Inhibition of bacterial growth by selective interference with the passage of basic amino acids into the cell.
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Amines↗
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Analysis of dansyl amino acids in feedstuffs and skin by micellar electrokinetic capillary chromatography.
Micellar electrokinetic capillary chromatography (MECC) using sodium dodecyl sulphate (SDS) and sodium cholate have been used for analyses of 30 dansylated (Dns) amino acids. The influences of sample preparation, Dns/amino acid ratio, sample solvent composition, and separation conditions including voltage, temperature, pH and buffer composition were investigated. Complete separations of acidic and neutral amino acids were obtained within 45 min in the SDS system. The efficiency expressed as number of theoretical plates for the applied capillary 0.52 m long were between 210,000 and 343,000, and the repeatability was very good with relative standard deviations on relative migration times between 0.09 and 0.70% and on relative normalised peak areas (RNPAs) between 0.85 and 3.41%. The linearity studies gave correlation coefficients between 0.9957 and 0.9993 for RNPAs against concentration. Detection limits were between 3 and 6 fmol or approximately 2 pg of each amino acid. Basic amino acids were separated in a MECC system using sodium cholate. Procedures and problems using Dns derivatisation for amino acids analysed by the MECC methods are described. Finally, examples of analyses of hydrolysates of real complex samples show, that this method can be applied to determine the amino acid composition of proteins in feedstuffs and skin.
Amino Acid Sequence↗
On-line desalting-mass spectrometry system for the structural determination of hydrophilic metabolites, using a column switching technique and a volatile ion-pairing reagent.
A novel desalting method, using a column switching technique and a volatile ion-pairing reagent, pentadecafluorooctanoic acid, was developed. This system allows hydrophilic and cationic compounds in a nonvolatile buffer to be directly introduced into a mass spectrometer for structural elucidation. The desalting procedure consists of four steps: (1) the fractionation of a target compound from a separation column, (2) the removal of salts with pentadecafluorooctanoic acid on the trap column, (3) the desorption of the compound from the trap column, and (4) the re-equilibration of the trap column with a pentadecafluorooctanoic acid solution. In this procedure, we investigated the methods for optimizing the desalting and re-equilibration steps. Various amino acids, including branched chain amino acids, aromatic amino acids, basic amino acids and methionine, after separation with phosphate buffer on a cation-exchange column, were successively desalted by this method, and were observed as protonated ions by mass spectrometry. This desalting system could be useful for the structural elucidation of unknown hydrophilic compounds eluted by conventional high-performance liquid chromatography methods, such as ion-exchange chromatography, with mobile phases containing nonvolatile salts. As an example, we present the structural elucidation of unknown metabolites in bovine serum.
Amino Acids↗
The 4F2hc/LAT1 complex transports L-DOPA across the blood-brain barrier.
L-DOPA is transported across the blood-brain barrier (BBB) by an amino acid transporter, system L. Recently, it has been demonstrated that system L consists of two subunits, 4F2hc and either LAT1 or LAT2. 4F2hc/LAT1 and 4F2hc/LAT2 show different transport characteristics, while their distribution in the brain has not been determined. To clarify whether 4F2hc/LAT1 participates in L-DOPA transport across the BBB, we first examined the expression of 4F2hc/LAT1 in the mouse brain capillary endothelial cell line, MBEC4, as an in vitro BBB model. Northern hybridization and immunoblotting revealed that both 4F2hc and LAT1 are expressed and form a heterodimer in MBEC4 cells. To confirm whether 4F2hc/LAT1 acts as system L to transport L-DOPA, we characterized L-DOPA uptake into the cells. The uptake process was time-dependent, temperature-sensitive, and Na(+)-independent. Neutral amino acids with bulky side chains and a bicyclic amino acid, 2-aminobicyclo-[2, 2,1]-heptane-2-carboxylic acid (BCH), inhibited L-DOPA uptake into MBEC4 cells to a great extent, while an acidic amino acid, basic amino acids, and glycine had no effect. Other neutral amino acids, such as alanine, asparagine, glutamine, serine, and threonine inhibited L-DOPA uptake by 40-70% at most. These characteristics are more compatible with those of 4F2hc/LAT1, rather than those of 4F2hc/LAT2. Finally, immunohistochemistry with anti-LAT1 antibody demonstrated that LAT1 is predominantly expressed in the microvessels of the central nervous system. This is the first report showing that the 4F2hc/LAT1 complex participates in L-DOPA transport across the BBB.
Amino Acid Transport Systems↗
Transport of sugars and amino acids in the intestine: evidence for a common carrier.
D-Galactose, L-arginine, and their respective actively transported analogs are partially competitive inhibitors of the active transport of neutral amino acids in the small intestine of hamsters. Since the aforesaid classes of compounds are all transported by similar, sodium-ion-dependent mechanisms and elicit countertransport of each other, all may share a common, polyfunctional carrier in which a series of separate binding sites, namely, one each for sugars, neutral amino acids, basic amino acids, and Na(+) are joined together, as in a mosaic.
Amino Acids↗
Stimulation of gonadotropin release by a non-GnRH peptide sequence of the GnRH precursor.
The human gonadotropin-releasing hormone (GnRH) precursor comprises the GnRH sequence followed by an extension of 59 amino acids. Basic amino acid residues in the carboxyl terminal extension may represent sites of processing to biologically active peptides. A synthetic peptide comprising the first 13 amino acids (H X Asp-Ala-Glu-Asn-Leu-Ile-Asp-Ser-Phe-Gln-Glu-Ile-Val X OH) of the 59-amino acid peptide was found to stimulate the release of gonadotropic hormones from human and baboon anterior pituitary cells in culture. The peptide did not affect thyrotropin or prolactin secretion. A GnRH antagonist did not inhibit gonadotropin stimulation by the peptide, and the peptide did not compete with GnRH for GnRH pituitary receptors, indicating that the action of the peptide is independent of the GnRH receptor. The GnRH precursor contains two distinct peptide sequences capable of stimulating gonadotropin release from human and baboon pituitary cells.
Animals↗
Metabolic fuel and amino acid transport into the brain in experimental hypothyroidism.
The effect of hypothyroidism in the adult rat on blood-brain barrier and muscle transport of hexoses, neutral amino acids, basic amino acids, monocarboxylic acids, and ketone bodies was examined using single arterial injection-tissue sampling technique. The cerebral blood flow and brain extraction of 3H2O (internal reference substance) was not altered in 3-month-old hypothyroid rats maintained on methimazole, 0.025% in the drinking water, for 7 weeks. The brain uptake index of D-beta-hydroxybutyrate was significantly reduced in hypothyroid rats (2.4 +/- 0.3 vs 4.6 +/- 0.6% p less than 0.001). Hypothyroid rats given thyroid hormone replacement therapy had normal brain uptake of D-beta-hydroxybutyrate (4.4 +/- 0.8%). The brain uptake index of butyrate was also significantly reduced in hypothyroid rats (39.3 +/- 2.1 vs 47.2 +/- 0.74%, p less than 0.001). The brain uptake index of other test substances and muscle uptake of nutrients examined were not altered in hypothyroid rats. These studies indicate that of the four transport systems examined in two tissues, the blood-brain barrier monocarboxylic acid transport system is most susceptible to the hypothyroidism-induced changes.
3-Hydroxybutyric Acid↗
Permeability changes in the blood-brain barrier: causes and consequences.
1. Generalized changes in blood-brain barrier (BBB) permeability are accompanied by extravasation of plasma proteins; thus, they are readily studied with protein markers or protein-dye complexes. Selective changes in permeability involve alterations in BBB transport systems; they are best studied with techniques which detect the qualitative hallmarks of carrier-mediated transport, namely saturation, competition, and stereospecificity. 2. Quantitative assessments of the selective permeability of the BBB can be made from the saturation data expressed in terms of Michaelis-Menten kinetics. The advantages of the latter are twofold: (a) alterations elicited by modified barrier affinity (Km) can be distinguished from alterations in carrier capacity (Vmax); (b) the relative rates of flux of a metabolite across the BBB can be placed in the perspective of cerebral metabolism. Kinetic data on transport processes in the BBB are obtained by either constant infusion or single injection techniques. Results obtained with both methodologies have been comparable. 3. Independent transport systems for glucose, neutral amino acids, basic amino acids, and monocarboxylic acids have been identified in the BBB. The description of these transport systems in kinetic terms provides a background of information on intact mechanisms to which altered transport can be compared. 4. Experimental evidence indicates that the availability of key metabolic substrates, such as glucose or essential amino acids, may be rate-limiting in cerebral metabolism. A working hypothesis was developed that the consequences of a selective change in barrier permeability to one or more of these essential substrates are directly related to altered rates of reaction in substrate-limited pathways, e.g., cerebral protein or neuro-transmitter biosynthesis. 5. Toxicological causes of generalized changes in BBB permeability include hypertonic solutions, organic solvents, surface-active agents, enzymes, and heavy metals. Some agents, e.g., mercury or hypertonic urea, induce selective changes in BBB transport at doses much lower than those required for nonspecific barrier break-down. Subtle changes in transport of metabolic substrates may remain unrecognized unless specifically investigated, yet may have profound consequences on brain metabolism. 6. Pathological processes can also induce selective changes in BBB permeability. Such changes often temporally precede the more generalized alterations in permeability that can occur during pathogenesis. For example, in brain edema due to an ischemic infarct, glucose transport increases during the early cytotoxic phase, whereas generalized changes are not detected until the later vasogenic phase.
Amines↗
[On the tissue non-specificity of basic amino acids and amines in the snail (Helix pomatia L.)].
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Amines↗
Transport of essential nutrients across the blood-brain barrier of individual structures.
The movement of essential substrates from plasma into cerebral structures has been studied in detail in normal alert rats as well as in rats with various metabolic abnormalities. Briefly, radioactive substrates were infused i.v. to rapidly establish and maintain a trace concentration in arterial blood. The rats were killed shortly thereafter, the brain was removed and frozen, and thin sections were cut for quantitative autoradiography. The permeability-to-surface area product (PA) was calculated from the amount of radioactivity accumulated by the brain and the integral of plasma radioactivity. Influx was calculated as the product of PA times plasma substrate concentration. This approach was used to measure the influx of glucose, neutral amino acids, basic amino acids, and ketone bodies. Studies were made of normal rats, rats with portacaval shunts (a model of hepatic encephalopathy), starved rats, diabetic rats, and normal rats infused with ammonium acetate. The results demonstrate specific changes in individual transport systems, which in most cases occurred throughout the brain, although some structures were affected more than others.
Amino Acids↗
Competitive inhibition and exchange transport between arginine and lysine in the process of absorption from the caecum in pigs.
Competitive inhibition and exchange transport between arginine and lysine in the process of absorption from the caecum in pigs. Acta Physiol. Pol., 1978, 29 (2): 161--165. Arginine competes with lysine in the process of transport across the mucosal epithelium of the pig caecum, and the ability of arginine absorption is much greater than that of lysine. Absorption of large amounts of arginine causes secretion of large amounts of lysine into the intestinal lumen in the process of primary active exchange transport. Lysine is better absorbed from solution containing no other amino acids than from solutions containing mixtures of amino acids. Basic amino acids (Arg, Lys, His) stimulate the absorption of serine and threonine from the caecum of pigs.
Animals↗
Comparison of procedures for extracting free amino acids from polymorphonuclear lekocytes.
We studies five methods for extracting amino acids from human polymorphonuclear leukocytes. Both the use of cell lysis and of a deproteinizing agent interfere with quantitative determination of the amino acids, basic amino acids being the most sensitive to the extraction procedure. Among the methods used, disruption of the cells by freezing-thawing is the best method for extracting all the amino acids. Taurine is the only amino acid extracted in the same amount by all the methods studied, and it represents half of the intracellular pool.
Adult↗
Contributions of multiple basic amino acids in the C-terminal region of yeast ribosomal protein L1 to 5 S rRNA binding and 60 S ribosome stability.
Previous studies suggest that the C-terminal region of ribosomal protein L1 from Saccharomyces cerevisiae is important for its interaction with the 5 S rRNA molecule. Within this region are several highly conserved basic amino acids including Lys276, Lys279, Lys289, Arg282, Arg285. To examine potential contributions of these amino acids to RNA-protein interaction and ribosomal assembly, effects of substitutions of these residues by methionine either individually or in combinations were examined. A methionine substitution of any one of the lysine residues did not significantly affect RNA binding in vitro. The mutant RNPs were as stable as the wild-type RNP. Yeast transformants expressing these mutant proteins grew at the same rate as the wild-type. However, mutant proteins containing substitutions of any two of these basic amino acids bound RNA weakly. The resultant RNPs were significantly less stable than the wild-type. Whereas cells expressing mutant L1 with a single substitution at 289 was not lethal, cells expressing mutant L1 with any double substitutions involving Lys289 as one of the substituted amino acids were lethal. These data suggest that Lys289 plays a key role in the binding of ribosomal protein L1 to 5 S rRNA. The other basic residues, particularly Arg282, and Arg285, in this region also contribute to RNA binding. These residues are predicted to locate on the same side of an alpha helix. We would like to propose a structural model for the yeast RNP that involves multiple contact sites located on one side of the helix in the C terminus of the protein and the 5 S rRNA. These basic amino acids also participate, directly or indirectly, in the interaction of the RNP complex with other components of the 60 S ribosomal subunit.
Amino Acid Sequence↗
An analysis of target preferences of Escherichia coli outer-membrane endoprotease OmpT for use in therapeutic peptide production: efficient cleavage of substrates with basic amino acids at the P4 and P6 positions.
The Escherichia coli outer-membrane endoprotease OmpT mainly cleaves peptide bonds between consecutive basic amino acids. The effect of adjacent residues on cleavage efficiency is currently unknown, except at positions P2 and P2'. Therefore we investigated the effects of amino acid residues upstream of the cleavage site on the ability of OmpT to cleave efficiently a fusion protein carrying human glucagon-like peptide-1 (7-37) in 4 M urea. The P1-P10 residues were replaced by Ala and each substrate was subjected to OmpT digestion. The replacement of Arg residue at P1 blocked the cleavage due to the loss of the cleavage site, and the replacement of Arg residue at P4 maximally reduced the cleavage rate. Conversely, cleavage efficiency increased on replacing Glu at P6. Substitution of the residues at P4 and P6 with several different amino acids showed that OmpT preferred basic residues at these positions, whereas acidic residues had a negative effect. This was also shown to be true with synthetic decapeptide substrates in the absence of urea. The k(cat)/ K(m) ratio increased with basic residues at P4 or P6, mainly due to a lower K(m) rather than an increase in k(cat). On the basis of these findings, we prepared a fusion protein carrying human atrial natriuretic peptide (ANP), a drug for acute congestive heart failure. OmpT released mature ANP from the E. coli-expressed fusion protein. As expected, the introduction of an Arg residue at P4 and P6 enhanced the release of ANP.
Amino Acid Sequence↗
Formation of cyanomethyl derivatives of basic amino acids and proteins with components in cigarette smoke.
A reaction of the basic amino acids, lysine and arginine, with components of cigarette smoke has been observed. The adducts produced have been identified as cyanomethyl derivatives. Both formaldehyde and cyanide, which are known to be present in cigarette smoke, are involved in the reaction with the primary amino group. The reaction is time-dependent and can be enhanced by an increase of temperature or by incubation under alkaline conditions. Cyanomethyl adduct formation was found to be increased when smoke from cigarettes with higher tar and nicotine content was used. When proteins, such as bovine serum albumin, trypsin inhibitors or crude rat lung proteins were incubated with the cigarette smoke solution, new protein adducts with increased pI values were produced which are separable from the original proteins by gel isoelectric focussing. Radioisotopically labelled cyanide can be irreversibly linked to protein and the linkage is enhanced in the presence of formaldehyde.
Arginine↗
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.
Amino Acid Sequence↗
Vascular activity of polycations and basic amino acids: L-arginine does not specifically elicit endothelium-dependent relaxation.
Irrespective of their stereochemistry (D- or L-form), polycations such as poly-lysine, poly-arginine and poly-histidine elicited endothelium dependent relaxation of pre-contracted rat aortic rings in a dose-dependent manner (ED50 less than or equal to 10-7 M). In contrast, the basic amino acids arginine, glutamine, histidine and lysine caused only endothelium-potentiated relaxation at high concentrations (ED 50 greater than 10-3 M). Both heparin (1U/ml) and dextran sulphate (10 microgram/ml) abolished relaxation by the polycations but had no effect on the responses to the basic amino acids or acetylcholine. These results indicate that the vasodilatory property of the polycations is due to an electrostatic interaction with anionic domains on the endothelial surface, whereas the basic amino acids elicit a non-specific relaxation. Therefore, L-arginine per se cannot be the immediate precursor of nitric oxide, the proposed endothelium-derived relaxing factor.
Acetylcholine↗
Na+-gradient dependence of basic amino acid transport into rat intestinal brush border membrane vesicles.
Uptake of L-arginine and L-lysine into brush border membrane (BBM) vesicles from rats fed either a high protein (HP) or a high carbohydrate (HC) diet was studied under conditions of a transmembrane Na+-gradient (Na+out greater than Na+in) or a Na+-equilibrium (Na+out = Na+in). The Na+-gradient caused a stronger stimulation of basic amino acid transport across the BBM in group HP than in group HC. Replacing Cl- as counter ion of Na+ by SCN- in the Na+-gradient experiments did not affect L-lysine uptake. In one series of experiments inhibitory effects of several amino acids and D-glucose on Na+-gradient-dependent L-arginine transport into BBM vesicles were tested. L-Lysine showed the strongest inhibition of L-arginine uptake, but also L-leucine, L-alanine and D-glucose caused a significant decrease of L-arginine uptake into BBM vesicles. The main conclusions are that a transmembrane Na+-gradient is able to energize the transport of basic amino acids across the intestinal BBM and that the transport mechanism of the intestinal BBM for basic amino acids adapts to the protein content of the diet.
Amino Acids↗