Soleimani M, Hattabaugh Y J, Bizal G L
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202-5116.
Biochim Biophys Acta. 1993 Jun 18;1149(1):127-34. doi: 10.1016/0005-2736(93)90033-v.
Brush-border membrane (BBM) proteins that bind the arginine-specific reagent phenylglyoxal (PG) and interact with stilbene disulfonic derivatives were identified in canine kidney cortex. Pretreatment of BBM vesicles with PG resulted in irreversible inhibition of anion exchange as assayed by 36Cl- influx mediated via Cl-/Cl- exchange. Cl-/Cl- exchange was reversibly inhibited by the disulfonic stilbene 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS). A stilbene-affinity matrix was prepared by immobilizing DNDS in polyacrylamide beads. Elution of the BBM proteins from a disulfonic stilbene (DNDS) affinity matrix revealed two proteins at 160 and 230 kDa that were significantly enriched compared to initial material. Radiolabeling of the eluted mixture with [14C]phenylglyoxal demonstrated covalent binding to several proteins, including the 160 kDa protein. Reconstitution of the proteins eluted from the affinity matrix into phosphatidylcholine demonstrated DIDS-sensitive 36Cl(-)-influx mediated via Cl-/Cl- exchange. Pretreatment of the BBM vesicles with PG selectively blocked binding of the 160 kDa protein to the DNDS affinity matrix. Radiolabelling of the PG-pretreated, affinity-purified membrane proteins showed selective prevention of [14C]phenylglyoxal binding to the 160 kDa protein. Reconstitution of the PG-pretreated proteins eluted from the affinity matrix demonstrated significant reduction in Cl-/Cl- exchange activity. These results suggest that a 160 kDa protein is a strong candidate for anion exchange transport in kidney proximal tubules.
在犬肾皮质中鉴定出了与精氨酸特异性试剂苯乙二醛（PG）结合并与二苯乙烯二磺酸衍生物相互作用的刷状缘膜（BBM）蛋白。用PG预处理BBM囊泡会导致阴离子交换的不可逆抑制，这通过经由Cl⁻/Cl⁻交换介导的³⁶Cl⁻内流来测定。Cl⁻/Cl⁻交换被二苯乙烯二磺酸4,4'-二硝基二苯乙烯-2,2'-二磺酸盐（DNDS）可逆抑制。通过将DNDS固定在聚丙烯酰胺珠粒中制备了二苯乙烯亲和基质。从二苯乙烯二磺酸（DNDS）亲和基质上洗脱BBM蛋白，发现了两种分子量分别为160 kDa和230 kDa的蛋白，与初始材料相比，它们显著富集。用[¹⁴C]苯乙二醛对洗脱混合物进行放射性标记，证明其与几种蛋白发生共价结合，包括160 kDa的蛋白。将从亲和基质上洗脱的蛋白重组成磷脂酰胆碱，显示出经由Cl⁻/Cl⁻交换介导的对DIDS敏感的³⁶Cl⁻内流。用PG预处理BBM囊泡可选择性地阻断160 kDa蛋白与DNDS亲和基质的结合。对PG预处理的、亲和纯化的膜蛋白进行放射性标记，显示[¹⁴C]苯乙二醛与160 kDa蛋白的结合被选择性阻止。将从亲和基质上洗脱的PG预处理蛋白进行重组，显示Cl⁻/Cl⁻交换活性显著降低。这些结果表明，160 kDa的蛋白是肾近端小管中阴离子交换转运的有力候选者。
