Talor Z, Gold R M, Yang W C, Arruda J A
Eur J Biochem. 1987 May 4;164(3):695-702. doi: 10.1111/j.1432-1033.1987.tb11182.x.
Binding of the anion-exchange inhibitor 3H2-labeled 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) to highly purified luminal and basolateral beef kidney tubular membranes was characterized. Specific binding of [3H2]DIDS is present in both luminal and basolateral membranes. Scatchard analysis revealed a Kd for [3H2]DIDS of 5.5 microM and 19.3 microM and a maximal number of binding sites of 10.9 nmol and 31.7 nmol DIDS/mg protein in basolateral and luminal membranes, respectively. To assess the role of this putative anion exchanger on transport we measured 35SO4 uptake by luminal and basolateral membranes. In both luminal and basolateral membranes sulfate uptake was significantly greater in the presence of an outward-directed Cl gradient, OH gradient or HCO3 gradient than in the absence of these gradients. There was an early anion-dependent sulfate uptake of five to ten times the equilibrium uptake at 60 min. The sulfate taken in could be released by lysis of the vesicles indicating true uptake and not binding of sulfate. No significant difference in SO4 uptake was found in the presence and in the absence of valinomycin, indicating that the anion exchanger is electroneutral. The anion-dependent sulfate uptake was completely inhibited by either DIDS or furosemide in both luminal and basolateral membranes. Dixon analysis of HCO3-dependent SO4 uptake by luminal membranes in the presence of different concentrations of DIDS revealed a Ki for DIDS of 20 microM. The similar values of the Kd for [3H2]DIDS binding and the Ki for DIDS inhibition of SO4 uptake might suggest an association between DIDS binding and the inhibition of SO4 transport. In addition, an inward-directed Na gradient stimulated sulfate uptake in luminal but not in basolateral membranes. The Na-dependent sulfate uptake in luminal membranes was also inhibited by DIDS. We conclude that, in addition to the well-known Na-dependent sulfate uptake in luminal membranes, there exists an anion exchanger in both basolateral and luminal membranes capable of sulfate transport.
对阴离子交换抑制剂3H2标记的4,4'-二异硫氰酸-2,2'-二苯乙烯二磺酸（DIDS）与高度纯化的牛肾管腔膜和基底外侧膜的结合特性进行了表征。[3H2]DIDS在管腔膜和基底外侧膜中均存在特异性结合。Scatchard分析显示，基底外侧膜和管腔膜中[3H2]DIDS的解离常数（Kd）分别为5.5微摩尔和19.3微摩尔，最大结合位点数分别为10.9纳摩尔和31.7纳摩尔DIDS/毫克蛋白。为了评估这种假定的阴离子交换体在转运中的作用，我们测量了管腔膜和基底外侧膜对35SO4的摄取。在管腔膜和基底外侧膜中，存在外向性Cl梯度、OH梯度或HCO3梯度时的硫酸盐摄取显著高于不存在这些梯度时。在60分钟时，存在早期阴离子依赖性硫酸盐摄取，其摄取量是平衡摄取量的五到十倍。摄取的硫酸盐可通过囊泡裂解释放，表明是真正的摄取而非硫酸盐结合。在存在和不存在缬氨霉素的情况下，SO4摄取未发现显著差异，表明阴离子交换体是电中性的。在管腔膜和基底外侧膜中，DIDS或呋塞米均可完全抑制阴离子依赖性硫酸盐摄取。对存在不同浓度DIDS时管腔膜中HCO3依赖性SO4摄取进行Dixon分析，结果显示DIDS的抑制常数（Ki）为20微摩尔。[3H2]DIDS结合的Kd值与DIDS抑制SO4摄取的Ki值相似，这可能表明DIDS结合与SO4转运抑制之间存在关联。此外，内向性Na梯度刺激管腔膜而非基底外侧膜摄取硫酸盐。管腔膜中Na依赖性硫酸盐摄取也受到DIDS抑制。我们得出结论，除了管腔膜中众所周知的Na依赖性硫酸盐摄取外，基底外侧膜和管腔膜中均存在能够转运硫酸盐的阴离子交换体。
