Characterization of the rat neutral and basic amino acid transporter utilizing anti-peptide antibodies.
Characterization of the rat neutral and basic amino acid transporter utilizing anti-peptide antibodies.
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利用抗肽抗体表征大鼠中性和碱性氨基酸转运蛋白。
DOI：
10.1073/pnas.90.9.4022
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发表时间：
1993
影响因子：
11.1
通讯作者：
S. Udenfriend
中科院分区：
文献类型：
--
作者：
R. Mosckovitz;N. Yan;E. Heimer;A. Felix;S. Tate;S. Udenfriend
我们已经针对大鼠肾广谱、不依赖钠的中性和碱性氨基酸转运蛋白 (NBAA-Tr) 产生了高滴度、位点特异性抗体，我们之前克隆了该蛋白的 cDNA。这些抗体使我们能够表征正常大鼠组织以及各种细胞和体外表达系统中的转运蛋白。 Western 分析检测到大鼠肾和空肠上皮细胞刷状缘膜中富含 84 至 87 kDa 糖基化物质。 NBAA-Tr 互补 RNA 在兔网织红细胞裂解物系统中的体外翻译产生了 78 kDa 的蛋白质，该分子量是通过从克隆的 cDNA 推导的氨基酸序列预测的。在粗糙微粒体膜存在的情况下进行翻译产生了糖基化的 89-kDa 物质。糖基化的 87-至 89-kDa 种类也在显微注射 NBAA-Tr 互补 RNA 的爪蟾卵母细胞和用 NBAA-Tr cDNA 转染的 COS-7 细胞中表达。 NBAA-Tr 在肾和肠刷状缘膜中的定位与其在氨基酸跨上皮转运中的作用一致。
High-titer, site-specific antibodies have been produced against the rat kidney broad-spectrum, sodium-independent neutral and basic amino acid transporter (NBAA-Tr) whose cDNA we cloned earlier. These antibodies have allowed us to characterize the transporter protein in normal rat tissues and in various cellular and in vitro expression systems. Western analysis detected 84- to 87-kDa glycosylated species enriched in rat renal and jejunal epithelial cell brush border membranes. In vitro translation of NBAA-Tr complementary RNA in the rabbit reticulocyte lysate system yielded a 78-kDa protein, a molecular mass that was predicted by the amino acid sequence deduced from the cloned cDNA. Translation in the presence of rough microsomal membranes yielded a glycosylated 89-kDa species. Glycosylated 87- to 89-kDa species were also expressed in Xenopus oocytes microinjected with NBAA-Tr complementary RNA and in COS-7 cells transfected with NBAA-Tr cDNA. Localization of NBAA-Tr in renal and intestinal brush border membranes is consistent with its proposed role in transepithelial transport of amino acids.