Peptide transport by germinating barley embryos: Uptake of physiological di- and oligopeptides

The uptake of a variety of physiological di- and oligopeptides by germinating barley (Hordeum vulgare L.) embryos is described. Peptides as large as pentaalanine can be absorbed. Evidence is presented suggesting the peptides are absorbed intact and subsequently undergo rapid intracellular hydrolysis. Uptake shows stereospecificity. The transport of peptides is generally faster than the transport of amino acids, making it likely that the former could play an important role in the mobilization of the protein storage reserves during germination. The peptide transport system in barley is compared with similar systems from other groups of organisms.Abbreviations Gly-sar glycylsarcosine - Gly-sar-sar glycylsarcosylsarcosine - Gly-sar-sar-sar Glycylsarcosylsarcosylsarcosine     

Characteristics of the active transport of peptides and amino acids by germinating barley embryos

Use of two different assays involving either radioactively labelled substrates or a fluorescent-labelling procedure, gave good agreement for the rates of transport of peptides and amino acids into the scutellum of germinating grains of barley (Hordeum vulgare cv. Maris Otter, Winter). However, evidence was obtained for the enzymic decarboxylation of transpored substrate, which can cause underestimates of transport rates when using radioactively labelled substrates. The peptide Gly-Phe, was shown to be rapidly hydrolysed after uptake, and autoradiography of transported Gly-[U-¹⁴C]Phe indicated a rapid distribution of tracer, i.e. [U-¹⁴C] phenylalanine into the epithelium and sub-epithelial layers of the scutellum. The developmental patterns of transport activity indicate that peptide transport is more important nutritionally during the early stages of germination (1–3 d) whereas amino acids become relatively more important later (4–6 d). A range of amino acids is shown to be actively transported and several compete for uptake. At physiological concentrations, e.g. 2mM, transport of peptides and amino acids is inhibited about 80% by protonophore uncouplers, but at higher concentrations (10–100 mM) passive uptake predominates.Abbreviations Gly glycine - Leu leucine - Phe phenylalanine - Pro proline     

Stereospecificity of peptide transport by germinating barley embryos

The stereospecific requirements for peptide transport in the scutellum of germinating barley (Hordeum vulgare) embryos are described. Replacement of an L-amino acid residue in a peptide by its D-stereoisomer decreases the affinity of the peptide for the transport site, leading to a reduction in transport. Substitution of a second D-residue reduces affinity still further. The extent to which transport is inhibited depends upon the position of the D-residue in the primary sequence, with D-residues at the C-terminus of the peptide having the greatest effect. Competition between D- and L-peptides indicates that they both enter via the same transport system. Although D-amino acids can be accumulated when presented as a peptide, these same D-residues are not transported when supplied as the free amino acids. L-Leu-D-leu is accumulated intact against a concentration gradient, indicating the operation of an active transport mechanism that can function without the involvement of peptidase activity.     

Characterization of a transport activity for long-chain peptides in barley mesophyll vacuoles

The plant vacuole is the largest compartment in a fully expanded plant cell. While only very limited metabolic activity can be observed within the vacuole, the majority of the hydrolytic activities, including proteolytic activities reside in this organelle. Since it is assumed that protein degradation by the proteasome results in the production of peptides with a size of 3-30 amino acids, we were interested to show whether the tonoplast exhibits a transport activity, which could deliver these peptides into the vacuole for final degradation. It is shown here that isolated barley mesophyll vacuoles take up peptides of 9-27 amino acids in a strictly ATP-dependent manner. Uptake is inhibited by vanadate, but not by NH(+)(4), while GTP could partially substitute for ATP. The apparent affinity for the 9 amino acid peptide was 15 μM, suggesting that peptides are efficiently transferred to the vacuole in vivo. Inhibition experiments showed that peptides with a chain length below 10 amino acids did not compete as efficiently as longer peptides for the uptake of the 9 amino acid peptide. Our results suggest that vacuoles contain at least one peptide transporter that belongs to the ABC-type transporters, which efficiently exports long-chain peptides from the cytosol into the vacuole for final degradation.     

Amino Acid and Peptide uptake in the scutella of germinating grains of barley, wheat, rice, and maize

Scutella separated from germinating grains of barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), rice (Oryza sativa L.), and maize (Zea mays L.) took up the four amino acids and the three peptides tested from incubation media. The uptake of amino acids by wheat scutella was similar to that of barley scutella and was via at least four uptake systems: two nonspecific amino acid uptake systems, one system specific for proline, and another system specific for basic amino acids. The scutellum of rice apparently has two nonspecific systems and a system specific for the basic amino acids, but the proline-specific system is lacking. The scutellum of maize seems to have the same systems as the scutellum of rice, but one (or both) of the nonspecific systems differs from that of the other species studied in taking up arginine only slowly. No great differences were observed in the uptake of peptides in the four species studied. The rates of uptake of different amino acids and peptides were of the same order of magnitude in the four cereals. The fact that carboxypeptidase activities in the endosperms of wheat and barley are 20-to 100-fold higher than those in rice and maize, does thus not seem to be reflected in the uptake properties of the scutella.     

Regulation of development of leucine uptake activity by glutamine in the scutellum of germinating barley grain

Scutella from ungerminated grains of barley (Hordeum vulgare L. cv Pirkka) take up leucine at a slow rate, which increases rapidly during germination. When endosperms were removed from the grains after imbibition for 4 hours or after germination for 12 or 72 hours, the increase in the rate of leucine uptake was greatly accelerated during subsequent incubation of the embryos or scutella. These increases were rapidly inhibited by cordycepin and cycloheximide, suggesting that protein synthesis, probably synthesis of the carrier protein, was required for the development of the uptake activity.

In separated embryos or scutella, the increases in the leucine uptake activity were inhibited by glutamine. The inhibitions caused by glutamine and cycloheximide were not additive, suggesting that glutamine did not interfere with the function of the carrier but repressed its synthesis. Glutamine did not inhibit the simultaneous increase in peptide uptake; in this respect, its effect was specific for leucine uptake, which appears to be due to a general amino acid uptake system.

Some other protein amino acids also inhibited the increase in leucine uptake without inhibiting the increase in peptide uptake. However, these effects were smaller than that of glutamine.

These results suggest that the transfer of leucine (and other amino acids) from the endosperm to the seedling in a germinating barley grain is regulated at the uptake step by repression of the synthesis of the amino acid carrier protein by glutamine and—possibly to a lesser extent—by some other amino acids taken up from the endosperm.

     

Peptide substrates rapidly modulate expression of dipeptide and oligopeptide permeases in Candida albicans

Previous studies showed that peptide transport activity in Candida albicans was completely repressed by NH4+, and that growth on amino acids as sole nitrogen source stimulated transport to a basal level. Here we show that addition of peptide mixtures to culture media gives a further 5-fold increase in transport of dipeptides and oligopeptides; the effect is specific for peptide transport, amino acid uptake being unaffected. Presence of peptides but not amino acids overrides NH4+ repression of peptide transport. Step-up activation of transport activity, caused by addition of peptides to incubation media, and step-down inhibition that accompanies removal of peptides, occurs rapidly (within 30 min at 28 degrees C). Step-up is independent of de novo protein synthesis. This substrate-induced regulation is compatible with a rapid, reversible activation of plasma membrane-bound peptide permease(s), or a mechanism of endocytosis involving a cycle of insertion and retrieval of preformed permease components. These results are considered in relation to the expression of peptide permeases in vivo, and the development of synthetic anticandidal peptide carrier prodrugs designed to exploit these systems.     

Characteristics of the protein carrier of the peptide-transport system in the scutellum of germinating barley embryos

Through the use of the protein reagents N-ethylmaleimide, p-chloromercuribenzenesulphonic acid and phenylarsine oxide, it is shown that in the scutellum of the germinating barley embryo, the transport of peptides, but not the transport of amino acids or glucose is specifically thiol-dependent. Furthermore, these essential thiol groups are shown to exist as redox-sensitive, vicinal-dithiols that lie at the substrate-binding sites of the peptide-transport proteins. The binding of N-ethylmaleimide to these dithiols is shown to be very fast, matching the kinetics of inhibition of peptide transport by this reagent. A technique for the specific labelling of the dithiols with N-ethyl[2,3-¹⁴C]maleimide is described, which allows the carrier proteins to be visualized at the scutellar epithelium using radioautography and permits calculation of the approximate amount of peptide-transport protein present per scutellum. In related studies, the importance of arginyl and histidyl residues to both amino-acid and peptide transport is shown, although other residues, e.g. carboxyl ligands do not seem to be critically involved.Abbreviations Ala alanine - Gly glycine - Leu Leucine - NEM N-ethylmaleimide - PAO phenylarsine oxide - PCMBS p-chloromercuribenzenesulphonic acid - Phe phenylalanine     

Purification and partial characterization of a 31-kDa cysteine endopeptidase from germinated barley

Proteolytic enzymes hydrolyze cereal seed storage proteins into small peptides and amino acids, which are very important for seed germination and the malting process. A cysteine-class endopeptidase was purified from 4-d-germinated barley (Hordeum vulgare L. cv. Morex). Four purification steps were used, carboxymethyl cellulose cation-exchange chromatography, chromatofocusing, size-exclusion chromatography, and electroelution from a polyacrylamide gel. The endopeptidase was most active at pH 4.5. It's isoelectric point (pI) was 4.4, as determined by isoelectric focusing, and it's SDS-PAGE molecular size was 31 kDa. The enzyme specifically hydrolyzed peptide bonds when the S₂ site contained relatively large hydrophobic amino acids. The N-terminal amino acid sequence residues (1–9) of the 31-kDa endopeptidase had high homology to those of the EP-A and EP-B cysteine proteinases reported previously. The 31-kDa endopeptidase had a hydrolytic specificity similar to that of the Morex green malt 30-kDa endopeptidase we characterized previously, and also reacted with the antibody raised against the purified 30-kDa proteinase, but the two had different mobilities on non-denaturing PAGE. The hydrolytic specificities of both 30- and 31-kDa endopeptidases are such that both would very quickly cleave hordein (barley storage) proteins to small glutamine- and proline-rich peptides that could be quickly degraded to amino acids by barley exopeptidases.Abbreviations CMC carboxymethyl cellulose - E-64 transepoxysuccinyl-l-leucylamido-(4-guanidino)butane - EMI N-ethylmaleimide - IEF isoelectricfocusing - Phen 1,10-phenanthroline - PI isoelectric point - PMSF phenylmethylsulfonyl fluoride We thank the American Malting Barley Association for partially funding this work. Germinated barley seeds were kindly prepared by Eddie D. Goplin. Special thanks to Laurie Marinac for her excellent technical assistance.     

Evidence for two different broad-specificity oligopeptide transporters in intestinal cell line Caco-2 and colonic cell line CCD841

Recently the existence of two different Na(+)-coupled oligopeptide transport systems has been described in mammalian cells. These transport systems are distinct from the previously known H(+)/peptide cotransporters PEPT1 and PEPT2, which transport only dipeptides and tripeptides. To date, the only peptide transport system known to exist in the intestine is PEPT1. Here we investigated the expression of the Na(+)-coupled oligopeptide transporters in intestinal cell lines, using the hydrolysis-resistant synthetic oligopeptides deltorphin II and [d-Ala(2),d-Leu(5)]enkephalin (DADLE) as model substrates. Caco-2 cells and CCD841 cells, both representing epithelial cells from human intestinal tract, were able to take up these oligopeptides. Uptake of deltorphin II was mostly Na(+) dependent, with more than 2 Na(+) involved in the uptake process. In contrast, DADLE uptake was only partially Na(+) dependent. The uptake of both peptides was also influenced by H(+) and Cl(-), although to a varying degree. The processes responsible for the uptake of deltorphin II and DADLE could be differentiated not only by their Na(+) dependence but also by their modulation by small peptides. Several dipeptides and tripeptides stimulated deltorphin II uptake but inhibited DADLE uptake. These modulating small peptides were, however, not transportable substrates for the transport systems that mediate deltorphin II or DADLE uptake. These two oligopeptide transport systems were also able to take up several nonopioid oligopeptides, consisting of 9-17 amino acids. This represents the first report on the existence of transport systems in intestinal cells that are distinct from PEPT1 and capable of transporting oligopeptides consisting of five or more amino acids.     

Evidence for amino-acid: proton cotransport in Ricinus cotyledons

During germination and early growth of the castor-bean (Ricinus communis L.), protein in the endosperm is hydrolyzed and the amino acids are transferred into the cotyledons and then via the translocation stream to the axis of the growing seedling. The cotyledons retain the ability to absorb amino acids after removal of the endosperm and hypocotyl, exhibiting rates of transport up to 70 mol g^-1 h^-1. The transport of L-glutamine was not altered by KCl or NaCl in low concentrations (0–20 mM). High concentrations of KCl (100 mM) inhibited transport, presumably by decreasing the membrane potential. An increase in the pH of the medium bathing the cotyledons was observed for 10 min following addition of L-glutamine but not with D-glutamine, which is not transported. The rate of proton uptake was dependent on the concentration of L-glutamine in the external solution. Inhibitors and uncouplers of respiration (azide, 2, 4-dinitrophenol, carbonyl cyanide phenylhydrazone and N-ethylmaleimide) inhibited both L-glutamine uptake and L-glutamine-induced proton uptake. Amino acids other than L-glutamine also caused a transient pH rise and the rate of proton uptake was proportional to the rate of amino-acid uptake. The stoichiometry was 0.3 protons per amino acid transported. Addition of sucrose also caused proton uptake but the alkalisation by sucrose and by amino acids were not additive. Nevertheless, when sucrose was added 60 min after providing L-glutamine at levels saturating its uptake system, a rise in pH was again observed. The results were consistent with amino-acid transport and sucrose transport in castor-bean cotyledons both occurring by a proton cotransport in the same membrane system but involving separate carriers.     

Analysis of the peptide carrier in the scutellum of barley embryos by photoaffinity labelling

The preparation of a phenylalanine analogue containing an azido group and its incorporation into dipeptides is described. Peptides modified in this way are taken up into barley (Hordeum vulgare L.) scutella via the previously characterized peptide-transport system. Photoactivation of modified peptides in the presence of isolated scutella resulted in irreversible inhibition of peptide uptake in a concentration-dependent manner. Transport of other solutes which share a common mechanism of energy coupling, but which are transported via distinct carriers, was not inhibited after photo-derivatization of scutella with the modified peptides. Derivatization of isolated scutellar tissue with a ¹⁴C-labelled peptide analogue, resulted in incorporation of label into two proteins of M_r = 54000 and 41000. Scutellar tissue from early-germinating seeds, which do not show active peptide uptake, did not incorporate label into these polypeptides. It is concluded that these proteins are components of the barley peptide-transport system.Abbreviations Ala alanine - Gly glycine - PAGE polyacrylamide gel electrophoresis - Phe phenylalanine - Pro proline - SDS sodium dodecyl sulphate This work was supported by a grant from the Agricultural and Food Research Council.     

Relationship between relative protein value and somein vitro indices of protein quality

Besides amino acid composition of a protein, their bioavailability is an important determinant of the protein quality. In view of the observations over the last decade or two, implicating the small peptide uptake by the mammalian intestine as a major route of protein absorption, a few animal and plant proteins were subjected to sequential enzymatic digestionin vitro with pepsin, pancreatin + trypsin and erepsin and the release of amino acids as small (including dipeptides) and large peptides and free amino acids, was determined. The relative protein values of α-lactalbumin, egg whites, casein, gluten, zein and protein isolates of soyabeans and groundnuts was determined using rat growth method. It was observed that relative protein value were positively correlated with the essential amino acid index of protein, quantity of essential amino acids released as small peptides and the dipeptide content of enzymatic digests, while there was a negative correlation between relative protein value and essential amino acid content of large peptide fraction.     

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.     

Reconstruction of the proteolytic pathway for use of β-casein by Lactococcus lactis

Amino acid auxotrophous bacteria such as Lactococcus lactis use proteins as a source of amino acids. For this process, they possess a complex proteolytic system to degrade the protein(s) and to transport the degradation products into the cell. We have been able to dissect the various steps of the pathway by deleting one or more genes encoding key enzymes/components of the system and using mass spectrometry to analyse the complex peptide mixtures. This approach revealed in detail how L . lactis liberates the required amino acids from β-casein, the major component of the lactococcal diet. Mutants containing the extracellular proteinase PrtP, but lacking the oligopeptide transport system Opp and the autolysin AcmA, were used to determine the proteinase specificity in vivo . To identify the substrates of Opp present in the casein hydrolysate, the PrtP-generated peptide pool was offered to mutants lacking the proteinase, but containing Opp, and the disappearance of peptides from the medium as well as the intracellular accumulation of amino acids and peptides was monitored in peptidase-proficient and fivefold peptidase-deficient genetic backgrounds. The results are unambiguous and firmly establish that (i) the carboxyl-terminal end of β-casein is degraded preferentially despite the broad specificity of the proteinase; (ii) peptides smaller than five residues are not formed in vivo  ; (iii) use of oligopeptides of 5–10 residues becomes only possible after uptake via Opp; (iv) only a few (10–14) of the peptides generated by PrtP are actually used, even though the system facilitates the transport of oligopeptides up to at least 10 residues. The technology described here allows us to monitor the fate of individual peptides in complex mixtures and is applicable to other proteolytic systems.     

Specificity of peptide transport systems in Lactococcus lactis: evidence for a third system which transports hydrophobic di- and tripeptides.

A proton motive force-driven di-tripeptide carrier protein (DtpT) and an ATP-dependent oligopeptide transport system (Opp) have been described for Lactococcus lactis MG1363. Using genetically well-defined mutants in which dtpT and/or opp were inactivated, we have now established the presence of a third peptide transport system (DtpP) in L. lactis. The specificity of DtpP partially overlaps that of DtpT. DtpP transports preferentially di- and tripeptides that are composed of hydrophobic (branched-chain amino acid) residues, whereas DtpT has a higher specificity for more-hydrophilic and charged peptides. The toxic dipeptide L-phenylalanyl-beta-chloro-L-alanine has been used to select for a di-tripeptide transport-negative mutant with the delta dtpT strain as a genetic background. This mutant is unable to transport di- and tripeptides but still shows uptake of amino acids and oligopeptides. The DtpP system is induced in the presence of di- and tripeptides containing branched-chain amino acids. The use of ionophores and metabolic inhibitors suggests that, similar to Opp, DtpP-mediated peptide transport is driven by ATP or a related energy-rich phosphorylated intermediate.     

Specificity and regulation of peptide transport on Neurospora crassa.

The tripeptide, glycyl-d,l-leucyl-l-tyrosine was chemically synthesized in radioactive form and used to directly study the specificity, regulation, and properties of an oligopeptide transport system in Neurospora. Transport activity is sensitive to azide but does not result in the accumulation of the intact peptide; rather, the radioactive label is accumulated as free tyrosine. Inhibition studies suggest that the transport system probably has a relatively wide range of specificity and is responsible for uptake of short oligopeptides of quite distinct sequences. However, free amino acids and dipeptides are not transported significantly, if at all, by the oligopeptide transport system. A free amino group appears to be a requirement for peptide transport. A mutant strain that is unable to use various peptides for growth is further described and shown to be reduced greater than 90% in transport of the tripeptide.     

Peptide uptake in germinating barley embryos involves a dithiol-dependent transport protein

An active transport system for small peptides occurs in the scutellar membrane of germinating barley and serves to move the products of partial hydrolysis of storage proteins from the endosperm into the growing embryo. Transport of peptides, but not amino acids or glucose, is inhibited by the thiol reagents, N-ethylmaleimide and p-chloromercuribenzene sulphonic acid (PCMBS). Peptide substrates protect against PCMBS inactivation. The dithiol-specific reagent, phenylarsine oxide (PAO) also inhibits. The reducing agent, dithiothreitol, reverses the inactivation caused by PCMBS and PAO. We conclude that the peptide transport system contains a redox-sensitive, dithiol-dependent protein.