Nørregaard A, Vilsen B, Andersen J P
Danish Biomembrane Research Centre, Institute of Physiology, University of Aarhus.
FEBS Lett. 1993 Dec 27;336(2):248-54. doi: 10.1016/0014-5793(93)80813-a.
Chimeric molecules consisting of parts from the sarcoplasmic reticulum Ca(2+)-ATPase and the Na+,K(+)-ATPase were expressed in COS-1 cells and analysed functionally. One chimera, in which most of the central cytoplasmic loop was derived from the Na+,K(+)-ATPase, while the transmembrane segments and the minor cytoplasmic loop came from the Ca(2+)-ATPase, was able to occlude Ca2+ and to be phosphorylated from ATP with normal apparent affinity for Ca2+ and ATP. This chimera also displayed normal sensitivity to thapsigargin, but was unable to undergo the transition from ADP-sensitive to ADP-insensitive phosphoenzyme and to transport Ca2+. The other chimera, which consisted of the NH2-terminal two-thirds of Na+,K(+)-ATPase and the COOH-terminal one-third of Ca(2+)-ATPase, was unable to phosphorylate from ATP, but phosphorylated from inorganic phosphate in a Ca(2+)-inhibitable and thapsigargin-insensitive reaction. These results can be explained in terms of a structural model in which the non-conserved residues in the central cytoplasmic domain of the Ca(2+)-ATPase are without major importance for the binding and occlusion of Ca2+, but are involved in the E1P-->E2P conformational changes of the phosphoenzyme, whereas residues in transmembrane segments on both sides of the central cytoplasmic domain are involved in formation of the Ca(2+)-binding sites. The data moreover show that thapsigargin sensitivity is dependent on residues in the NH2-terminal one-third of the Ca(2+)-ATPase molecule.
由肌浆网Ca(2+)-ATP酶和Na+,K(+)-ATP酶的部分组成的嵌合分子在COS-1细胞中表达并进行了功能分析。一种嵌合体，其大部分中央胞质环来自Na+,K(+)-ATP酶，而跨膜片段和较小的胞质环来自Ca(2+)-ATP酶，能够封闭Ca2+并以对Ca2+和ATP正常的表观亲和力从ATP进行磷酸化。这种嵌合体对毒胡萝卜素也表现出正常的敏感性，但无法从ADP敏感的磷酸酶转变为ADP不敏感的磷酸酶，也不能转运Ca2+。另一种嵌合体由Na+,K(+)-ATP酶的氨基末端三分之二和Ca(2+)-ATP酶的羧基末端三分之一组成，不能从ATP进行磷酸化，但在Ca(2+)-抑制且对毒胡萝卜素不敏感的反应中从无机磷酸进行磷酸化。这些结果可以用一种结构模型来解释，即Ca(2+)-ATP酶中央胞质结构域中的非保守残基对Ca2+的结合和封闭没有重要影响，但参与磷酸酶的E1P→E2P构象变化，而中央胞质结构域两侧跨膜片段中的残基参与Ca(2+)-结合位点的形成。此外，数据表明毒胡萝卜素敏感性取决于Ca(2+)-ATP酶分子氨基末端三分之一中的残基。
