Characterization of the rat neutral and basic amino acid transporter utilizing anti-peptide antibodies.

Abstract

High-titer, site-specific antibodies have been produced against the rat kidney broad-spectrum, sodiumindependent neutral and basic amino acid transporter (NBAATr) 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.

Oligomeric structure of a renal cystine transporter: implications in cystinuria

“…Six synthetic peptides representing sequences within rat NBAT were used as antigens for the production of rabbit antisera directed against NBAT. Details of peptide synthesis, production of the antisera, and evaluation of their specificity have been described previously [9,14]. The immunoglobulin (IgG) fraction from each antiserum was purified using a rProtein A antibody purification kit from Repligen (Cambridge, MA).…”

“…The membranes were subjected to SDS-PAGE under both reducing and non-reducing conditions. Under reducing conditions, only the 87 and 89 kDa bands representing the glycosylated NBAT monomers produced in oocytes [14] are seen (Fig. 3, lane 1).…”

“…Under non-reducing conditions (i.e. when the BBMs were treated with SDS in the absence of MSH), Western analysis gave two major immunoreactive bands, one at 85 kDa corresponding to the glycosylated NBAT monomer [14], and another, representing about 20% of the total NBAT-containing species, at about 135 kDa (Fig. 1A, lane 1).…”

The rBAT Gene Is Responsible for L-Cystine Uptake via the bo,+-like Amino Acid Transport System in a “Renal Proximal Tubular” Cell Line (OK Cells)

“…No functional rBAT Is Responsible for L-Cystine Uptake in OK Cellsexpression of the amino acid transport activity associated with rBAT has been obtained in mammalian cells (e.g. COS cells) (30). All the data previously reported on the amino acid transport activity associated with rBAT were obtained in Xenopus oocytes (reviewed in Ref.…”

“…Indirect evidence suggested that rBAT forms a heterodimeric structure of 125 kDa with an unidentified protein of 40 -50 kDa in renal brush border membranes and oocytes (29). 2 In addition, transient expression of rBAT in COS cells revealed either expression of rBAT in the cell surface without concomitant amino acid transport activity (30) or a protein product that does not reach the plasma membrane. 3 Thus, no cell system other than oocytes has shown expression of amino acid transport activity associated with rBAT.…”