Levy R, Livne A
Biochim Biophys Acta. 1984 Nov 7;777(2):157-66. doi: 10.1016/0005-2736(84)90417-6.
Human erythrocytes were treated by a series of SH-reagents, including maleimides, iodo compounds, mercurials and oxidizing agents. Rates of Li efflux into Na-rich medium, Li leak and Lii-Nao countertransport were then determined. Of the 13 different reagents studied, only N-ethylmaleimide, iodoacetamide and iodoacetate inhibited selectively the countertransport activity. The effect of the various reagents indicates that the sensitive SH-groups of the countertransport system are not externally exposed. N-Ethylmaleimide was used to probe for changes elicited by substrate cations in Lii-Nao countertransport. In Na- and Li-free medium, inhibition of Lii-Nao countertransport by N-ethylmaleimide of 35% was reached within 2 s. In Na or Li medium, maximal inhibition was twice as great, but was attained much more slowly, within 10 min. Kinetic data and Hill plot analysis indicate the involvement of two classes of SH-groups: one expressed in the various media with and without substrate cations, and an additional one, which becomes specifically available to N-ethylmaleimide in the presence of external Na or Li. The affinity of Na to the site promoting inhibition by N-ethylmaleimide (apparent Km = 12 mM) is higher than the affinity of Na to its external countertransport site (apparent Km = 25 mM, as reported by Sarakadi, B., Alifimoff, J.K., Gunn, R.B. and Tosteson, D.C. (1978) J. Gen. Physiol. 72, 249-265). Reactivity of N-ethyl[14C]maleimide was not modified by the media tested. It is concluded that external Na and Li cause a conformational change in the protein(s) of the countertransport system in human erythrocytes.
用人红细胞与一系列含巯基（SH）的试剂进行处理，这些试剂包括马来酰亚胺、碘化合物、汞制剂和氧化剂。然后测定锂向富含钠的介质中的流出速率、锂泄漏以及锂 - 钠逆向转运。在所研究的13种不同试剂中，只有N - 乙基马来酰亚胺、碘乙酰胺和碘乙酸选择性地抑制了逆向转运活性。各种试剂的作用表明，逆向转运系统中敏感的巯基并非暴露于外部。N - 乙基马来酰亚胺用于探究底物阳离子在锂 - 钠逆向转运中引发的变化。在无钠和无锂的介质中，N - 乙基马来酰亚胺在2秒内对锂 - 钠逆向转运的抑制率达到35%。在有钠或锂的介质中，最大抑制率是前者的两倍，但达到最大抑制的速度要慢得多，在10分钟内才能达到。动力学数据和希尔图分析表明涉及两类巯基：一类在有无底物阳离子的各种介质中均有表达，另一类在外部存在钠或锂时，会对N - 乙基马来酰亚胺变得特别敏感。钠对促进N - 乙基马来酰亚胺抑制作用的位点的亲和力（表观Km = 12 mM）高于钠对其外部逆向转运位点的亲和力（表观Km = 25 mM，如Sarakadi, B., Alifimoff, J.K., Gunn, R.B.和Tosteson, D.C.（1978年）《普通生理学杂志》72, 249 - 265所报道）。所测试的介质并未改变N - 乙基[14C]马来酰亚胺的反应活性。结论是，外部的钠和锂会导致人红细胞逆向转运系统蛋白质的构象发生变化。
