Immunodetection of a type III sodium-dependent phosphate cotransporter in tissues and OK cells
Immunodetection of a type III sodium-dependent phosphate cotransporter in tissues and OK cells
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DOI：
10.1016/s0005-2736(97)00159-4
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发表时间：
1998-01-05
影响因子：
3.4
通讯作者：
Béliveau, R
中科院分区：
文献类型：
--
作者：
Boyer, CJC;Baines, AD;Béliveau, R
在兔子体内产生了针对长臂猿白血病病毒人膜受体 Glvr-1 的 14 个氨基酸部分的多克隆抗体。该表位还含有两性鼠逆转录病毒 Ram-1 受体共有的 7 个氨基酸。通过蛋白质印迹法测定 Glvr-1/Ram-1 相关蛋白的抗体特异性和分子大小。使用标准 Laemmli 缓冲系统，在还原条件下，在负鼠肾 (OK) 细胞制备的膜以及大鼠、兔子和仓鼠的脑膜中免疫检测到约 85 kDa（指定为 p85）的单条带。在小鼠大脑中，p85 以及 70-72 kDa 的蛋白质被免疫检测到。这种蛋白质也存在于其他几种小鼠组织中。 p85 和来自小鼠的 70-72kDa 蛋白的有限蛋白水解产生相似的肽片段，表明这两种蛋白是相关的。蛋白水解后，在 OK 细胞膜中也检测到相同分子质量的片段，表明两种模型（小鼠脑和 OK 细胞）中的 p85 具有相似的序列。 p85 未被 N-糖基化，因为使用糖苷内切酶 F/N-糖苷酶 F 进行的测定并未改变 p85 的电泳迁移率。我们还观察到，通过培养不含任何磷酸盐的 OK 细胞或通过 PTH 处理来调节磷酸盐转运，p85 的量不会发生任何变化。总之，这些数据首次证明使用多克隆抗体对 III 型磷酸盐转运蛋白进行蛋白质印迹检测。他们还表明，与位于肾脏的 I 型和 II 型磷酸盐转运蛋白相反，第三种类型的转运蛋白是普遍存在的，并且可能从间质液中吸收容易获得的磷酸盐，以实现许多物种和组织中的正常细胞功能，充当管家 Na+/P-i 协同转运系统。这也是第一份表明 p85 的调节方式与 II 型磷酸盐转运蛋白不同的报告。 (C) 1998 Elsevier Science B.V.
Polyclonal antibodies were raised in rabbits against a 14-amino acid portion of the gibbon ape leukemia virus human membrane receptor Glvr-1. This epitope also contained seven amino acids common to the receptor for the amphotropic murine retrovirus Ram-1. Antibody specificity and molecular size of Glvr-1/Ram-1-related proteins were assayed by Western blot. Using a standard Laemmli buffer system, under reducing conditions, a single band of approximately 85 kDa (designated p85) was immunodetected in membranes prepared from opossum kidney (OK) cells and in brain membranes from rat, rabbit and hamster. In mouse brain, p85 as well as a protein of 70-72 kDa were immunodetected. This protein was also present in several other mouse tissues. Limited proteolysis of p85 and the 70-72kDa-protein from mouse yielded similar peptide fragments, suggesting that both proteins are related. Fragments of the same molecular masses were also detected in OK cell membranes following proteolysis, showing that p85 in both models (mouse brain and OK cell) share a similar sequence. p85 is not N-glycosylated since an assay using endoglycosidase F/N-glycosidase F did not alter the electrophoretic mobility of p85. We also observed that regulation of phosphate transport by incubating OK cells without any phosphate or by PTH treatment occurs without any changes in the amount of p85, In conclusion, these data demonstrate for the first time a Western blot detection of a type III phosphate transporter using polyclonal antibodies. They also suggest that, conversely to type I and type II phosphate transporters which are localized in the kidney, this third type of transporter is ubiquitous and probably absorbs the readily available phosphate from interstitial fluid for normal cellular functions in many species and tissues, serving as a housekeeping Na+/P-i cotransport system. This is also the first report showing that p85 is not regulated in the same manner as type II phosphate transporters. (C) 1998 Elsevier Science B.V.