MONOCLONAL-ANTIBODY TO NA,K-ATPASE - IMMUNOCYTOCHEMICAL LOCALIZATION ALONG NEPHRON SEGMENTS
MONOCLONAL-ANTIBODY TO NA,K-ATPASE - IMMUNOCYTOCHEMICAL LOCALIZATION ALONG NEPHRON SEGMENTS
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DOI：
10.1038/ki.1985.216
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发表时间：
1985-01-01
影响因子：
19.6
通讯作者：
FORBUSH, B
中科院分区：
文献类型：
--
作者：
KASHGARIAN, M;BIEMESDERFER, D;FORBUSH, B
为了获得可用于 Na,K-ATP 酶超微结构定位的高度特异性试剂，使用富含 Na,K-ATP 酶的狗和大鼠外肾髓质微粒体制备物制备单克隆抗体。单克隆抗体 (C62.4) 针对狗抗原产生，从用 35S 蛋氨酸或 3H NAB 哇巴因标记的膜中免疫沉淀 96,000 道尔顿蛋白。与 C62.4 反应后，Na、K-ATP 酶、Na-ATP 酶和 KpNPP 酶活性分别为最大 25%、60% 和 100%。用 SDS 激活的 Na,K-ATP 酶受到抑制，但右侧紧密膜囊泡中的 Na,K-ATP 酶没有受到抑制。 C62.4 在 Mg 和 Pi 存在下抑制哇巴因结合。使用胶体金标记的 C62.4 可以很容易地看到破损膜的标记。完整的右侧向外囊泡没有显示标记的证据，表明抗体针对酶的细胞质结构域的表位。确定了 C62.4 沿肾单位的差异定位。除了系膜区域的偶尔细胞外，肾小球没有显示出显着的抗体结合。仅来自所有小管段的细胞的基底侧膜用 C62.4 标记。没有特定顶端标记的证据。亨利氏环的粗升肢表现出最大浓度的抗体结合。在皮质和外髓集合管中，只有主细胞在任何表面上都显示出丰富的抗体结合。这些研究表明，Na,K-ATP酶仅定位于肾小管上皮细胞的基底侧膜，并且在不同肾单位节段中的密度和分布各不相同。
To obtain a highly specific reagent that could be utilized for ultrastructural localization of Na,K-ATPase, monoclonal antibodies were produced using microsomal preparations of outer renal medulla of dog and rat enriched for Na,K-ATPase. The monoclonal antibody (C62.4) raised agaisst dog antigen, immunoprecipitated a 96,000 Dalton protein from membranes labeled either with 35S methionine or 3H NAB ouabain. Na,K-ATPase, Na-ATPase, and KpNPPase activity were 25, 60, and 100% maximal after reaction with C62.4. Na,K-ATPase activated with SDS was inhibited, but Na,K-ATPase in tight right-side-out membrane vesicles was not. C62.4 inhibited ouabain binding in the presence of Mg and Pi. Labeling of broken membranes was readily seen using C62.4 labeled with colloidal gold. Intact right-side-out vesicles showed no evidence of labeling, demonstrating that the antibody is directed to an epitope of the cytoplasmic domain of the enzyme. Differential localization of C62.4 along the nephron was identified. Glomeruli showed no significant antibody binding except by occasional cells in the mesangial regions. Only basal lateral membranes of cells from all tubule segments labeled with C62.4. There was no evidence of specific apical labeling. The thick ascending limb of Henle''s loop demonstrated the greatest concentration of antibody binding. In the cortical and outer medullary collecting duct, only principal cells showed abundant antibody binding on any surface. These studies demonstrate that Na,K-ATPase is localized exclusively to the basal lateral membrane of renal tubular epithelial cells and varies in density and distribution in different nephron segments.