Utilization of amino acids and dipeptides by Lactobacillus plantarum from orange in nutritionally stressed conditions

Aims:  To investigate amino acid and dipeptide utilization by Lactobacillus plantarum N4 isolated from orange peel, in a nutritionally depleted medium based on MRS (Mann, Rogosa, Sharpe).
Methods and Results:  In MRS with 0·1 g l⁻¹ of meat extract and without peptone and yeast extract, growth increased when essential and stimulatory amino acids and nonessential amino acid were added to the medium. Replacement of the essential amino acid, leucine, and the nonessential amino acid, glycine, by leucyl-leucine (Leu-Leu) and/or glycyl-glycine (Gly-Gly) significantly enhanced growth. Essential amino acids were mainly consumed and the dipeptides were almost completely used at the end of growth. Leucine and glycine accumulated internally from the peptides were higher than from the free amino acids. Glucose utilization increased in the media containing dipeptides compared with the medium containing free amino acids.
Conclusions:  In a N-depleted medium, Leu-Leu and/or Gly-Gly were more effective than the respective amino acids in supporting growth of the micro-organism. The more efficient internal accumulation of glycine and especially leucine from dipeptides confirmed the ability of the strain to assimilate mainly complex nitrogen molecules rather than simple ones.
Significance and Impact of the Study:  The ability of Lact. plantarum N4 to efficiently use dipeptides could contribute to spoilage development in the natural medium of the organism, orange juice.     

Inhibition by Peptides of Amino Acid Uptake by Bacterial Populations in Natural Waters: Implications for the Regulation of Amino Acid Transport and Incorporation

To investigate the regulatory interactions of amino acid transport and incorporation, we determined the effects of dipeptides on amino acid uptake by bacteria in an estuary and a freshwater lake. Dipeptides noncompetitively inhibited net transport and incorporation of amino acids into macromolecules but had no effect on the ratio of respiration to incorporation. Nearly maximum inhibition occurred at peptide concentrations of <10 nM. In contrast, the initial uptake rate of glycyl-[¹⁴C]phenylalanine was not affected by glycine or phenylalanine. Net amino acid transport appeared to be inhibited by the increased flux into the intracellular pools, whereas the incorporation of labeled monomers into macromolecules was isotopically diluted by the unlabeled amino acids resulting from intracellular hydrolysis of the dipeptide. Chloramphenicol, sodium azide, and dinitrophenol all inhibited the initial uptake rate of leucine and phenylalanine. These results suggest that in aquatic environments amino acids are taken up by active transport which is coupled closely to protein synthesis.     

Effect of Dipeptides on the Growth of <Emphasis Type="Italic">Oenococcus oeni</Emphasis> in Synthetic Medium Deprived of Amino Acids

Oenococcus oeni has numerous amino acid requirements for growth and dipeptides could be important for its nutrition. In this paper the individual or combined effect of dipeptides on growth of O. oeni X₂L in synthetic media deficient in one or more amino acids with L-malic acid was investigated. Utilization of dipeptides, glucose, and L-malic acid was also analyzed. Dipeptides were constituted by at least one essential amino acid for growth. Dipeptides containing two essential amino acids, except leucine, had a more favorable effect than free amino acids on the growth rate. Gly-Gly was consumed to a greater extent than Leu-Leu and a rapid exodus of glycine to the extracellular medium accompanied it. The microorganism could use glycine in exchange for other essential amino acids outside the cell, favoring growth. In the presence of Leu-Leu, the increase in glucose consumption rate could be related to the additional energy required for dipeptide uptake.     

Studies on a wide-spectrum intestinal dipeptide uptake system in the monkey and in the human.

1. The intestinal transport of glycine and leucine residues of glycyl-L-leucine was studied in the monkey and in the human in vitro. Uptake of both [14C]glycyl-L-leuine and glycyl-L-[14C]leucine show similar Kt values, but there is a marked difference in the Vmax. values. Preliminary studies suggest that this anomalous difference in the Vmax. values may be due to the greater efflux rate of glycine from the tissue. 2. Arrhenius plots of both [14C]glycyl-L-leucine uptake and glycyl-L-[14C]leucine uptake in the monkey intestine show a discontinuity at about 20 degrees C. The activation energies above and below the discontinuity are similar for both [14C]glycyl-L-leucine uptake and glycyl-L-[14C]leucine uptake. These similarities in uptake characteristics suggest that the dipeptide glycyl-L-leucine is transported as one unit. 3. In the monkey intestine, glycyl-L-leucine uptake is inhibited by a wide variety of dipeptides, including those containing acidic and basic amino acids. The inhibition was shown to be competitive by using four representative dipeptides namely: L-alanyl-L-alanine, L-alanyl-L-leucine, L-glutamyl-L-glutamic acid and L-lysyl-L-lysine. The results strongly suggest that in the monkey intestine there may be a dipeptide-uptake system with an extremely broad specificity. These results were also confirmed in the human in a limited way.     

Identification of histidyl and thiol groups at the active site of rabbit renal dipeptide transporter

Active transport of dipeptides in rabbit renal brush-border membrane vesicles is energized by an inward-directed H+ gradient rather than a Na+ gradient. We examined the effects of treatment of membrane vesicles with diethylpyrocarbonate (DEP), a reagent specific for histidyl groups, on this H+ gradient-dependent dipeptide uptake. DEP inhibited the uptake of all three dipeptides studied, Gly-sarcosine, Gly-Gly, and Gly-Pro (Ki = 0.6-0.9 mM), and the inhibition was noncompetitive. The dipeptide transporter could be protected from DEP inhibition by the presence of dipeptide substrates during the treatment of the vesicles with the inhibitor, whereas leucine plus Na+ failed to offer the protection. Na+-dependent leucine uptake was also inhibited by DEP (Ki = 2.5 mM) and the amino acid transporter could be protected from the inhibition by leucine plus Na+, but not by dipeptides. Treatment of membrane vesicles with the thiol group-specific reagents, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole,3-bromopyruvate, p-chloromercuribenzenesulfonic acid, and N-ethylmaleimide, also inhibited the H+ gradient-dependent dipeptide uptake. The potency of their inhibition was in the order: 7-chloro-4-nitrobenz-2-oxa-1,3-diazol greater than p-chloromercuribenzenesulfonic acid greater than 3-bromopyruvate greater than N-ethylmaleimide. The inhibition could be reversed in some cases by treatment of the membrane vesicles with reducing agents such as 2,3-dimercaptopropanol following incubation with the inhibitors. Dipeptide substrates could protect the dipeptide transporter from the inhibition. We conclude that histidyl and thiol groups are present at or near the substrate-binding site of the rabbit renal dipeptide transporter.     

Fungicidal Activity of Oxadiazolyl Sulfides

Glycyl-l-leucine is one of the best substrates for peptide hydrolases in the intestinal mucosa. Its absorption and hydrolysis were investigated in epithelial cells isolated from the rat intestine in the presence of bestatin, a specific inhibitor of certain peptide hydrolases, Bestatin competitively inhibited dipeptide hydrolase activities in isolated cells with a K_i value of 10^?8 m, but noncompetitively inhibited, and less significantly, the dipeptide absorption by isolated cells. At 10^?4 m bestatin inhibited half the dipeptide absorption, but only minimally inhibited the absorption of constituent amino acids. In the presence of bestatin at substantial concentrations the isolated cells took up a significant amount of the intact dipeptide, which otherwise appeared entirely in the form of free amino acids. These results are interpreted to substantiate a notion that a dual mechanism is operative for the absorption of readily-hydrolysable peptides: the peptide hydrolysis followed by uptake of thereby released amino acids, and the peptide transport followed by cytosolic hydrolysis.     

Evidence for a dipeptide transport system in renal brush border membranes from rabbit

Papain treatment of renal brush border vesicles was carried out as a successful first step towards the purification of the membrane components involved in dipeptide transport. The treated vesicles exhibited increased specific transport activity of glycyl-l-proline. In contrast, the specific transport activity of l-alanine in the treated vesicles was less than that in the control vesicles. Papain treatment resulted in the solubilization of 38% of protein, 55% of alkaline phosphatase, 90% of γ-glutamyltransferase and 95% of leucine aminopeptidase. There was no change in the intravesicular volume nor was there any increase in vesicular permeability. Glycyl-l-proline transport was Na⁺-independent in the control and papain-treated vesicles. Diamide reduced the Na⁺-dependent l-alanine transport while glycyl-l-proline transport remained unaffected in the presence of Na⁺. Many dipeptides inhibited glycyl-l-proline transport both in the presence and absence of Na⁺. The inhibition by dipeptides was greater than the inhibition by equivalent concentrations of free amino acids. These data demonstrate that renal brush border vesicles can efficiently handle dipeptides by a mechanism completely different from that of amino acid transport.     

Thermostable dipeptidase from Bacillus stearothermophilus: its purification, characterization, and comparison with aminoacylase

Dipeptidase (dipeptide hydrolase [EC 3.4.13.11]) has been purified to homogeneity and crystallized from the cell extract of Bacillus stearothermophilus IFO 12983. The enzyme has a molecular weight of about 86,000, and is composed of two subunits identical in molecular weight (43,000). The enzyme contains 2 g atoms of zinc per mol of protein. A variety of dipeptides consisting of glycine or only L-amino acids serve as substrates of the enzyme; Km and Vmax values for L-valyl-L-alanine are 0.5 mM and 68.0 units/mg protein, respectively. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturants such as urea and guanidine hydrochloride. The enzyme also catalyzes hydrolysis of several N-acylamino acids with Vmax values 3-30% of those for the hydrolysis of dipeptides. The thermostable dipeptidase shares various properties with bacterial aminoacylase [EC 3.5.1.14]: their subunit molecular weight, metal content and requirement, amino acid composition, and amino acid sequence in the N-terminal region are very similar.     

A Phosphate-Bond-Driven Dipeptide Transport System in Streptococcus cremoris Is Regulated by the Internal pH

The uptake of amino acids and peptides by Streptococcus cremoris is mediated by different highly specific transport systems. The leucine transport system has a high affinity only for leucine, isoleucine, and valine and no affinity for leucyl-peptides. The transport system for leucyl-leucine is strongly inhibited by several dipeptides with hydrophobic, neutral, N-terminal amino acids but not by leucine. The leucyl-leucine transport system has a high affinity for dipeptides containing β-methyl groups in the side chain; the C terminus of the dipeptide affects the affinity to a much lower extent. Leucyl-leucine transport in whole cells was studied as a function of the internal pH at different external pH values in the presence and absence of nigericin. The internal pH was shown to be an important controlling factor in leucyl-leucine uptake, but the ΔpH was not involved as a driving force. At increasing external pH values, the affinity of the transport system for leucyl-leucine decreased. Uptake of leucyl-leucine was also studied in the presence of arsenate, which inhibited ATP synthesis by substrate-level phosphorylation. The rate of leucyl-leucine transport appeared to be dependent on the intracellular ATP concentrations. These results indicate that the energy for the leucyl-leucine transport is directly supplied by ATP.     

Peptide Utilization by Amino Acid Auxotrophs of Neurospora crassa

The ability of auxotrophs of Neurospora crassa to grow on certain tripeptides, despite the presence of excess competing amino acids, suggests it has an oligopeptide transport system. In general, dipeptides did not support growth except in those instances where extracellular hydrolysis occurred, or where the dipeptide appeared to be accumulated by an uptake system which is sensitive to inhibition by free amino acids. Considerable intracellular peptidase activity toward a large number of peptides was demonstrated, including a number of peptides which could not be utilized for growth. The intracellular peptidase activity was shown to be selective for amino acid composition and sequence (N-terminal or C-terminal) within the peptide; glycine-containing peptides were particularly poor substrates for peptidase activity. Only a small amount of extracellular peptidase activity could be detected.     

NATURE OF THE INHIBITION OF PROTEIN SYNTHESIS BY ETHIONINE AND AMINO ACID UPTAKE IN EARLY SEA URCHIN EMBRYOS

The inhibition of protein synthesis by ethionine reported previously was found to be apparent, and ethionine inhibited only amino acid uptake like other usual amino acids. Even under such strong inhibition of the uptake, the syntheses of protein and DNA remained almost undiminished. The uptake of amino acid mixture by sea urchin embryos in the early cleavage stage was found to be carried out by active transport, since it was temperature-sensitive and was inhibited by 2,4-dinitrophenol. The uptake of an amino acid mixture or of single amino acids, e.g., valine, leucine and phenylalanine, was inhibited nonspecifically by an excess amount of other single amino acids added exogenously. Reflecting the inhibition of amino acid uptake, in vivo incorporation of amino acids into the protein fraction was apparently inhibited by excess amounts of other amino acids. As far as tested, the inhibition seems to be nonspecific and competitive for all amino acid species. The uptakes of leucine and phenylalanine were inhibited mutually by competition, with almost the same Km and Ki.     

Peptide utilization by Pseudomonas putida and Pseudomonas maltophilia.

Pseudomonas putida assimilates peptides and hydrolyses them with intracellular peptidases. Amino acid auxotrophs (his, trp, thr or met) grew on a variety of di- and tripeptides up to twice as slowly as with free amino acids. Pseudomonas putida has separate uptake systems for both dipeptides and oligopeptides (three or more residues). Although the dipeptide system transported a variety of structurally diverse dipeptides it did not transport peptides having either unprotonatable N-terminal amino groups, blocked C-terminal carboxyl groups, D-residues, three or more residues, N-methylated peptide bonds, or beta-amino acids. Oligopeptide uptake lacked amino acid side-chain specificity, required a free N-terminal L-residue and had an upper size limit. Glycylglycyl-D,L-p-fluorophenylalanine inhibited growth of P. putida. Uptake of glycylglycyl[I-14C]alanine was rapid and inhibited by 2,4-dinitrophenol. Both dipeptide and oligopeptide uptake were constitutive. Dipeptides competed with oligopeptides for oligopeptide uptake, but oligopeptides did not compete in the dipeptide system. Final bacterial yields were 5 to 10 times greater when P. putida his was grown on histidyl di- or tripeptides rather than on free histidine because the histidyl residue was protected from catabolism by L-histidine ammonia-lyase. Methionine peptides could satisfy the methionine requirements of P. maltophilia. Generation times on glycylmethionine and glycylmethionylglycine were equal to those obtained with free methionine. Methionylglycylmethionylmethionine gave a generation time twice that of free methionine. Growth of P. maltophilia was inhibited by glycylglycyl-D,L-p-fluorophenylalanine.     

Glutamine-stimulated amino acid and peptide incorporation in Bacteroides melaninogenicus.

The uptake of a number of amino acids and dipeptides by cells and spheroplasts of Bacteroides melaninogenicus was stimulated by the presence of glutamine; 50 mM glutamine induced maximum uptake of glycine or alanine, and glutamine stimulated the uptake of glycine over a wide concentration range (0.17 to 170 mM). Glutamine stimulated the uptake of the dipeptides glycylleucine and glycylproline at significantly faster rates compared with glycine and leucine. The amino acids whose uptake was stimulated by glutamine were incorporated into trichloroacetic acid-precipitable material, and the inclusion of chloramphenicol or puromycin did not affect this incorporation. The uptake of glutamine by cells was concentration dependent. In contrast, in the absence of chloramphenicol 79% of the glutamine taken up by cells supplied with a high external concentration (4.4 mM) was trichloroacetic acid soluble. Glutamate and alpha-ketoglutarate were identified in the intracellular pool of glutamine-incubated spheroplasts. The amino acids and peptides were incorporated into cell envelope material, and a portion (30 to 50%) of the incorporated amino acids could be removed by trypsinization or treatment with papain. The effect of glutamine was depressed by inhibitors of energy metabolism, suggesting that glutamine-stimulated incorporation is an energy-mediated effect.     

Characterization of an oligopeptide transporter in renal lysosomes

Renal lysosomes play a major role in catabolism of plasma proteins. Final products of this catabolism include dipeptides and tripeptides that must be exported to the cytosol for hydrolysis. The aim of the present study was to determine whether an oligopeptide transporter is present in the renal lysosomal membrane that could mediate this export. The existence of an oligopeptide transporter was probed with the uptake of glycylglutamine (Gly-Gln) by membrane vesicles prepared from renal lysosomes. Kinetic analysis showed the presence of a single transporter with a K(m) of 8.77 mM for the uptake of Gly-Gln. The Gly-Gln uptake was energized by the imposition of an inwardly directed proton gradient (pH(out) 5.0/pH(in) 7.3) and membrane potential (outside positive/inside negative) resulting in overshoot. The Gly-Gln uptake was inhibited by the presence of dipeptides and tripeptides, but not amino acids. Western blot analysis of lysosomal membrane proteins with Pept-1 (an oligopeptide transporter) antibody as the probe showed the presence of an immunoreactive protein. This immunoreaction was abolished when the antiserum was preabsorbed with the Pept-1 epitope (0.5 microg/ml). In conclusion, the present data show the existence of a low-affinity dipeptide transporter in the renal lysosomal membrane that appears to belong to the Pept family of transporters. The function of this transporter appears to be to prevent accumulation of dipeptides in renal lysosomes.     

The sites of hydrolysis of dipeptides containing leucine and glycine by rat jejunum in vitro

1. The effect of peptides containing leucine and glycine on accumulation of leucine and glycine by everted jejunal rings was studied. 2. It was shown that, on a molar basis, leucyl-leucine is a more effective inhibitor of uptake of [(14)C]leucine than is either leucylglycine or glycyl-leucine. These latter dipeptides behave alike. 3. The concentration of the dipeptides and their constituent amino acids in both the incubation medium and the tissue has been followed in these experiments by amino acid analysis. No leucine-containing peptides were observed in the tissue. 4. The inhibitory effects of the mixed dipeptides are altered by pH changes in an analogous way to the alterations in peptidase activity. 5. The experimental results indicate that leucine-containing peptides are hydrolysed before the transport step. 6. Glycylglycine, on the other hand, has only a small effect on the accumulation of glycine, although large amounts of the peptide accumulate unchanged in the tissue. This suggests that glycylglycine is taken up by a different mechanism to that for the leucine dipeptides.     

Mechanism of hepatic assimilation of dipeptides. Transport versus hydrolysis

To investigate dipeptide assimilation by the liver, a series of interrelated experiments were performed in rats. Partial hepatectomy prolonged the plasma half-life (min) of Gly-Ala (3.42 +/- 0.22 versus 4.90 +/- 0.35, p less than 0.05) but had no significant effect on plasma half-life of Gly-Leu, Gly-Pro, or Gly-Sar. We then investigated the rate of disappearance (mumol X (g liver X h)-1) of the above four dipeptides (initial concentration = 1 mM) from the medium during isolated liver perfusion. The order of dipeptide disappearance was: Gly-Leu (8.75 +/- 0.65) greater than Gly-Ala (3.36 +/- 0.46) greater than Gly-Pro (1.29 +/- 0.54) greater than Gly-Sar (0.35 +/- 0.12). This order of dipeptide disappearance corresponded exactly to the order of the rates of glycine accumulation in the medium during liver perfusion with the four dipeptides. Addition of glucagon had no effect on the disappearance rate of Gly-Ala from the medium, but reduced accumulation rates of glycine (3.39 +/- 0.30 versus 1.42 +/- 30, p less than 0.01) and alanine (4.42 +/- 0.66 versus 1.35 +/- 0.39, p less than 0.01). Finally, we found that hydrolysis by the liver plasma membranes and/or perfusion medium accounted for disappearance of dipeptides. In conclusion, the liver does not appear to have a transport system for dipeptides, but assimilates dipeptides by extracellular hydrolysis. Hydrolysis is achieved by enzymes either located on the plasma membranes or released from the cytosol. The amino acid residues released as the result of dipeptide hydrolysis are then taken up by the liver.     

Transport of [14C]Gly-Pro in a proline peptidase mutant of Salmonella typhimurium.

The transport of [14C]Gly-Pro was examined using a mutant of Salmonella typhimurium (strain TN87) deficient in an X-Pro dipeptidase and an X-Pro-Y iminopeptidase. The dipeptide was taken up by one saturable transport system having a Km of 5.3-10(-7)M and a V of 1.4 nmol/mg dry wt cell per min. The uptake of Gly-Pro was not inhibited by amino acids or tripeptides and the transport system exhibited a rather broad side chain specificity for dipeptides. Dipeptides containing hydrophobic residues were the most potent inhibitors of this dipeptide transport system exhibiting Ki values between 10(-8) and 10(-7) M. In contrast, dipeptides containing glycine residues were particularly weak inhibitors. Finally, Gly-Pro was found to be in the intact form inside the cell and was concentrated more than 1000-fold.     

Characterization of system L and system y+ amino acid transport activity in cultured vascular smooth muscle cells

The uptake of L-leucine and L-lysine into vascular smooth muscle cells cultured from the aortas of rats has been investigated. Both amino acids are taken up by saturable systems that are independent of the presence of a ^·Na⁺ gradient and can be stimulated in trans by neutral bulky amino acids for leucine and cationic amino acids for lysine. Leucine uptake is inhibited competitively in cis by several neutral amino acids, whereas lysine uptake is inhibited strongly by other cationic amino acids but also significantly by neutral amino acids such as leucine. The leucine inhibition is noncompetitive. Cells preloaded with leucine and lysine could also export these amino acids and the rate of efflux was stimulated by the presence of appropriate amino acids in trans. These data are all consistent with leucine being transported largely if not entirely by System L and lysine by the System y⁺ transporter. © 1993 Wiley-Liss, Inc.     

Regulation of chemoautotrophic metabolism

Summary Incorporation of ¹⁴C-phenylalanine by T. neapolitanus was inhibited competitively by relatively low concentrations of glycine, serine, alanine, valine, leucine, isoleucine, tryptophan, tyrosine, histidine, threonine, and methionine (Group I amino acids), but not greatly depressed by aspartate, glutamate, lysine, arginine, cysteine (Group II amino acids) and proline at similar concentrations. Group I acids competed with each other for incorporation but were little affected by Group II acids. Similarly Group I acids little depressed the incorporation of Group II acids, among which, however, some mutual inhibition occurred. Incorporation of proline was depressed by both Group I and II acids. Two main permeation mechanisms are proposed, one transporting Group I acids, the other Group II acids, but some overlapping of function probably occurs. Proline may be transported by a third permease, which is subject to inhibition by both Group I and II acids. T. concretivorus also has a common transport mechanism for some amino acids. Less interaction between amino acids was found using two heterotrophic pseudomonads.Exogenous phenylalanine inhibited both the biosynthesis and the uptake of tyrosine and tryptophan by T. neapolitanus. High phenylalanine concentrations depressed the assimilation of ¹⁴C-labelled tyrosine and tryptophan less than low ones, suggesting that the bacteria developed a requirement for external tyrosine and tryptophan when exposed to highly inhibitory concentrations of phenylalanine.     

The influence of peptides on the uptake of amino acids inFusobacterium; predicted interactions withPorphyromonas gingivalis

A study of the uptake of amino acids and its influence by a peptide source was carried out withFusobacterium varium as a convenient representative of the genus. Reference strains and a clinical isolate had similar amino acid uptake profiles, but most amino acids were incorporated at lower concentrations by the latter. In general, high levels of serine, asparagine, glutamate, cysteine, and arginine were incorporated by all species. Histidine, lysine, threonine, and aspartate were taken up at lower levels, whereas the nonpolar neutral amino acids such as alanine, valine, leucine, isoleucine, glycine, proline, phenylalanine, and methionine were poorly metabolized. Yeast extract, as a source of peptides, stimulated the uptake of several amino acids such as histidine and glutamate, whereas others such as methionine, threonine, and asparagine were repressed. The incorporation of some amino acids such as aspartate, ornithine, lysine, and arginine was unaffected by the presence of peptides. Equimolar nitrogen concentrations of amino acids or ammonia could not replace the peptide requirement, emphasizing the importance of peptides as an energy source. The limited capacity ofFusobacterium spp. to hydrolyze proteins increased approximately 30% in the presence of the proteolytic species,Porphyromonas gingivalis, and may represent one bacterial interaction in which peptides may become available toFusobacterium species in vivo.