Na+,K(+)-ATPase-derived part consisted of the region corresponding to the third transmembrane segment. These proteins phosphorylated from ATP in the presence of a high Ca2+ concentration and from Pi in the absence of Ca2+, but the phosphorylation displayed strongly reduced sensitivity to inhibition by thapsigargin. In two other chimeras, the N-terminal two-thirds of the molecule were derived from Na+,K(+)-ATPase, except the region containing the fourth transmembrane segment. These proteins were unable to phosphorylate from ATP but phosphorylated from Pi, the latter reaction being insensitive to inhibition by thapsigargin. In the last chimera, the N-terminal two-thirds of the molecule were derived from Na+,K(+)-ATPase, except the region corresponding to both the third and the fourth transmembrane segments. This chimera phosphorylated from Pi in the reaction that was fully sensitive to inhibition by thapsigargin. It can be concluded that the third transmembrane segment is indispensable to thapsigargin sensitivity of the Ca(2+)-ATPase and most likely, therefore, constitutes a major binding region for thapsigargin.
在COS-1细胞中表达了肌浆网Ca(2+)-ATP酶与大鼠肾脏Na+,K(+)-ATP酶α1亚型之间的五种不同嵌合融合蛋白，并对其功能进行了分析。在两种嵌合体中，唯一源自Na+,K(+)-ATP酶的部分由对应于第三个跨膜段的区域组成。这些蛋白在高Ca2+浓度存在时从ATP磷酸化，在无Ca2+时从Pi磷酸化，但磷酸化对毒胡萝卜素抑制的敏感性显著降低。在另外两种嵌合体中，分子的N端三分之二源自Na+,K(+)-ATP酶，但不包括包含第四个跨膜段的区域。这些蛋白不能从ATP磷酸化，但能从Pi磷酸化，后一反应对毒胡萝卜素抑制不敏感。在最后一种嵌合体中，分子的N端三分之二源自Na+,K(+)-ATP酶，但不包括对应于第三个和第四个跨膜段的区域。这种嵌合体在对毒胡萝卜素抑制完全敏感的反应中从Pi磷酸化。可以得出结论，第三个跨膜段对于Ca(2+)-ATP酶对毒胡萝卜素的敏感性是不可或缺的，因此很可能构成了毒胡萝卜素的主要结合区域。
