Expression of normal and mutant GFP-tagged y(+)L amino acid transporter-1 in mammalian cells

Lysinuric protein intolerance (LPI; MIM 222700) is an autosomal recessive disorder characterized by defective transport of cationic amino acids lysine, arginine and ornithine. The defect is localized in the basolateral membrane of polar epithelial cells of the renal tubules and intestine. The SLC7A7 (solute carrier family 7, member 7) gene that encodes y(+)LAT-1 (y(+)L amino acid transporter-1) is mutated in LPI, and leads to the malfunction of the heterodimer composed of y(+)LAT-1 and 4F2hc (4F2 heavy chain) responsible for the system y(+)L amino acid transport activity at the membrane. In this study, the intracellular trafficking and membrane expression of wild type and four mutant y(+)LAT-1 proteins (LPI(Fin), G54V, 1548delC, W242X) was studied in two human cell lines by expressing green fluorescent protein (GFP) tagged proteins. Different SLC7A7 mutations influenced the trafficking of y(+)LAT-1 in the cells differently, as the wild type and missense mutant fusion proteins localized to the plasma membrane, while the frameshift and nonsense mutants sequestered to the cytoplasmic membranes, never reaching the target areas of the cell.     

Characterization of cationic amino acid transporters (hCATs) 1 and 2 in human skin

In the present study, we characterized the distribution of human cationic amino acid transporters 1 (hCAT1) and 2 (hCAT2) in healthy skin and compared it to psoriatic skin lesions by means of immunohistochemistry. Moreover, we tested the hypothesis that l-arginine and l-ornithine influence the expression and synthesis of hCAT1 and hCAT2 in cell culture experiments by means of real-time-PCR and Western blot. Immunohistochemical comparison between healthy and psoriatic skin revealed a decreased amount of hCAT1, especially in the stratum granulosum of psoriatic skin; the distribution pattern of hCAT2 was not significantly affected in psoriatic skin. Cell culture experiments showed that supraphysiological concentrations of 15 mM l-arginine (72 h) lead to a significant increase of the hCAT1-mRNA and protein expression, whereas other concentrations had no significant influence. In contrast, l-arginine concentrations of 2 mM led to a significant increase of the hCAT2B mRNA-expression after 24 h. However, 48 and 72 h revealed no significant changes and high concentrations (15 mM l-arginine) led to a significant downregulation of the hCAT2B transporter over all time points analyzed. l-ornithine had no effect on the hCAT1 expression of mRNA and protein level. On the other hand the expression of hCAT2B was significantly up regulated at a 5-mM concentration of l-ornithine at all analyzed time points. Other concentrations had no effect. For the first time, the findings yield data about hCAT1 and hCAT2 on protein-level and suggest that l-arginine is a worthwhile object of studies, which investigated l-arginine as a possible therapeutic agent to reduce psoriatic symptoms.     

Polyamines are absorbed through a y+ amino acid carrier in rat intestinal epithelial cells

Summary. Due to the similarity in transport characteristics of polyamines and the y⁺ basic amino acid system, we hypothesized that both substrates could be moving through a common carrier site. Competitive and cross inhibition experiments in intestinal epithelial cells revealed the possibility of a common transport site. N-ethylmalemide (NEM) inhibited both lysine and putrescine transport, confirming that both were carried by a y⁺ transporter. Overexpressing the y⁺ transporter CAT-1 in a polyamine transport-deficient cell line, CHO-MG, did not reconstitute polyamine-transport. Thus, polyamines are not traveling through CAT-1. To determine if lysine is carried by a polyamine transport site, an antizyme-overexpressing cell line was used. Antizyme overexpression decreased polyamine uptake by 50%; in contrast, lysine transport was unaffected. Therefore, lysine is not traveling through a polyamine transport site. It appears that polyamines and lysine are likely traveling through a common unknown y⁺ transport site.     

Analysis of the genomic organization of the human cationic amino acid transporters CAT-1, CAT-2 and CAT-4

Summary. By screening nucleotide databases, sequences containing the complete genes of the human cationic amino acid transporters (hCATs) 1, 2 and 4 were identified. Analysis of the genomic organization revealed that hCAT-2 consists of 12 translated exons and most likely of 2 untranslated exons. The splice variants hCAT-2A and hCAT-2B use exon 7 and 6, respectively. The hCAT-2 gene structure is closely related to the structure of hCAT-1, suggesting that they belong to a common gene family. hCAT-4 consists of only 4 translated exons and 3 short introns. Exons of identical size and highly homologous to exon 3 of hCAT-4 are present in hCAT-1 and hCAT-2. Received September 8, 2000 Accepted January 8, 2001     

Effect of chemical modifiers of amino acid residues on proton conduction by the H+-ATPase of mitochondria

The effect of chemical modifiers of amino acid residues on the proton conductivity of H⁺-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H⁺ in ESMP as well as in particles deprived of F₁ (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H⁺ exhibited two first-order phases, both an expression of the H⁺ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H⁺ conduction caused by respiratory H⁺ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H⁺ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H⁺ conduction. In USMP, H⁺ release followed simple first-order kinetics. Oligomycin depressed H⁺ release, enhanced respiratory H⁺, and restored the biphasicity of H⁺ release. Phenylglyoxal and tetranitromethane inhibited H⁺ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H⁺ release from USMP, introducing a very rapid phase of H⁺ release. Addition of soluble F₁ to USMP also restored biphasicity of H⁺ release. A mechanism of proton conduction by F _o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H⁺ conduction, and those modified by butanedione, which retard H⁺ diffusion.     

Effects of Eu3+ on the metabolism of amino acid and protein in xerophytic Lathyrus sativus L.

The contents of amino acids and proteins and the activity of Na⁺, K⁺-ATPase were determined in roots, stems, and leaves of Eu³⁺-treated Lathyrus sativus L. The results showed that the treatment of Eu³⁺ made the contents of amino acid and protein and the activity of Na⁺, K⁺-ATPase change. The first possible mechanism was that Eu³⁺ directly made the electric potential of −NH₂ or −COOH of amino acid change. The second possible mechanism was that Eu³⁺ played a role in metallic-activated factors of certain enzymes, which catalyze the catabolism and anabolism of protein. Then, the contents of amino acids and proteins were relatively changed. The third possible mechanism was that Eu³⁺ regulated the activity of ATPase through changing the Na⁺/K⁺ ratio. The energy released by ATPase was the driving force for the translocation of amino acids and proteins in the plant cell. Because of the changeability of its valence, Eu played an, important role in regulating certain physiological reactions to increase the adaptability of L. sativus in arid environment. These authors contributed equally to this work.     

Purification,characterization and partial amino acid sequencing of a 70 kD inhibitor protein of Na+,K+-ATPase from goat testis cytosol

A protein isolated from goat testis cytosol is found to inhibit Na⁺,K⁺-ATPase from rat brain microsomes. The inhibitor has been purified by ammonium sulphate precipitation followed by hydroxyapatite column chromatography. The purified fraction appears as a single polypeptide band on 10% SDS-PAGE of approximate molecular mass of 70 kDa. The concentration at which 50% inhibition (I₅₀) occurs is in the nanomolar range. The inhibitor seems to bind Na⁺,K⁺-ATPase reversibly at ATP binding site in a competitive manner with ATP, but away from ouabain binding site. It does not affect p-nitrophenyl-phosphatase activity. The inhibitor is found to inhibit the phosphorylation step of the Na⁺,K⁺-ATPase. The enhancement of tryptophan fluorescence and changes in CD pattern suggest conformational changes of Na⁺,K⁺-ATPase on binding to the inhibitor. Amino acid sequence of the trypsinised fragments show some homology with aldehyde reductase.     

Complexes of the {ReOX2} (X = Cl, Br) core with single amino acid chelate derivatives

The reactions of 1 equiv. of the ligand 3-(ethoxycarbonylmethyl-pyridin-2-ylmethyl-amino)-propionic acid methyl ester (2) with the Re(V) starting materials [ReOX₃(PPh₃)₂] (X = Cl, Br) in refluxing chloroform yielded the Re(V)-oxo dihalide complexes [ReOX₂{(C₅H₄NCH₂)N(CH₂CO₂)(C₂H₄CO₂CH₃)}] (X = Cl, 3; X = Br, 4). The complexes were characterized by elemental analysis, NMR and IR spectroscopy, cyclic voltammetry and X-ray crystallography. Complex 3 displays distorted octahedral coordination geometry with the tridentate ligand coordinating facially to the Re(V) center. The carboxylate oxygen atom occupies an axial site trans to the ReO bond. The two chlorine atoms consequently adopt a cis configuration.     

Influence of an extracellular volume expansion (ECVE) on renal amino acid- and sodium handling in patients with autosomal dominant polycystic kidney disease (ADPKD)

Summary Although ADPKD is one of the first kidney diseases to be understood from the gene to the pathogenesis of clinical abnormalities, there were no data concerning the renal handling of amino acids and possible disorders of amino acid (AA) pattern in these patients. Therefore, in 9 patients suffering from ADPKD and in 8 healthy normal persons (NP) renal amino acid excretion was measured before and after extracellular volume expansion (ECVE) (21 of physiological electrolyte solution). Renal function was stable in both groups (serum creatinine: ADPKD: 85.1 ± 18.4 vs. NP 84.4 ± 13.5 mol/l; GFR: 93.8 ± 16.4 vs. 104.4 ± 9.4 ml/min/1.73 m²). Mean blood pressure was higher in ADPKD patients than in NP (99.4 ± 2.6 vs. 85.5 ± 2.4 mmHg), but did not change after ECVE. After ECVE in both groups, urine volume increased distinctly, whereas GFR was only slightly enhanced. The plasma concentrations of leucine, glycine, valine, threonine, glutamine, and alanine were significantly higher in controls than in ADPKD patients. The amino acid reabsorption capacity was reduced in ADPKD patients in 12 of 21 amino acids before ECVE. After ECVE, the fractional excretion of amino acids (FE_AA) increased only in NP. In parallel with changes in amino acid handling, the FE_Na (%) after ECVE increased both in ADPKD patients and in NP (before ECVE - ADPKD: 1.22 ± 0.23 vs. NP: 1.53 ± 0.23; after ECVE: 3.17 ± 0.25 (ADPKD) vs. 2.74 ± 0.22/NP; (ADPKD p 0.01, NP p 0.02) whereas FE_Li (%) increased significantly only in ADPKD (p 0.045) range (before ECVE - ADPKD: 25.8 ± 8.9 vs. NP: 20.5 ± 4.0; after ECVE: 41.4 ±15.4 vs. 25.2 ± 3.9). Furthermore, concentrations of cGMP (pmol/ml) in plasma increased after ECVE (before ECVE - ADPKD: 5.31 ± 0.56 vs. NP: 6.65 ±0.79; after ECVE: 11.31 ± 1.66 vs. 11.30 ± 1.91; p 0.05). Na⁺-dependent and, perhaps, NO-mediated processes in the reabsorption of AA in the proximal tubule seem to be different in ADPKD and may be related to different distributions of receptors and ATP-dependent transport systems with pathogenetic impact on abnormal transtubular fluid transport in ADPKD.     

structure of ascorbic acid and its biological function

Ascorbic acid/isoascorbic acid are present as radicals at physiological pH with the unpaired electron located in the C(4) region. Since a distinction can be made between both types of radicals, the electron spin resonance technique can be used for discrimination between the epimers of vitamin C. The radical has a cyclic side-chain structure which is formed by the hydrogen bond C(3)-O^– ... HO-C(6) ( 2.7 kJ) and which engulfs Na⁺ or K⁺ in the case of the ascorbyl or the isoascorbyl radical, respectively. The radicals Na-ASC and K-Iso-ASC are electroneutral. Red. glutathione affects both types of radicals by restoring the original electronic configuration at C(4) without changing the electroneutral bicyclic structure. In this way, the mobile carriers Na-ASC and K-Iso-ASC can transport Na⁺ and K⁺ across membranes. Its highest efficiency is around 37 C and pH 7, that is, at physiological values. The biological importance of the side chain of vitamin C is outlined and a possible transport mechanism proposed.     

Identification of Cysteine Residues in Human Cationic Amino Acid Transporter hCAT-2A That Are Targets for Inhibition by N-Ethylmaleimide

In most cells, cationic amino acids such as l-arginine, l-lysine, and l-ornithine are transported by cationic (CAT) and y⁺L (y⁺LAT) amino acid transporters. In human erythrocytes, the cysteine-modifying agent N-ethylmaleimide (NEM) has been shown to inhibit system y⁺ (most likely CAT-1), but not system y⁺L (Devés, R., Angelo, S., and Chávez, P. (1993) J. Physiol. 468, 753–766). We thus wondered if sensitivity to NEM distinguishes generally all CAT and y⁺LAT isoforms. Transport assays in Xenopus laevis oocytes established that indeed all human CATs (including the low affinity hCAT-2A), but neither y⁺LAT isoform, are inhibited by NEM. hCAT-2A inhibition was not due to reduced transporter expression in the plasma membrane, indicating that NEM reduces the intrinsic transporter activity. Individual mutation of each of the seven cysteine residues conserved in all CAT isoforms did not lead to NEM insensitivity of hCAT-2A. However, a cysteine-less mutant was no longer inhibited by NEM, suggesting that inhibition occurs through modification of more than one cysteine in hCAT-2A. Indeed, also the double mutant C33A/C273A was insensitive to NEM inhibition, whereas reintroduction of a cysteine at either position 33 or 273 in the cysteine-less mutant led to NEM sensitivity. We thus identified Cys-33 and Cys-273 in hCAT-2A as the targets of NEM inhibition. In addition, all proteins with Cys-33 mutations showed a pronounced reduction in transport activity, suggesting that, surprisingly, this residue, located in the cytoplasmic N terminus, is important for transporter function.     

Characterization of chicken interleukin 2 receptor alpha chain, a homolog to mammalian CD25

To identify chicken IL-2R alpha chain (chCD25), the cDNA of chCD25 was cloned and mapped onto chicken chromosome 1. The polyclonal and monoclonal antibodies raised from the recombinant chCD25 specifically bound to the cell surface of splenic mononuclear cells (SMC) and inhibited chicken IL-2-dependent proliferation of T cells. Flow cytometry analysis revealed that chCD25 molecules could be expressed on the surface of monocytes/macrophages, thrombocytes, CD4+ and CD8+ cells as well as tissue cells. Importantly, the CD4+CD25+ and CD8+CD25+ cells were upregulated dramatically in chickens infected with H9N2 avian influenza virus. These results confirm that the cloned cDNA is the nucleotide sequence of chicken IL-2R, and suggest that chicken CD4+CD25+ and CD8+CD25+ cells may play an important role in immune responses induced by H9N2 virus, and the monoclonal antibodies to chCD25 may be useful for investigating biological functions of chicken regulatory T cells.     

Molecular and clinical evaluation of Turkish patients with lysinuric protein intolerance

Lysinuric protein intolerance is an autosomal recessive metabolic disorder caused by defective transport of the cationic amino acids lysine, arginine and ornithine in the epithelial cells of the basolateral membrane in the small intestine and renal tubules. Mutations in the solute carrier family 7, member 7, SLC7A7, gene cause this multisystemic disease with a variety of clinical symptoms such as hepatosplenomegaly, osteoporosis, hypotonia, developmental delay, pulmonary insufficiency or end-stage renal disease. In the present study, genomic structure of SLC7A7 in six Turkish patients with lysinuric protein intolerance was examined in order to detect disease causing mutations by denaturing high pressure liquid chromatography and direct sequencing. Four novel mutations were identified in SLC7A7: c.223insGTC, p.Val74_Ile75insVal; c.283insTGG, p.Glu94_Thr95insTrp; c.344_347delTTGC, p.Leu115LeufsX53; and c.1099insT, p.Ile367TyrfsX16. Clinical and biochemical findings were evaluated together with these molecular analyses.     

Transport of glutamine inXenopus laevis oocytes: Relationship with transport of other amino acids

Summary We have investigated transport of the amino acid glutamine across the surface membranes of prophase-arrestedXenopus laevis oocytes. Glutamine accumulation was linear with time for 30 min; it was stereospecific with aK _m of 0.12±0.02mm andV _max of 0.92±0.17 pmol/oocyte · min forl-glutamine. Transport ofl-glutamine was Na⁺-dependent, the cation not being replaceable with Li⁺, K⁺, choline, tris(hydroxymethyl)-aminomethane (Tris), tetramethylammonium (TMA) or N-methyld-glucamine NMDG); external Cl^– appeared to be necessary for full activation of Na⁺-dependent glutamine transport. Two external Na⁺ may be required for the transport of one glutamine molecule.l-glutamine transport (at 50 m glutamine) was inhibited by the presence of other amino acids:l-alanine,d-alanine,l-leucine,l-asparagine andl-arginine (about 60% inhibition at 1mm);l-histidine,l-valine and glycine (25 to 40% inhibition at 1mm);l-serine,l-lysine,l-phenylalanine andl-glutamate (45 to 55% inhibition at 10mm). N-methylaminoisobutyric acid (meAIB) had no effect at 10mm, but 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) inhibited Na⁺/glutamine transport by about 50% at 10mm.l-glutamine was a competitive inhibitor of the Na⁺-dependent transport ofl-alanine,d-alanine andl-arginine; this evidence is consistent with the existence of a single system transporting all four amino acids. Glutamine uptake in oocytes appears to be catalyzed by a transport system distinct from the cotransport Systems A, ASC, N and Gly, although it resembles System B^0,+.     

Nitric oxide enhances salt tolerance in maize seedlings through increasing activities of proton-pump and Na+/H+ antiport in the tonoplast

Nitric oxide (NO), an endogenous signaling molecule in animals and plants, mediates responses to abiotic and biotic stresses. Our previous work demonstrated that 100 μM sodium nitroprusside (SNP, an NO donor) treatment of maize seedlings increased K⁺ accumulation in roots, leaves and sheathes, while decreasing Na⁺ accumulation (Zhang et al. in J Plant Physiol Mol Biol 30:455–459, 2004b). Here we investigate how NO regulates Na⁺, K⁺ ion homeostasis in maize. Pre-treatment with 100 μM SNP for 2 days improved later growth of maize plants under 100 mM NaCl stress, as indicated by increased dry matter accumulation, increased chlorophyll content, and decreased membrane leakage from leaf cells. An NO scavenger, methylene blue (MB-1), blocked the effect of SNP. These results indicated that SNP-derived NO enhanced maize tolerance to salt stress. Further analysis showed that NaCl induced a transient increase in the NO level in maize leaves. Both NO and NaCl treatment stimulated vacuolar H⁺-ATPase and H⁺-PPase activities, resulting in increased H⁺-translocation and Na⁺/H⁺ exchange. NaCl-induced H⁺-ATPase and H⁺-PPase activities were diminished by MB-1. 1-Butanol, an inhibitor of phosphatidic acid (PA) production by phospholipase D (PLD), reduced NaCl- and NO-induced H⁺-ATPase activation. In contrast, applied PA stimulated H⁺-ATPase activity. These results suggest that NO acts as a signal molecule in the NaCl response by increasing the activities of vacuolar H⁺-ATPase and H⁺-PPase, which provide the driving force for Na⁺/H⁺ exchange. PLD and PA play an important role in this process.