Intestinal handling of two tetrapeptides by rodent small intestine in vitro

Uptake of free Leu and Ala and uptake of these amino acids from the tetrapeptides Leu-Gly-Gly-Gly and Ala-Gly-Gly-Gly has been studied in rings of everted rodent jejunum in vitro. When mediated uptake of free Leu was virtually saturated addition of Leu-Gly-Gly-Gly gave no significant increase in uptake of Leu. Uptake of Leu and of Ala from the tetrapeptides was strongly inhibited by Met, as was uptake of these amino acids from free solution. The results did not suggest that either tetrapeptide was taken up intact by the jejunum.     

Modulation of glutamine uptake and phosphate-activated glutaminase activity in rat brain mitochondria by amino acids and their synthetic analogues

Uptake of L-[14C]Gln and phosphate-activated glutaminase (PAG) activity were measured in nonsynaptic mitochondria isolated from rat cerebral hemispheres, in the presence of protein and nonprotein amino acids and their synthetic structural analogues and derivatives. The uptake was inhibited by > 50% in the presence of a 10-fold excess of His, homocysteine (Hcy), Trp, Leu, Tyr, Ile, Thr, Ala, Phe, Met, Ser, by > 20% in the presence of a 10-fold excess of Val, Arg, Glu, and was not affected by a 10-fold excess of Orn, alpha-ketoglutarate, Tau and Pro. Uptake of L-[14C] Leu differed from Gln uptake by its resistance to Arg, Glu, and a relatively high sensitivity to the reference inhibitor of the plasma membrane transport of large neutral amino acids (L-system)--BCH (2-aminobicyclo[2.2.1]heptane-2-carboxylic acid), and a number of natural L-system substrates. A newly synthesized alanine analogue, 2'-cyano-(biphenyl) alanine, referred to as MRC01, was the only compound tested that inhibited Gln uptake more strongly than Leu uptake. The strongest Gln uptake inhibitors: MRC01, His, Hcy and Leu, inhibited PAG activity by > 50% when added at the inhibitor/Gln concentration ratio of 1:2. PAG activity was not affected by Tau, Lys or Pro, compounds which did affect Gln uptake. The results suggest that a number of natural amino acids function as common endogenous modulators of cerebral mitochondrial Gln uptake and its degradation. MRC01, because of its inhibitory potency towards both mitochondrial Gln uptake and PAG activity, may become a convenient tool in studying the role of Gln transport in its mitochondrial metabolism in intact CNS cell and tissues.     

Uptake, metabolism and distribution of organic and inorganic nitrogen sources by Pinus sylvestris

Although an increasing number of studies show that many plant species have the capacity to take up amino acids from exogenous sources, the importance of such uptake for plant nitrogen nutrition is largely unknown. Moreover, little is known regarding metabolism and distribution of amino acid-N following uptake or of the regulation of these processes in response to plant nitrogen status. Here results are presented from a study following uptake, metabolism, and distribution of nitrogen from NO(3)(-) NH(4)(+), Glu, or Ala in Scots pine (Pinus sylvestris L). In a parallel experiment, Ala uptake, processing, and shoot allocation were also monitored following a range of pretreatments intended to alter plant C- and N-status. Uptake data, metabolite profiles, N fluxes through metabolite pools and tissues, as well as alanine aminotransferase activity are presented. The results show that uptake of the organic N sources was equal to or larger than NH(4)(+) uptake, while NO(3)(-) uptake was comparatively low. Down-regulation of Ala uptake in response to pretreatments with NH(4)NO(3) or methionine sulphoximine (MSX) indicates similarities between amino acid and inorganic N uptake regulation. N derived from amino acid uptake exhibited a rapid flux through the amino acid pool following uptake. Relative shoot allocation of amino acid-N was equal to that of NH(4)(+) but smaller than for NO(3)(-) Increased N status as well as MSX treatment significantly increased relative shoot allocation of Ala-N suggesting that NH(4)(+) may have a role in the regulation of shoot allocation of amino acid-N.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

Amplification of bacterial plasmids without blocking protein biosynthesis

The effect of amino acids (presence or absence from the growth media) and metal ions on the replication of Escherichia coli plasmids in rel A+ strains was studied. It was found that: (i) The absence of one amino acid from the growth media had no effect on the plasmid copy number in prototrophic E. coli strains: (ii) The presence of only one amino acid in artificial media free of amino acids had a negligible effect on the plasmid copy number for the amino acids Ala, Arg, Glu, His, Leu, Phe, Thr, Trp, and Tyr: (iii) The combination of Met and Thr caused a rise in pBR322 plasmid copy number up to 90-100 plasmid copies per cell: (iv) The Fe3+ concentration had an amplification effect on E. coli plasmids. The pBR322 plasmid copy number for media free of amino acids and supplemented with 0.2-0.4 mM FeCl3 was 60-80 plasmid copies per cell: (v) The combination of Fe3+ with certain amino acids (Ala, Arg, Glu, Leu, Thr, and Trp) leads to a dramatic increase in the plasmid copy number reaching 180-270 plasmid copies per cell for the plasmid pBR322 and 20-24 for the plasmid pR100.     

METABOLISM OF AMINO ACIDS IN INCUBATED SLICES OF MOUSE BRAIN1

• 1 Slices of mouse brain were incubated with [U-¹⁴C]alanine, valine, leucine, phenylalanine, proline, histidine, lysine, arginine or aspartic acid, and the extent of metabolism was estimated by analyses utilizing paper chromatography of the tissue extracts and with an amino acid analyser.

• 2 The metabolism of Ala and Asp was high; of Leu and Pro, moderate; and of Lys, Arg and Phe, low; the metabolism of Val and His was not significant. The time-course of metabolism in most cases showed varying rates, indicating heterogeneous metabolic compartments for the amino acids.

• 3 Production of CO₂ was high from Asp, moderate from Ala, and low from Leu; the other amino acids were not oxidized to CO₂ to any significant extent. A large portion of the metabolized label was trapped in the form of Glu or Asp.

• 4 Metabolism increased with increasing concentration of amino acid to some extent and was largely inhibited by omission of glucose, by anaerobic conditions, or by cyanide. Although these conditions also inhibit uptake, the time-course and extent of inhibition uptake and metabolism were different.

• 5 With Asp, Ala and Phe, metabolism was lowest in slices from pons-medulla; the brain area exhibiting the highest metabolism differed for each amino acid. The metabolism of Asp was lower in brain samples from newborn than in those from adults; the metabolism of Leu was higher in slices from newborn brain.

• 6 The results indicate that the majority of the amino acids can be metabolized in brain tissue and that the metabolic rates are influenced by a number of factors, among them the level of amino acids and the level of available energy.

     

Structural and functional analysis of critical amino acids in TMVI of the NHE1 isoform of the Na+/H+ exchanger

The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) resides on the plasma membrane and exchanges one intracellular H(+) for one extracellular Na(+). It maintains intracellular pH and regulates cell volume, and cell functions including growth and cell differentiation. Previous structural and functional studies on TMVI revealed several amino acids that are potentially pore lining. We examined these and other critical residues by site-directed mutagenesis substituting Asn227→Ala, Asp, Arg; Ile233→Ala; Leu243→Ala; Glu247→Asp, Gln; Glu248→Asp, Gln. Mutant NHE1 proteins were characterized in AP-1 cells, which do not express endogenous NHE1. All the TMVI critical amino acids were highly sensitive to substitution and changes often lead to a dysfunctional protein. Mutations of Asn227→Ala, Asp, Arg; Ile233→Ala; Leu243→Ala; Glu247→Asp; Glu248→Gln yielded significant reduction in NHE1 activity. Mutants of Asn227 demonstrated defects in protein expression, targeting and activity. Substituting Asn227→Arg and Ile233→Ala decreased the surface localization and expression of NHE1 respectively. The pore lining amino acids Ile233 and Leu243 were both essential for activity. Glu247 was not essential, but the size of the residue at this location was important while the charge on residue Glu248 was more critical to NHE1 function. Limited trypsin digestion on Leu243→Ala and Glu248→Gln revealed that they had increased susceptibility to proteolytic attack, indicating an alteration in protein conformation. Modeling of TMVI with TMXI suggests that these TM segments form part of the critical fold of NHE1 with Ile233 and Leu465 of TMXI forming a critical part of the extracellular facing ion conductance pathway.     

Postnatal amino acid uptake by the rat small intestine. Energetics of membrane transport systems for amino acids in the developing jejunum

The energetics of amino acid uptake by the developing small intestine was investigated in vitro. L-valine, L-leucine, L-phenylalanine, L-methionine, L-lysine and L-arginine were all actively transported by the newborn rat jejunum. Metabolic inhibitors (e.g. 2,4-dinitrophenol) significantly reduced uptake of all amino acids but uptake against a concentration gradient was not totally abolished. Uptake of all amino acids was reduced at low[Na+]. Inhibition of transport of neutral amino acids by reduced luminal [Na+] was greater than that of basic amino acids, and the tissue was barely able to concentrate the neutral amino acids. [Na+] affected the Michaelis constant (Km) of neutral transport systems for their substrates; for the basic amino acids Km values were unaffected by the presence or absence of Na+. Ouabain significantly inhibited neutral amino acid uptake but had no effect on L-lysine or L-arginine uptake. These results are discussed in terms of the Na+ gradient hypothesis for amino acid transport, and the site of energy input to active transport. The role of glycolysis in providing energy for intestinal transport in the neonatal rat and the efficiency of Na+ dependent and independent transport mechanisms are considered. It is concluded that the energetics of amino acid transport systems in neonatal and adult rats are essentially similar.     

Amino acid uptake systems in Bacteroides ruminicola

Uptake of amino acids by Bacteroides ruminicola was observed in cells grown in a complete defined medium, containing ammonia as the nitrogen source. A high rate of uptake occurred only in fresh medium, as an inhibitory substance, possibly acetate, apparently accumulated during growth. All amino acids except proline were taken up and incorporated into cold trichloroacetic acid precipitable material. Different patterns of incorporation and different responses to 2,4-dinitrophenol and potassium ferricyanide indicated multiple uptake systems were involved. Kinetic inhibition patterns suggested six distinct systems were present for amino acid uptake, with specificities related to the chemical structures of the amino acids. Thus, the failure of free amino acids to act as sole nitrogen sources for growth of B. ruminicola is not due to the absence of transport systems for these compounds.     

Formation of N-[2(3-Alkylpyrazin-2-on-l-yl)acyl]amino Acids or -peptides on Heating Tri- or Tetrapeptides with Glyoxal

The reaction of tri- and tetrapeptides with glyoxal at 100°C and pH 5.0 was studied. A series of new pyrazinone compounds was isolated from the reaction solutions of tri- and tetrapeptides with glyoxal: N-[2(3-alkylpyrazin-2-on-l-yl)acyl] amino acids (I) and 2-(3-alkyl- pyrazin-2-on-l-yl)aIkyl acids (II) from tripeptides, (I), (II) and N-[2(3~alkylpyrazin-2-on-l-y]) acyl]-dipeptides (III) from tetrapeptides. Their chemical structures were determined by UV, IR, MS and NMR spectroscopy.

Aldehydes and free amino acids were also detected as reaction products. The amino acids were proved to be derived from the C-terminals of the peptides. Reaction mechanisms for the reaction of tri- and tetrapeptides with glyoxal were also proposed.     

Leishmania amazonensis: uptake and hydrolysis of 3H-amino acid methyl esters by isolated amastigotes

Intracellular and isolated amastigotes of Leishmania amazonensis can be destroyed by L-amino acid methyl esters known to disrupt mammalian lysosomes. To evaluate the mechanism(s) involved in the leishmanicidal activity, we examined the uptake and hydrolysis of tritiated esters by isolated amastigotes. After incubation with the labeled compounds, parasites were recovered, were washed on filters, and their radioactivity was determined. Alternatively, amastigotes were separated from the medium by centrifugation through oil, and the radioactivity associated with free or esterified amino acids was measured after thin-layer chromatography. The results showed that the methyl esters of Trp, Leu, and Met, which are leishmanicidal, accumulated in and were rapidly hydrolysed by the amastigotes. [3H]Leu derived from [3H]Leu-OMe remained associated with the amastigotes even after a 1-hr chase in label-free medium, but the ester species was rapidly lost upon washing of the parasites. In contrast, the esters of Ile and Ala, which are not leishmanicidal, were only slowly hydrolysed, and most of the radioactivity was lost upon washing. We have previously shown that certain amino acid esters and weak bases protect Leishmania from damage by leucine methyl ester (Leu-OMe). In the present experiments, these compounds reduced, in concentration-dependent fashion, the hydrolysis of [3H]Leu-OMe and the accumulation of [3H]Leu in the amastigotes. Overall, the results indicate that, as in lysosomal disruption, leishmanicidal activity is associated with ester hydrolysis and amino acid accumulation in the parasites. The nature and location of the parasite esterolytic enzymes requires additional investigation.     

A judgment on postmortem aging in longissimus dorsi based on a peptide substrate library

We attempted to develop a method to determine easily and effectively the degree of postmortem aging of pork longissimus dorsi (LD) by measuring the activity of proteases in the LD using fluorogenic peptide substrates. LD was used to measure the change with time in the protease activity detected with these substrates. Determining the variations within the LD muscles, strong positive correlations were found between changes in hardness and fluorescence intensities against Ac-Ala-MCA, Ac-Met-MCA, Ac-Ser-MCA, Ac-Thr-MCA, and Ac-Ala-Phe-MCA (P<0.005), and strong negative correlations were found between changes in total amounts of free amino acids and Ac-Ala-MCA, Ac-Met-MCA, Ac-Ser-MCA, Ac-Thr-MCA, and Ac-Ala-Phe-MCA (P<0.001). Negative correlations were also observed between changes in the amounts of free Ala, Arg, Lys, Leu, Met, Phe, and Tyr and the fluorescence intensities against Ala, Arg, Lys, Leu, Met, Phe, and Tyr-MCA respectively (P<0.001).     

Analyses of Soluble Protein,Total and Free Amino Acids in Leaves of Different Ecotypes of Phragmites communis Growing in the Hexi Corridor

The difference of total and free amino acids and protein extracted from the leaves of four different reed ecotypes growing in Hexi corridor of Gansu Province were investigated. In all of the different reed ecotypes, the content of Asp, Glu, Gly, Leu and Ala in total amino acids were high, while the contents of Ala, Phe, Met and Thr, Pro in total amino acids varied among different reed ecotypes. Albeit Ala, Glu, Asp, Gly and Ser were the chief composition of free amino acids in leaves of all reed ecotypes, but temarkble difference was found in the content of each free amino acid from different reed ecotypes. The content of free Pro in leaves of salt meadow and salt meadow-sand dune transitional zone reed were 3.5 and 1.6 times respectively as much as in leaves of swamp reed. Swamp reed had 11 soluble proteins whereas other three reed ecotypes show that each has 13 soluble proteins. Three “salt adaptation proteins” (66 kD, 40.3 kD, 16.5 kD) were found in leaves of three reed ecotypes with varying degree of salt stress, however, the contents of 3 “salt adaptation protens” showed a negative correlation with the degree of salt stress. There was a large amount of “special protein” (11.7 kD) in leaves of sand dune reeds. These results suggest that the difference in cytogene expression takes a priority basis of adaptation of reed plants to different habitats, while a closer relationship of reeds tolerance to salt or drought stress with Pro accumulation in cells is seen than with the of accumulation stress adaptation protein.     

Synthesis of Photolabile Caged Amino Acids for Measuring Amino Acid Transporters

Photolabile precursors (caged compounds) of amino acids such as Ala, Leu, Lys, and Ser were prepared by some simple reactions. These compounds were designed for the rapid, photochemically initiated release of amino acids. These amino acid transporters were expressed in Xenopus oocyte by injecting mRNA prepared from rat kidney. The electrical response of each transporter was examined by applying the amino acids and caged compounds before and after photolysis. Photolysis of the caged amino acids increased the electrical response of the facilitated amino acid transporters expressed in the oocyte. Consequently, these synthesized caged amino acids would be applicable to kinetic investigations on the transporters when combined with a pulsed laser or xenon arc flash lamp.     

烧伤患者血浆和红细胞游离氨基酸变化及输注氨基酸对其变化的影响

动态测定烧伤患者血浆及红细胞内游离氨基酸的含量 ,探讨输入外源性氨基酸后对血及红细胞内游离氨基酸的影响。以日立 835— 5 0型氨基酸自动分析仪测定烧伤患者血浆及红细胞内游离氨基酸含量。结果发现烧伤患者血浆总游离氨基酸浓度从伤后到 2 1天均显著降低 (P <0 .0 5～ 0 .0 1) ;赖、苯丙和苯丙 酪氨酸比值显著升高 (P <0 .0 5～ 0 .0 1) ;色、组、精、丙、甘、苏、脯和丝氨酸比值显著降低 (P <0 .0 5～ 0 .0 1) ;缬、亮、异亮、酪、胱和支链氨基酸伤后早期降低。烧伤患者红细胞内总游离氨基酸含量不同程度降低 ,其中 1、3、7天降低显著 (P <0 .0 5～ 0 .0 1) ;红细胞内苯丙和苯丙 酪氨酸比值未见显著性升高 ;色、蛋、精、脯氨酸含量很低或基本未测出。输注复合氨基酸注射液后未能显著改善患者血及红细胞内游离氨基酸含量。结果提示烧伤患者红细胞内游离氨基酸含量的变化趋势与血浆游离氨基酸变化趋势基本一致 ;烧伤后红细胞内苯丙氨酸及苯丙 酪氨酸比值有别于血浆变化。本研究条件下补充外源性氨基酸未能显著改变烧伤患者血浆及红细胞内游离氨基酸的含量     

Metal ion-binding ability of tetrapeptides containing alpha-aminoisobutyric acid.

alpha-Aminoisobutyric acid (Aib), one of the Calpha,alpha-disubstituted glycines, is a sterically hindered amino acid that acts as a conformational constraint in peptides. However, studies for the application of the ability of Aib to control conformation are quite few. The paper focuses on the molecular recognition ability of acyclic oligopeptides containing Aib. Liquid-liquid extraction of nine kinds of metal ions from aqueous layers to nonpolar organic layers with acyclic tetrapeptides, X-Trp-Xaa2-Gly-Xaa4-NH-Ar (X = H or C6H5CH2OCO (Z), Xaa2 = Aib or Gly, Xaa4 = Leu or Ala, Ar = phenyl or 3,5-dimethylphenyl) was examined using picrate as the anion of ion pairs. The extraction behaviour of the metal ions with the tetrapeptides was investigated in the pH range from 3 to 9. In the case of basic pH regions, Cu(II) and Ag(I) were effectively extracted with Trp-Aib-Gly-Leu-NH-Ar. Pd(II) was specifically extracted with Trp-Aib-Gly-Leu-NH-Ar in acidic pH regions. The extraction percent (%E) of the peptide host, which has a 3,5-dimethylphenyl group, was even larger than that of the host, which has a phenyl group. Moreover, Pd(II) was extracted with a peptide host which has Leu and a 3,5-dimethylphenyl group in the absence of picrate as the anion of ion pairs. The free alpha-amino group, the turn conformation and the hydrophobicity of peptide molecules were important factors for the extraction of the metals.     

癌肿与氨基酸代谢的研究

研究了癌肿与氨基酸代谢的关系。这些癌肿包括喉癌HepⅡ细胞 ,急性非淋巴细胞白血病和急性淋巴细胞白血病 ,结果表明 :( 1 )喉癌细胞株培养过程中亮氨酸、赖氨酸、丝氨酸、天冬酰胺、异亮氨酸、甘氨酸以及苏氨酸等水平明显降低 ,而色氨酸水平明显增加 ,说明喉癌细胞的生长繁殖必须依赖以上 7种氨基酸同时释放了色氨酸 ;( 2 )急性非淋巴细胞白血病 (ANLL)患者血浆中的谷氨酸、甘氨酸、亮氨酸、苯丙氨酸、酪氨酸和色氨酸等水平明显升高 ,而苏氨酸、组氨酸、丙氨酸等水平明显降低 ,这些结果与国际报道相一致 ;( 3)经治疗后 ,ANLL患者血浆中甘氨酸、色氨酸和苯丙氨酸等水平明显降低 ,而丙氨酸、组氨酸等水平明显升高 ,表明肿瘤细胞处在无氧代谢。患者经治疗后色氨酸和苯丙氨酸水平降低和组氨酸水平的升高对患者预后是有益的 ;( 4)急性淋巴细胞白血病患者血浆中苯丙氨酸、赖氨酸、色氨酸和酪氨酸水平提高 ,这些氨基酸能促进肿瘤生长 ,而门冬酰胺、谷氨酰胺以及天冬氨酸水平降低 ,说明这 3种氨基酸为肿瘤生长所必须。此外还发现ALL患者外周淋巴细胞中精氨酸水平增加 ,精氨酸对癌肿细胞有直接杀伤作用。     

Differential effect of amino acid residues on the stability of double helices formed from polyribonucleotides and its possible relation to the evolution of the genetic code

The interaction of amino acid residues with polyribonucleotides was characterized by measurements of melting temperatures (tm) for poly(A).poly(U) and poly(I).poly(C) as functions of the concentrations of various amino acid amides. The amides of hydrophilic amino acids lead to a continuous increase of tm with increasing concentration, whereas amides of hydrophobic amino acids induce a decrease of tm at low concentrations (approximately 1 mM) followed by an increase at higher concentrations. Analysis of the data by a simple site model provides the affinity of each ligand for the double helix relative to that for the single strands. This parameter decreases in the order Ala greater than Gly greater than Ser greater than Asn greater than Pro greater than Met, Val greater than Ile, Leu for poly(A).poly(U) and Ala, Gly, Ser greater than Asn greater than Pro greater than Val greater than Ile, Met, Leu for poly(I).poly(C). The special effects of hydrophobic amino acids may be related to the similarity of the codons for these amino acids. A simple model for assignment of codons to amino acids is proposed.     

Bicyclic peptides. VI. Synthesis, conformation, and ion-binding of two bicyclic nonapeptides

Two related homodetic bicyclic nonapeptides (cyclo Glu-X-Pro-Gly-Lys-X-Pro-Gly)-cyclo (l gamma----5 epsilon), X = Ala(BCP2), X = Leu(BCP3) have been synthesized using conventional solution phase methods involving mixed anhydride coupling reactions starting with appropriately protected naturally occurring amino acids. The conformation and ion binding properties of BCP2 have been studied by nuclear magnetic resonance and circular dichroism techniques. The results of these studies have been compared to those of BCP3. The presence of Ala caused both Ala-Pro bonds to be trans in free BCP2. This characteristic imparted subtle differences to the ion-binding properties of BCP2 as compared to free BCP3 which has one cis Leu-Pro bond and one trans Leu-Pro bond.     

The ontogeny of the uptake systems for glutamate,GABA, and glycine in synaptic vesicles isolated from rat brain

The ontogeny of the uptake of glutamate, GABA and glycine into synaptic vesicles isolated from rat brain has been investigated. The vesicular uptake of the three amino acids increased with developmental age in parallel with synaptogenesis, indicating a functional role of uptake of the amino acids by synaptic vesicles in the nerve terminals. Uptake of the amino acids by plasma membrane particles (synaptosomes) in brain homogenate showed a somewhat different developmental profile. The uptake of glutamate increased markedly with developmental time, while the uptake of GABA showed only a slight increase. Uptake of glycine by plasma membrane particles was very low and therefore not registered. The observed developmental increase in uptake of glycine by synaptic vesicles isolated from brain, supports previous reports indicating that glycine can be taken up by vesicles from non-glycine terminals.Special issue dedicated to Dr. Morris H. Aprison.