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Basic amino acid transport in renal papilla: microinfusion of Henle's loops and vasa recta.

To determine whether basic amino acids, like acidic and neutral amino acids, could be reabsorbed distal to tips of Henle's loops and recycled between loops and vasa recta in the renal papilla, we continuously microinfused ascending Henle's loops and vasa recta with 14C-labeled L-lysine (L-Lys; 1.28 mM) or L-arginine (L-Arg; 1.17 mM) and 3H-labeled inulin. We also determined percent of recovered radiolabel as intact amino acid. Like acidic and neutral amino acids, relative to inulin, approximately 30% of L-Lys and approximately 45% of L-Arg microinfused into Henle's loops were reabsorbed. However, whereas radiolabeled L-Lys reabsorption, like reabsorption of acidic and neutral amino acids, was not readily inhibited, radiolabeled L-Arg reabsorption was reduced to approximately 25% by addition of unlabled L-Arg (50 mM) or L-homoarginine (L-Homo-Arg) (50 mM) to infusate. This observation provides greater evidence for specific, carrier-mediated reabsorption for L-Arg than for acidic or neutral amino acids. About 36% (relative to inulin) of each of these amino acids microinfused into ascending vasa recta apparently was transferred directly into ipsilateral tubular structures (probably thin descending limbs of Henle's loops). Transfer of radiolabeled L-Arg was reduced to approximately 8% by the inclusion of unlabeled L-Arg (50 mM) in infusate. Transfer of unlabeled L-Lys was unaffected by inclusion of unlabeled L-Lys (50 mM) in infusate but was reduced to approximately 20% by inclusion of unlabeled L-Arg (50 mM) or L-Homo-Arg (50 mM) in infusate. (ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

Nuclear localization of the Hermes transposase depends on basic amino acid residues at the N-terminus of the protein.

For the Hermes transposable element to be mobilized in its eukaryotic host, the transposase, encoded by the element, must make contact with its DNA. After synthesis in the cytoplasm, the transposase has to be actively imported into the nucleus because its size of 70.1 kDa prevents passive diffusion through the nuclear pore. Studies in vitro using transient expression of a Hermes-EGFP fusion protein in Drosophila melanogaster Schneider 2 cells showed the transposase was located predominantly in the nucleus. In silico sequence analysis, however, did not reveal any nuclear localization signal (NLS). To identify the sequence(s) responsible for localization of Hermes transposase in the nucleus, truncated or mutated forms of the transposase were examined for their influence on sub-cellular localization of marker proteins fused to the transposase. Using the same expression system and a GFP-GUS fusion double marker, residues 1-110 were recognized as sufficient, and residues 1-32 as necessary, for nuclear localization. Amino acid K25 greatly facilitated nuclear localization, indicating that at least this basic amino acid plays a significant role in this process. This sequence overlaps the proposed DNA binding region of the Hermes transposase and is not necessarily conserved in all members of the hAT transposable element family.

Amino Acid Sequence↗

Protein D2 channel of the Pseudomonas aeruginosa outer membrane has a binding site for basic amino acids and peptides.

Protein D2 of Pseudomonas aeruginosa outer membrane is known to facilitate the specific permeation of imipenem (N-formimdoylthienamycin) across this membrane barrier. We have characterized the binding site in the protein D2 channel by studying the competitive inhibition, by various solutes, of imipenem diffusion into the periplasm. We found that basic amino acids, lysine, arginine, histidine, and ornithine, were effective inhibitors. L- and D-lysine were found to be competitive inhibitors with approximate Ki values of 0.6 and 0.3 mM, respectively. Peptides containing L-lysine at the carboxyl terminus, as well as dipeptides containing L-lysine at the amino terminus, were also able to inhibit the transport. Wild type cells transported tripeptide Thr-Ser-Lys into the periplasm three to four times as rapidly as the mutant cells lacking the D2 protein. These results suggest that protein D2 plays a physiologically significant role in the uptake of basic amino acids and peptides containing these amino acids across the outer membrane of P. aeruginosa.

Amino Acid Sequence↗

APL1, a yeast gene encoding a putative permease for basic amino acids.

A Saccharomyces cerevisiae gene (1722 bp), encoding a protein (574 aa) highly homologous to the basic-amino-acid permeases LYP1 and CAN1, was sequenced. The gene, which was named APL1 (Amino-acid Permase Like), is located 881 bp upstream from LYP1 (lysine-specific permease), and in head-to-head orientation to it. These sequence data have been deposited in the EMBL/GenBank/DDBJ nucleotide sequence data libraries under Accession Number X74069.

Amino Acid Sequence↗

Analysis of the S(2) subsite specificities of the recombinant cysteine proteinases CPB of Leishmania mexicana, and cruzain of Trypanosoma cruzi, using fluorescent substrates containing non-natural basic amino acids.

We have explored the specificity of the S(2) subsite of recombinant cysteine proteinases from Leishmania mexicana (CPB2.8 Delta CTE) and from Trypanosoma cruzi (cruzain) employing a series of fluorogenic substrates based on the peptide Bz-F-R-MCA, in which Bz is the benzoyl group and the Phe residue has been substituted for by Arg, His and non-natural basic amino acids that combine a basic group with an aromatic or hydrophobic group at the side chain: 4-aminomethyl-phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Ppa), 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca). Bz-F-R-MCA was hydrolyzed well by CPB2.8 Delta CTE and cruzain, but all the substitutions of Phe resulted in less susceptible substrates for the two enzymes. CPB2.8 Delta CTE has a restricted specificity to hydrophobic side chains as with cathepsin L. However, the peptides with the residues Amf and Ama presented higher affinity to CPB2.8 Delta CTE, and the latter was an inhibitor of the enzyme. Although, cruzain accepts basic as well as hydrophobic residues at the S(2) subsite, it is more restrictive than cathepsin B and no inhibitor was found amongst the examined peptides.

Amino Acids, Basic↗

A C-terminal basic amino acid motif of Zaire ebolavirus VP35 is essential for type I interferon antagonism and displays high identity with the RNA-binding domain of another interferon antagonist, the NS1 protein of influenza A virus.

The ebolavirus VP35 protein antagonizes the cellular type I interferon response by blocking phosphorylation of IRF-3, a transcription factor that turns on the expression of a large number of antiviral genes. To identify the domain of VP35 responsible for interferon antagonism, we generated mutations within the VP35 gene and found that a C-terminal basic amino acid motif is required for inhibition of ISG56 reporter gene expression as well as IFN-beta production. Remarkably, this basic amino acid motif displayed high sequence identity with part of the N-terminal RNA-binding domain of another interferon-antagonist, the NS1 protein of influenza A virus.

Amino Acid Motifs↗

Expression control and specificity of the basic amino acid exporter LysE of Corynebacterium glutamicum.

LysE of Corynebacterium glutamicum belongs to a large new superfamily of translocators whose members are probably all involved in the export of small solutes. Here, the transcript initiation site of lysE, and its divergently transcribed regulator gene, lysG, are identified. Single-copy transcriptional fusions of lysE with lacZ, and titration experiments, show that LysG is the positive regulator of lysE expression enabling its up to 20-fold induction. This induction requires the presence of a coinducer, which is either intracellular L-lysine, or L-arginine. A competition experiment showed that LysE exports these two basic amino acids at comparable rates of about 0.75 nmol min(-1) (mg dry wt)(-1). Although L-histidine and L-citrulline also act as coinducers of lysE expression, these two amino acids are not exported by LysE. As is evident from the analysis of a lysEG deletion mutant, the physiological role of the lysEG system is to prevent bacteriostasis due to elevated L-lysine or L-arginine concentrations that arise during growth in the presence of peptides or in mutants possessing a deregulated biosynthesis pathway. C. glutamicum has additional export activities other than those of LysE for exporting L-histidine, L-citrulline and L-ornithine.

Amino Acid Transport Systems, Basic↗

Replacements of single basic amino acids in the pleckstrin homology domain of phospholipase C-delta1 alter the ligand binding, phospholipase activity, and interaction with the plasma membrane.

The pleckstrin homology (PH) domain of phosphatidylinositol-specific phospholipase C-delta1 (PLC-delta1) binds to both D-myo-inositol 1,4, 5-trisphosphate (Ins(1,4,5)P3) and phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) with high affinities. We have previously identified a region rich in basic amino acids within the PH domain critical for ligand binding (Yagisawa, H., Hirata, M., Kanematsu, T., Watanabe, Y., Ozaki, S., Sakuma, K., Tanaka, H., Yabuta, N., Kamata, H., Hirata, H., and Nojima, H. (1994) J. Biol. Chem. 269, 20179-20188; Hirata, M., Kanematsu, T., Sakuma, K., Koga, T., Watanabe, Y., Ozaki, S., and Yagisawa, H. (1994) Biochem. Biophys. Res. Commun. 205, 1563-1571). To investigate the role of these basic residues, we have performed site-directed mutagenesis replacing each of the basic amino acid in the N-terminal 60 residues of PLC-delta1 (Lys24, Lys30, Lys32, Arg37, Arg38, Arg40, Lys43, Lys49, Arg56, Lys57, and Arg60) with a neutral or an acidic amino acid. The effects of these mutations on the PH domain ligand binding properties and their consequence for substrate hydrolysis and membrane interactions of PLC-delta1 were analyzed using several assay systems. Analysis of [3H]-Ins(1,4,5)P3 binding, measurement of the binding affinities, and measurements of phospholipase activity using PtdIns(4,5)P2-containing phospholipid vesicles, demonstrated that residues Lys30, Lys32, Arg37, Arg38, Arg40, and Lys57 were required for these PLC-delta1 functions; in comparison, other mutations resulted in a moderate reduction. A subset of selected mutations was further analyzed for the enzyme activity toward substrate present in cellular membranes of permeabilized cells and for interaction with the plasma membrane after microinjection. These experiments demonstrated that mutations affecting ligand binding and PtdIns(4,5)P2 hydrolysis in phospholipid vesicles also resulted in reduction in the hydrolysis of cellular polyphosphoinositides and loss of membrane attachment. All residues (with the exception of the K43E substitution) found to be critical for the analyzed PLC-delta1 functions are present at the surface of the PH domain shown to contain the Ins(1,4,5)P3 binding pocket.

Amino Acid Sequence↗

cDNA and gene structure for a human subtilisin-like protease with cleavage specificity for paired basic amino acid residues.

A cDNA encoding the human fur gene product was isolated from a human hepatoma cell line. The cDNA encodes a protein with significant amino acid sequence identity to the prokaryotic subtilisin family of serine proteases. More extensive sequence identity was found when the protein was compared with eukaryotic proteases such as PRB1 of Saccharomyces cerevisiae, and with PC2 and PC3, the only other known mammalian subtilisin-like proteases. In contrast to these proteins, however, the fur gene product shares a more extensive topographic and functional homology with the KEX2 endoprotease of S. cerevisiae. Each protease contains a signal peptide, a glycosylated extra cytoplasmic domain, a hydrophobic membrane-spanning region, and a short, hydrophilic "tail" sequence. As with KEX2, the expressed human protease was shown to cleave mammalian proproteins at their paired basic amino acid processing sites. We have, therefore, proposed the function-based acronym PACE (paired basic amino acid cleaving enzyme) for this prototypic mammalian proprotein processing enzyme.

Amino Acid Sequence↗

Lysine transport in two barley mutants with altered uptake of basic amino acids in the root.

Amino acid uptake was examined in two barley (Hordeum vulgare L.) mutants R906 and R4402 which had been selected as resistant to the lysine analog S-(2-aminoethyl)-cysteine. The mutants were found to be allelic by crossing and examination of F(1) and F(2) progeny. The mutant genes were designated aec1a and aec1b, respectively. The uptake of the basic amino acids lysine, arginine, and ornithine from 50 micromolar solutions was strongly decreased in roots of the mutants, whereas uptake of neutral and acidic amino acids was unaffected. The pattern of uptake of lysine over the range 10(-7) to 10(-2) molar was consistent with there being, principally, two uptake systems operating for basic amino acids in roots and that a low-concentration, high-affinity system is reduced or lacking in the mutants. The residual transport activity in the mutants had a different relative affinity for lysine and arginine to the wild-type system. Uptake of lysine by leaf slices was unimpaired in the mutants suggesting that the leaf uptake system is unaffected by the aec1 gene.

Journal Article↗

N-nitrosations of basic amino acid residues in polypeptide.

Changes in the electrophoretic pattern were noted in the products of polypeptides of identical basic amino acids preincubated with reactive or degraded PN, suggesting the occurrence of N-nitrosation of the epsilon-amino group of lysine, the guanido group of arginine and the imidazole group of histidine. Additionally, increase in the N-nitroso immunoreactivity of preincubated histones H2A and H2B was detected by Western blot analysis.

Animals↗

Basic amino acid residues in the beta-structure region contribute, but not critically, to presynaptic neurotoxicity of ammodytoxin A.

The molecular mechanism of action of presynaptically toxic secreted phospholipases A2 (sPLA2s) isolated from snake venoms is not completely understood. It has been proposed that the positive charge in the beta-structure region is important for their toxic activity. To test this hypothesis, we characterised several mutants of ammodytoxin A (AtxA) possessing substitution of all five basic residues in this region. The mutations had relatively little influence on the catalytic activity of AtxA, either on charge-neutral or anionic phospholipid vesicles. An exception was R72 when replaced by a hydrophobic (higher activity) or an acidic (lower activity) residue. Lethal potencies of the eight single site mutants were up to four times lower than that of the wild-type, whereas the triple mutant (K74S/H76S/R77L) was 13-fold less toxic. The substitutions also lowered the affinity of the toxin, slightly to moderately, for the neuronal receptors R25 and R180. Interaction with calmodulin was only slightly affected by substitutions of K86, more by those of the K74/H76/R77 cluster and most by those of R72 (up to 11-fold lower binding affinity). The results clearly indicate that the basic amino acid residues in the beta-region of AtxA contribute to, but are not necessary for, its neurotoxic effect.

Amino Acid Sequence↗

Substrate specificity of an adenohypophyseal endopeptidase capable of hydrolyzing luteinizing hormone-releasing hormone: preferential cleavage of peptide bones involving the carboxyl terminus of hydrophobic and basic amino acids.

The substrate specificity of a peptidase from anterior pituitaries that is capable of hydrolyzing luteinizing hormone-releasing hormone (LH-RH; less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) at the Tyr5-Gly6 peptide bond has been investigated by using inhibitors and model substrates. While trypsin and chymotrypsin inhibitors from plants and animals are without any effect, many microbial protease inhibitors and synthetic peptides containing hydrophobic and basic amino acids inhibit the degradation of radiolabeled LH-RH by this enzyme. The model substrates N-acetyl-Phe-Gly-Leu-beta-naphthylamide, N-acetyl-Leu-Gly-Leu-beta-naphthylamide, and N alpha-benzoyl-Arg-Gly-Leu-beta-naphthylamide are hydrolyzed at the X-Gly peptide bonds; N-acetyl-Gly-Gly-Leu-beta-naphthylamide is not degraded. Hydrolysis of typical amino- and carboxypeptidase substrates was not observed. Degradation of the general protease substrates insulin B chain and denatured hemoglobin also could not be detected. Thus, the enzyme is not LH-RH specific but may be characterized as an endopeptidase that hydrolyzes peptides preferentially at the carboxyl terminus of hydrophobic and basic amino acids.

Amino Acid Sequence↗

Sequence requirements for proteolytic cleavage of precursors with paired basic amino acids.

When expressed in COS cells, human prorenin was secreted into the medium without being processed to an active renin. Co-expression of furin, a mammalian homologue of the yeast KEX2 gene product, did not affect proteolytic processing of prorenin. A mutant proreninR-4 constructed by site-directed mutagenesis of Pro (-4) to Arg was not cleaved by an endoprotease in the COS cell. However, proreninR-4 was detectably cleaved to yield the active renin upon co-transfection with furin DNA, indicating that Arg at position -4 is important for recognition and processing by furin in addition to the absolute requirement for paired basic amino acids. Another mutant precursor in which Leu (+1) of proreninR-4 was replaced with Ser was found to be much more efficiently processed than proreninR-4, regardless of co-expression of furin. The results suggest that not only a basic amino acid at position -4 but also Leu at position +1 significantly affect the processing of prorenin catalyzed by the COS cell endoprotease or furin.

Amino Acid Sequence↗

Effects of dietary sodium and chloride on growth, efficiency of feed utilization, plasma electrolytes and plasma basic amino acids in young pigs.

The metabolic and physiological consequences of three dietary levels of sodium (0.02, 0.11 and 0.18%) and chloride (0.10, 0.22 and 0.33%) were investigated. Corn, soybean meal and corn-gluten meal diets were formulated with varying levels of sodium and chloride. Pigs fed 0.02% sodium gained slower and less efficiently than pigs fed higher levels of sodium. Blood pH was not affected by either dietary sodium or chloride. Linear (P less than 0.01) increases in HCO3, base excess (BE) and plasma sodium were observed when dietary sodium increased. hemoglobin, plasma potassium, urea nitrogen, ornithine, lysine and total basic amino acids decreased linearly as dietary sodium increased. Blood hematocrit decreased curvilinearly (P less than 0.05) with increasing dietary sodium. In addition, increasing dietary chloride linearly increased plasma potassium and decreased blood HCO3 and BE. A curvilinear response to dietary chloride was observed for hemoglobin. Dietary sodium influenced pigs' growth and feed utilization as well as plasma electrolytes and basic amino acids. Although dietary chloride did influence plasma potassium and the buffering capacity of the blood, it had no effect on the rate and efficiency of weight gain of the pigs.

Amino Acids↗

Reactivity of consecutive basic amino acid residues in peptides.

Different tetrapeptides of general formula L-Ala-X-X-Gly, possessing a basic doublet in the second and third position (X = Arg or Lys), have been synthesized as free or N-acetylated molecules. The chemical reactivity of the arginine guanidino group and of the lysine epsilon-amino group were studied using respectively the Sakaguchi and the ortho-diacetylbenzene reactions, in the tetrapeptides as well as in related molecules. In both cases, the colour yield is markedly influenced by the length of the polypeptide chain and by the relative positions of the arginine and lysine residues, suggesting the occurrence of intramolecular bonds within the tetrapeptide molecule. Tryptic hydrolysis of the tetrapeptides was followed by evaluating the amino acids or peptides which appear to be specific for the different possible cleavages at the arginyl or at the lysyl bonds. The susceptibility to trypsin of the carboxylic group of the second basic amino acid decreases progressively in the order Lys-Arg greater than Arg-Arg much greater than Lys-Lys greater than Arg-Lys, which shows a fair correlation with the intra-cellular cleavage of the bonds observed during the processing of preproteins of of the precursors of several physiologically active peptides.

Amino Acid Sequence↗

The basic amino acid transporter (rBAT)-like immunoreactivity in paraventricular and supraoptic magnocellular neurons of the rat hypothalamus.

In the rat hypothalamus, the basic amino acid transporter (rBAT)-like immunoreactivity was analyzed by immunohistochemistry using an antibody against the 15-amino acid sequence of the deduced rat rBAT protein. In the supraoptic and the paraventricular nuclei, magnocellular neurons exhibited the marked rBAT-like immunoreactivity in intracellular structures but not in the plasma membrane. The results suggest that the rBAT serves as an intracellular amino acid transport system in magnocellular neurons.

Amino Acid Sequence↗

Implications of a non-lamellar lipid phase for the tight junction stability. Part II: Reversible modulation of transepithelial resistance in high and low resistance MDCK-cells by basic amino acids, Ca2+, protamine and protons.

The transepithelial resistance of confluent epithelial cell monolayers was monitored to investigate the influence of basic amino acids, Ca2+, protamine and protons on tight junction electrical resistance. In an accompanying paper we investigated the effect of these substances on the lamellar/hexagonal II phase transition in reconstituted phospholipid membranes containing phosphatidylserine and phosphatidylethanolamine. We conclude that the permeability of tight junctions may be described by a lipid phase equilibrium where the lamellar phase corresponds to an open state and the hexagonal lipid phase to the closed state of the cell contact. This dynamic lipid model is well suited to describe the morphological as well as functional properties of the tight junctions.

Amino Acids↗