Calmodulin activation of the Ca2+ pump revealed by fluorescent chelator dyes in human red blood cell ghosts.
Calmodulin activation of the Ca2+ pump revealed by fluorescent chelator dyes in human red blood cell ghosts.
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DOI：
10.1085/jgp.99.1.41
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发表时间：
1992-01
期刊：
影响因子：
--
通讯作者：
James-Kracke MR
中科院分区：
文献类型：
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作者：
James-Kracke MR
在红细胞血影中的Ca2+转运用fura 2或quin2作为游离酸在重新密封期间掺入来监测。这是第一次报告的主动运输监测的荧光强度的螯合剂染料fura 2（5 - 50微M）或quin2（250微M）在血红蛋白耗尽的鬼。由于血影中没有细胞内区室，并且可以设置所有测定螯合剂物质（包括钙调蛋白（CaM）、染料和ATP）的细胞内浓度，因此可以计算转运期间游离和总Ca [（Cafree] i和[Catotal] i）的细胞内浓度。用或不用CaM制备的鬼快速挤出Ca2+至60 - 100 nM的稳态浓度。在含有1 mM Ca2+的培养基中常规产生Ca2+的10（4）倍梯度。在活性Ca 2+挤出过程中，d [Cafree] i/dt是[Cafree] i的二阶函数，并且与染料浓度无关，而d [Catotal] i/dt作为[Cafree] i和Ca：染料复合物浓度的一阶函数增加。CaM（5 μ M）在1 μ M [Cafree] i时使d [Catotal] i/dt增加400%，而d [Cafree] i/dt仅增加25%。从一系列的实验中，我们得出结论，螯合形式的Ca 2+作为基板的泵下允许控制的[Cafree] i，这种双重效应可以解释协同性。游离的Ca2+被挤出，并且可能也与CaM或其他螯合剂结合的Ca2+，而CaM和螯合剂保留在细胞中。
Ca2+ transport in red blood cell ghosts was monitored with fura2 or quin2 incorporated as the free acid during resealing. This is the first report of active transport monitored by the fluorescent intensity of the chelator dyes fura2 (5-50 microM) or quin2 (250 microM) in hemoglobin-depleted ghosts. Since there are no intracellular compartments in ghosts and the intracellular concentrations of all assay chelator substances including calmodulin (CaM), the dyes, and ATP could be set, the intracellular concentrations of free and total Ca [( Cafree]i and [Catotal]i) could be calculated during the transport. Ghosts prepared with or without CaM rapidly extruded Ca2+ to a steady- state concentration of 60-100 nM. A 10(4)-fold gradient for Ca2+ was routinely produced in medium containing 1 mM Ca2+. During active Ca2+ extrusion, d[Cafree]i/dt was a second order function of [Cafree]i and was independent of the dye concentration, whereas d[Catotal]i/dt increased as a first order function of both the [Cafree]i and the concentration of the Ca:dye complex. CaM (5 microM) increased d[Catotal]i/dt by 400% at 1 microM [Cafree]i, while d[Cafree]i/dt increased by only 25%. From a series of experiments we conclude that chelated forms of Ca2+ serve as substrates for the pump under permissive control of the [Cafree]i, and this dual effect may explain cooperativity. Free Ca2+ is extruded, and probably also Ca2+ bound to CaM or other chelators, while CaM and the chelators are retained in the cell.