N-乙基马来酰亚胺对人红细胞血影中哇巴因不敏感阳离子通量的影响。

Effects of N-ethylmaleimide on ouabain-insensitive cation fluxes in human red cell ghosts.

作者信息

Smith D K, Lauf P K

出版信息

Biochim Biophys Acta. 1985 Aug 27;818(2):251-9. doi: 10.1016/0005-2736(85)90565-6.

Abstract

In red cells of several species, the sulfhydryl reagent N-ethylmaleimide activates a Cl- -dependent, ouabain-resistant K+ transport pathway. Here we report our attempts to demonstrate ouabain-resistant Cl- -dependent K+ fluxes stimulated by N-ethylmaleimide in resealed human red cell ghosts using Rb+ as a K+ analogue. In contrast to intact cells, the rate constants of the base level Rb+ efflux in ghosts were similar in NaNO3 and NaCl (okRb = 0.535 +/- 0.079 h-1 and 0.534 +/- 0.085 h-1, respectively), while 1 mM N-ethylmaleimide stimulated Rb+ efflux strongly in NaNO3 (okRb = 14.26 +/- 1.32 h-1) and moderately in NaCl (okRb = 2.73 +/- 0.54 h-1). This effect was dependent on the presence of internal ATP. Stimulation of Rb+ efflux was observed in the presence of greater than or equal to 0.2 mM N-ethylmaleimide and increased at pH values approaching 8.0, consistent with titration of SH groups. N-Ethylmaleimide-stimulated Rb+ efflux was approx. 50% inhibited by 100 microM quinine sulfate whereas 1 microM bumetanide had no effect. In NaCl the N-ethylmaleimide-stimulated efflux saturated with initial internal ghost Rb+ concentration, but rates increased linearly in NaNO3. Replacement of external Na+ with glucamine or choline decreased the N-ethylmaleimide-stimulated Rb+ efflux, suggesting a role for external Na+. N-Ethylmaleimide-stimulated Rb+ efflux was greater in buffers with lipophilic anions such as SCN- or NO3- than in solutions with Cl- or acetate. However, the cation selectivity of the pathway studied was low, as Li+ efflux was also stimulated by N-ethylmaleimide. We conclude that the effect of N-ethylmaleimide on ouabain-resistant cation effluxes of human red cell ghosts is very different from the selective action of N-ethylmaleimide on Rb+ influxes in intact red cells.

摘要

在几种物种的红细胞中，巯基试剂N - 乙基马来酰亚胺激活了一种依赖于Cl⁻且对哇巴因耐药的K⁺转运途径。在此我们报告我们尝试使用Rb⁺作为K⁺类似物，在重封的人红细胞血影中证明N - 乙基马来酰亚胺刺激的依赖于Cl⁻且对哇巴因耐药的K⁺通量。与完整细胞不同，血影中基础水平的Rb⁺外流速率常数在NaNO₃和NaCl中相似（分别为okRb = 0.535 ± 0.079 h⁻¹和0.534 ± 0.085 h⁻¹），而1 mM N - 乙基马来酰亚胺在NaNO₃中强烈刺激Rb⁺外流（okRb = 14.26 ± 1.32 h⁻¹），在NaCl中适度刺激（okRb = 2.73 ± 0.54 h⁻¹）。这种效应依赖于细胞内ATP的存在。在存在大于或等于0.2 mM N - 乙基马来酰亚胺时观察到Rb⁺外流受到刺激，并且在pH值接近8.0时增加，这与SH基团的滴定一致。N - 乙基马来酰亚胺刺激的Rb⁺外流约50%被100 μM硫酸奎宁抑制，而1 μM布美他尼没有作用。在NaCl中，N - 乙基马来酰亚胺刺激的外流随初始细胞内血影Rb⁺浓度饱和，但在NaNO₃中速率呈线性增加。用葡糖胺或胆碱替代外部Na⁺会降低N - 乙基马来酰亚胺刺激的Rb⁺外流，表明外部Na⁺起作用。在含有亲脂性阴离子如SCN⁻或NO₃⁻的缓冲液中，N - 乙基马来酰亚胺刺激的Rb⁺外流比在含有Cl⁻或乙酸盐的溶液中更大。然而，所研究途径的阳离子选择性较低，因为Li⁺外流也受到N - 乙基马来酰亚胺的刺激。我们得出结论，N - 乙基马来酰亚胺对人红细胞血影中对哇巴因耐药的阳离子外流的影响与N - 乙基马来酰亚胺对完整红细胞中Rb⁺内流的选择性作用非常不同。