Characterization of the rat neutral and basic amino acid transporter utilizing anti-peptide antibodies.
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.
Mosckovitz
R
R
Department of Neurosciences, Roche Institute of Molecular Biology, Nutley, NJ 07110.
Yan
N
N
Heimer
E
E
Felix
A
A
Tate
S S
SS
Udenfriend
S
S
D000595:Amino Acid Sequence; D026923:Amino Acid Transport Systems, Basic; D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D000906:Antibodies; D001692:Biological Transport; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D007583:Jejunum / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009865:Oocytes / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D014176:Protein Biosynthesis; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D014162:Transfection; D014981:Xenopus
D026923:Amino Acid Transport Systems, Basic; D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids; D000906:Antibodies; D002352:Carrier Proteins; D010455:Peptides; D011994:Recombinant Proteins; C081009:Slc3a1 protein, rat