Use of ionophore A23187 to measure cytoplasmic Ca buffering and activation of the Ca pump by internal Ca
Use of ionophore A23187 to measure cytoplasmic Ca buffering and activation of the Ca pump by internal Ca
复制标题
使用离子载体 A23187 测量细胞质 Ca 缓冲和内部 Ca 对 Ca 泵的激活
作者：
H. G. Ferreira;V. Lew
在成熟的哺乳动物红细胞形成过程中，网织红细胞的细胞核、内质网和线粒体脱落，留下一个无特征的细胞质，细胞质中血红蛋白含量高，被质膜包围。所有的钙积累细胞器丢失和成熟的红细胞保持几乎无钙在其整个生命1的低钙渗透性的膜2可能有助于一个强大的钙挤压泵3。我们对红细胞中钙转运的大部分知识来自于对重封血影的研究，但在这种制备中，结果往往受到细胞膜和代谢改变的影响2 -4。在生理条件下使用完整的新鲜细胞的主要困难是不可能以受控的方式增加它们的Ca含量和评估电离的Ca的分数。然而，使用二价阳离子离子载体A23187（参考文献5），我们克服了这些困难，发现细胞质钙缓冲的发生就好像细胞有一个单一的大容量，低亲和力的钙缓冲液，有两个钙转运网站的亲和力相等的钙泵的内表面。
DURING the formation of the mature mammalian erythrocyte, the reticulocyte sheds its nucleus, endoplasmic reticulum and mitochondria leaving a featureless cytoplasm with a high haemoglobin content surrounded by a plasma membrane. All Ca-accumulating organelles are lost and the mature red cell is maintained virtually Ca-free throughout its life1 by the low Ca permeability of its membrane2 perhaps aided by a powerful Ca-extrusion pump3. Most of our knowledge of Ca transport in red cells comes from studies with resealed ghosts, but in this preparation the results are often affected by alterations to the membrane and metabolism of the cell2–4. The main difficulty of using intact fresh cells in physiological conditions has been the impossibility of increasing their Ca content in a controlled way and of assessing the fraction of Ca which is ionised. Using a divalent-cation ionophore, A23187 (ref. 5), however, we have overcome these difficulties and found that cytoplasmic Ca buffering occurs as if the cell had a single large-capacity, low-affinity Ca buffer and that there are two Ca-translocating sites of equal affinity at the internal surface of the Ca pump.