Mosckovitz R, Yan N, Heimer E, Felix A, Tate S S, Udenfriend S
Department of Neurosciences, Roche Institute of Molecular Biology, Nutley, NJ 07110.
Proc Natl Acad Sci U S A. 1993 May 1;90(9):4022-6. doi: 10.1073/pnas.90.9.4022.
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.
我们此前已克隆出大鼠肾脏广谱、不依赖钠的中性和碱性氨基酸转运体（NBAA-Tr）的cDNA，并制备出了针对该转运体的高效、位点特异性抗体。这些抗体使我们能够在正常大鼠组织以及各种细胞和体外表达系统中对该转运体蛋白进行表征。蛋白质印迹分析检测到在大鼠肾脏和空肠上皮细胞刷状缘膜中富集的84至87 kDa糖基化蛋白。在兔网织红细胞裂解物系统中对NBAA-Tr互补RNA进行体外翻译产生了一种78 kDa的蛋白，该分子量与从克隆的cDNA推导的氨基酸序列预测的分子量一致。在粗面微粒体膜存在的情况下进行翻译产生了一种糖基化的89 kDa蛋白。糖基化的87至89 kDa蛋白也在注射了NBAA-Tr互补RNA的非洲爪蟾卵母细胞和用NBAA-Tr cDNA转染的COS-7细胞中表达。NBAA-Tr在肾脏和肠道刷状缘膜中的定位与其在氨基酸跨上皮转运中的假定作用一致。
