Immunolocalization of the Na+/Ca2+ exchanger in rabbit kidney.
Immunolocalization of the Na+/Ca2+ exchanger in rabbit kidney.
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兔肾中 Na /Ca2 交换器的免疫定位。
DOI：
10.1152/ajprenal.1993.265.2.f327
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Biemesderfer,D
中科院分区：
文献类型：
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作者：
Reilly,RF;Shugrue,CA;Lattanzi,D;Biemesderfer,D
我们最近从兔肾中分离到一个编码Na+/Ca2+交换器的cDNA，该交换器与犬心脏肌层Na+/Ca2+交换器高度相似。在本研究中，我们使用两种不同的抗体来鉴定交换蛋白，并建立其在肾脏中的细胞和亚细胞定位。第一种抗体是针对一种融合蛋白制备的，该融合蛋白由190个氨基酸组成，可能是兔肾交换器的细胞内环与麦芽糖结合蛋白融合。第二种是针对分离纯化的犬心肌肌基质交换剂产生的单克隆抗体。为了鉴定Na+/Ca2+交换蛋白，我们对兔肾皮质膜泡进行了免疫印迹分析。两种抗体都免疫印迹了已知与交换剂相关的120和70 kDa的蛋白。间接免疫荧光显示，这两种抗血清标记了连接小管（CNT）中大多数细胞的基底外侧表面。由于碳纳米管中的相密集（插层）细胞未被染色，这表明标记的细胞是碳纳米管细胞。除了皮质集管的大多数细胞群偶尔出现微弱的染色外，其他肾元节段未检测到标记。我们没有在其他肾元片段中检测到标记的事实与以下情况是一致的：1)这些片段中Na+/Ca2+交换器的表达缺失，2)交换器的表达水平低于本研究中使用的两种抗体的检测阈值，或者3)这些片段中的交换器由不同的异构体代表。（摘要删节250字）
We recently isolated a cDNA encoding a Na+/Ca2+ exchanger from rabbit kidney that was highly similar to the canine cardiac sarcolemmal Na+/Ca2+ exchanger. In the present study, we used two different antibodies to the exchanger to identify the protein and establish its cellular and subcellular localization in the kidney. The first antibody was prepared against a fusion protein consisting of 190 amino acids of the large, presumably intracellular loop of the rabbit renal exchanger fused to the maltose-binding protein. The second was a monoclonal antibody generated against the isolated purified canine cardiac sarcolemmal exchanger. To identify the Na+/Ca2+ exchanger protein, we performed immunoblot analysis against a membrane vesicle preparation from rabbit kidney cortex. Both antibodies immunoblotted proteins of 120 and 70 kDa that are known to be associated with the exchanger. Indirect immunofluorescence revealed that both antisera labeled the basolateral surface of the majority of cells in the connecting tubule (CNT). Since the phase-dense (intercalated) cells in the CNT were not stained, this suggested that the labeled cells were CNT cells. No labeling was detected in other nephron segments with the exception of occasional faint staining of the majority cell population of the cortical collecting duct. The fact that we did not detect labeling in other nephron segments is consistent with either 1) the absence of expression of the Na+/Ca2+ exchanger in these segments, 2) the expression of the exchanger in levels below the threshold of detection of the two antibodies used in this study, or 3) the exchanger in these segments is represented by a different isoform.(ABSTRACT TRUNCATED AT 250 WORDS)