Role of water and electrolyte influxes in anoxic plasma membrane disruption

The role of water and electrolyte influxes in anoxia-inducedplasma membrane disruption was investigated using rabbit proximal tubule suspension. The results indicated that normal proximal tubule(PT) cells have a great capacity for expanding cell volume in responseto water influx, whereas anoxia increases the susceptibility to waterinflux-induced disruption, and this was attenuated by glycine. However,resistance of anoxic plasma membranes to water influx-induced stress isnot lost, although their mechanical strength was diminished, comparedwith normoxic membranes. Anoxic membranes did not disrupt under anintra-to-extracellular osmotic difference as great as 150 mosM.Potentiating or attenuating water influx by incubating PT cells inhypotonic or hypertonic medium, respectively, during anoxia, did notaffect anoxia-induced membrane disruption. After the transmembraneelectrolyte concentration gradient was eliminated by a"intracellular" buffer or by permeabilizing the plasma membraneto molecules <4 kDa using -toxin, anoxia still caused furthermembrane disruption that was prevented by glycine or low pH. Theseresults demonstrate that 1) water ornet electrolyte influxes are probably not a primary cause foranoxia-induced membrane disruption and2) glycine could prevent the plasmamembrane disruption during anoxia independently from its effect ontransmembrane electrolyte or water influxes. The present data support abiochemical rather than a mechanical alteration of the plasma membraneas the underlying cause of membrane disruption during anoxia.

     

The ATP/substrate stoichiometry of the ATP-binding cassette (ABC) transporter OpuA

ATP-binding cassette (ABC) transport proteins catalyze the translocation of substrates at the expense of hydrolysis of ATP, but the actual ATP/substrate stoichiometry is still controversial. In the osmoregulated ABC transporter (OpuA) from Lactococcus lactis, ATP hydrolysis and substrate translocation are tightly coupled, and the activity of right-side-in and inside-out reconstituted OpuA can be determined accurately. Although the ATP/substrate stoichiometry determined from the uptake of glycine betaine and intravesicular ATP hydrolysis tends to increase with decreasing average size of the liposomes, the data from inside-out reconstituted OpuA indicate that the mechanistic stoichiometry is 2. Moreover, the two orientations of OpuA in proteoliposomes allowed possible contributions from substrate (glycine betaine) inhibition on the trans-side of the membrane and inhibition by ADP to be determined. Here we show that OpuA is not inhibited by up to 400 mm glycine betaine on the trans-side of the membrane. ADP is an inhibitor, but accumulation of ADP was negligible in the assays with inside-out-oriented OpuA, and potential effects of the ATP/ADP ratio on the ATP/substrate stoichiometry determinations could be eliminated.     

Multiplicity of peptide permeases in Candida albicans: evidence from novel chromophoric peptides.

Evidence is presented for the presence of multiple peptide permeases in the eucaryotic organism Candida albicans. Instrumental in these studies were the peptides L-alanyl-L-2-thiophenylglycine (Ala-alpha-TPG) and L-alanyl-L-2-thiophenylglycyl-L-alanine (Ala-alpha-TPG-Ala), which contain thiophenol attached to the alpha-carbon of glycine. Subsequent to transport into the fungal cell, enzymatic hydrolysis of these peptides resulted in the release of free thiophenol, which was quantified by using Ellman reagent. Thiophenol release was shown to be directly correlated to peptide transport and hydrolysis, with transport being the rate-limiting step in intact cells. These peptides, whose uptake showed Michaelis-Menten kinetics, have been used to determine peptide uptake in C. albicans. In addition, we found that the intracellular peptidases can readily be assayed in permeabilized cells and that bestatin, an aminopeptidase inhibitor, inhibits all detectable peptidase activity. C. albicans 124 was able to transport and hydrolyze both Ala-alpha-TPG and Ala-alpha-TPG-Ala, whereas the mutant (124NIK5) was able to transport only the tripeptide. The intracellular peptidases of this mutant were unaffected. In wild-type C. albicans 124, oligopeptides were able to compete with uptake of Ala-alpha-TPG-Ala to a far greater extent than with that of Ala-alpha-TPG; dipeptides inhibited uptake of both Ala-alpha-TPG and Ala-alpha-TPG-Ala. These results provide complementary evidence for the existence of distinct transport systems.     

The requirement for the protonated α-amino group for the transport of peptides in Escherichia coli

Many glycine peptides support growth of a glycine auxotroph of Escherichia coli. If the alpha-amino group of these peptides is methylated, the products are still utilized for growth, and also retain comparable ability with the unsubstituted peptides to compete with natural peptides for transport into the cell. In contrast, glycine peptides devoid of an alpha-amino group, or that have the alpha-amino group substituted by one of a number of acyl groups are not utilized, although E. coli possesses intracellular enzymic activity able to release glycine from such compounds; further, these derivatives do not compete with natural peptides for transport into the cell.     

Selective and ATP-dependent translocation of peptides by the homodimeric ATP binding cassette transporter TAP-like (ABCB9)

The transporter associated with antigen processing (TAP)-like (TAPL, ABCB9) belongs to the ATP-binding cassette transporter family, which translocates a vast variety of solutes across membranes. The function of this half-size transporter has not yet been determined. Here, we show that TAPL forms a homodimeric complex, which translocates peptides across the membrane. Peptide transport strictly requires ATP hydrolysis. The transport follows Michaelis-Menten kinetics with low affinity and high capacity. Different nucleotides bind and energize the transport with a slight predilection for purine bases. The peptide specificity is very broad, ranging from 6-mer up to at least 59-mer peptides with a preference for 23-mers. Peptides are recognized via their backbone, including the free N and C termini as well as side chain interactions. Although related to TAP, TAPL is unique as far as its interaction partners, transport properties, and substrate specificities are concerned, thus excluding that TAPL is part of the peptide-loading complex in the classic route of antigen processing via major histocompatibility complex class I molecules.     

Structure and Mode-of-Action of the Two-Peptide (Class-IIb) Bacteriocins

This review focuses on the structure and mode-of-action of the two-peptide (class-IIb) bacteriocins that consist of two different peptides whose genes are next to each other in the same operon. Optimal antibacterial activity requires the presence of both peptides in about equal amounts. The two peptides are synthesized as preforms that contain a 15–30 residue double-glycine-type N-terminal leader sequence that is cleaved off at the C-terminal side of two glycine residues by a dedicated ABC-transporter that concomitantly transfers the bacteriocin peptides across cell membranes. Two-peptide bacteriocins render the membrane of sensitive bacteria permeable to a selected group of ions, indicating that the bacteriocins form or induce the formation of pores that display specificity with respect to the transport of molecules. Based on structure–function studies, it has been proposed that the two peptides of two-peptide bacteriocins form a membrane-penetrating helix–helix structure involving helix–helix-interacting GxxxG-motifs that are present in all characterized two-peptide bacteriocins. It has also been suggested that the membrane-penetrating helix–helix structure interacts with an integrated membrane protein, thereby triggering a conformational alteration in the protein, which in turn causes membrane-leakage. This proposed mode-of-action is similar to the mode-of-action of the pediocin-like (class-IIa) bacteriocins and lactococcin A (a class-IId bacteriocin), which bind to a membrane-embedded part of the mannose phosphotransferase permease in a manner that causes membrane-leakage and cell death.     

Effects of amino acid substitutions at glycine 420 on SR-BI cholesterol transport function

Scavenger receptor class B type I (SR-BI) facilitates the uptake of HDL cholesteryl esters (CEs) in a two-step process involving binding of HDL to its extracellular domain and transfer of HDL core CEs to a metabolically active membrane pool, where they are subsequently hydrolyzed by a neutral CE hydrolase. Recently, we characterized a mutant, G420H, which replaced glycine 420 in the extracellular domain of SR-BI with a histidine residue and had a profound effect on SR-BI function. The G420H mutant receptor exhibited a reduced ability to mediate selective HDL CE uptake and was unable to deliver HDL CE for hydrolysis, despite the fact that it retained the ability to bind HDL. This did not hold true if glycine 420 was replaced with an alanine residue; G420A maintained wild-type HDL binding and cholesterol transport activity. To further understand the role that glycine 420 plays in SR-BI function and why there was a disparity between replacing glycine 420 with a histidine versus an alanine, we generated a battery of point mutants by substituting glycine 420 with amino acids possessing side chains that were charged, hydrophobic, polar, or bulky and tested the resulting mutants for their ability to support HDL binding, HDL cholesterol transport, and delivery for hydrolysis. The results indicated that substitution with a negatively charged residue or a proline impaired cell surface expression of SR-BI or its interaction with HDL, respectively. Furthermore, substitution of glycine 420 with a positively charged residue reduced HDL CE uptake as well as its subsequent hydrolysis.     

Kinetic analysis of glycine and glycylglycine absorption in rat small intestine in chronic experiment

Kinetics of glycylglycine hydrolysis and absorption as well as that of free glycine absorption in isolated loop of the small intestine was studied in chronic experiments in two groups of rats. In the 1st group (n = 5), the isolated loop daily received for 1 or two hours a glucose load (25 mM), whereas in the 2nd group (n = 4)--a glutamic acid load (25 mM). The "true" values (i.e. corrected for the influence of the pre-epithelial layer) of the Michaelis constant for dipeptide transport were lower than those for the free glycine transport: 16 +/- 1.8 versus 36.3 +/- 3.7 mM (in the 1st group) and 15.9 +/- 2.2 versus 34.0 +/- 3.7 mM (in the 2nd group), whereas values of the maximal rate of active transport as calculated per 1 cm of the intestine length were, on the contrary, higher: 0.64 +/- 0.06 versus 0.42 +/- 0.10 mumol/(min.cm) 1st group and 0.86 +/- 0.13 versus 0.56 +/- 0.04 mumol/(min.cm) in the in the 2nd group. It has been shown that, under these conditions, regarded as the most physiological, over 90% of glycylglycine is absorbed via the peptide transport system. Only a small part of this dipeptide amount (less than 10%) splits during membrane hydrolysis with subsequent absorption of the derived glycine. It has also been found that glutamic acid solution as a regular substrate load is more effective (as compared with the glucose solution) in retarding the atrophic changes occurring in the isolated intestine loop and in preserving its structural and functional parameters on a higher level.     

A revision of current data and views on membrane hydrolysis and transport in the mammalian small intestine based on a comparison of techniques of chronic and acute experiments: experimental re-investigation and critical review

Literature data and the results of our investigations using both generally accepted and original perfusion techniques of the isolated loop of the rat small intestine in in vivo experiments are reviewed. Significant differences in the functioning of the small intestine under conditions of acute and chronic experiments are revealed. It has been established that in chronic experiments as compared to acute ones: (a) the absorption of glucose, galactose, fructose and glycine is 2-5 times higher; (b) Kt as well as Jmax values of the transport of these nutrients differ considerably; (c) Na+-independent mechanism of glucose and glycine transport predominates; (d) higher rates of membrane hydrolysis and more effective interactions between enzyme and transport systems of the enterocyte brush border membranes are observed; (e) functional characteristics of the small intestine affected by various experimental factors are more stable. The conclusion is made that it is necessary to revise current views of the scale and regularities of digestive-transport processes in the small intestine under physiological conditions. The importance of the suggested approaches for general and comparative physiology and biochemistry is discussed.     

ATP-dependent ferric hydroxamate transport system in Escherichia coli: periplasmic FhuD interacts with a periplasmic and with a transmembrane/cytoplasmic region of the integral membrane protein FhuB, as revealed by competitive peptide mapping

The Escherichia coli iron transport system via ferrichrome belongs to the group of ATP-dependent transporters that are widely distributed in prokaryotes and eukaryotes. Transport across the cytoplasmic membrane is mediated by three proteins: FhuD in the periplasm, FhuB in the cytoplasmic membrane and FhuC (ATPase) associated with the inside of the cytoplasmic membrane. Interaction of FhuD with FhuB was studied in vitro with biotinylated synthetic 10 residue and 20–24 residue peptides of FhuB by determining the activity of β-galactosidase linked to the peptides via streptavidin. Peptides identical in sequence to only one of the four periplasmic loops (loop 2), predicted by a transmembrane model of FhuB, and peptides representing a transmembrane segment and part of the adjacent cytoplasmic loop 7 of FhuB bound to FhuD. Decapeptides were transferred into the periplasm of cells through a FhuA deletion derivative that forms permanently open channels three times as large as the porins in the outer membrane. FhuB peptides that bound to FhuD inhibited ferrichrome transport, while peptides that did not bind to FhuD did not affect transport. These data led us to propose that the periplasmic FhuD interacts with a transmembrane region and the cytoplasmic segment 7 of FhuB. The transmembrane region may be part of a pore through which a portion of FhuD inserts into the cytoplasmic membrane during transport. The cytoplasmic segment 7 of FhuB contains the conserved amino acid sequence EAA…G (in FhuB DTA…G) found in ABC transporters, which is predicted to interact with the cytoplasmic FhuC ATPase. Triggering of ATP hydrolysis by substrate-loaded FhuD may occur by physical interaction between FhuD and FhuC, which bind close to each other on loop 7. Although FhuB consists of two homologous halves, FhuB(N) and FhuB(C), the sites identified for FhuD-mediated ferrichrome transport are asymmetrically arranged.     

Arachidonic acid inhibits glycine transport in cultured glial cells.

The effects of arachidonic acid on glycine uptake, exchange and efflux in C6 glioma cells were investigated. Arachidonic acid produced a dose-dependent inhibition of high-affinity glycine uptake. This effect was not due to a simple detergent-like action on membranes, as the inhibition of glycine transport was most pronounced with cis-unsaturated long-chain fatty acids, whereas saturated and trans-unsaturated fatty acids had relatively little or no effect. Endogenous unsaturated non-esterified fatty acids may exert a similar inhibitory effect on the transport of glycine. The mechanism for this inhibitory effect has been examined in a plasma membrane vesicle preparation derived from C6 cells, which avoids metabolic or compartmentation interferences. The results suggest that part of the selective inhibition of glycine transport by arachidonic acid could be due to the effects of the arachidonic acid on the lipid domain surrounding the carrier.     

Apoplastic pH during low-oxygen stress in Barley

BACKGROUND AND AIMS: Anoxia leads to an energy crisis, tolerance of which varies from plant to plant. Although the apoplast represents an important storage and reaction space, and engages in the mediation of membrane transport, this extracellular compartment has not yet been granted a role during oxygen shortage. Here, an attempt is made to highlight the importance of the apoplast during oxygen stress and to test whether information about it is transferred systemically in Hordeum vulgare. METHODS: Non-invasive ion-selective microprobes were used which, after being inserted through open stomata, directly contact the apoplastic fluid and continuously measure the apoplastic pH and changes to it. KEY RESULTS: (a) Barley leaves respond to oxygen stress with apoplastic alkalinization and membrane depolarization. These responses are persistent under anoxia (N2; O2 < 3%) but transient under hypoxia. (b) Being applied to the root, the information 'anoxia' is signalled to the leaf as an increase in pH, whereas 'hypoxia' is not: flooding of the roots within the first 2 h has no effect on the leaf apoplastic pH, whereas anoxia (N2) or chemical anoxia (NaCN/salicylic hydroxamic acid) rapidly increase the leaf apoplastic pH. (c) Under anoxia, the proton motive force suffers a decrease by over 70 %, which impairs H(+) -driven transport. CONCLUSIONS: Although anoxia-induced apoplastic alkalinization is a general response to stress, its impact on the proton motive force (reduction) and thus on transport mediation of energy-rich compounds is evident. It is concluded that anoxia tolerance depends on how the plant is able to hold the proton motive force and H(+) turnover at a level that guarantees sufficient energy is harvested to overcome the crisis.     

Transport of a tripeptide, Gly-Pro-Hyp, across the porcine intestinal brush-border membrane.

The transcellular transport of oligopeptides across intestinal epithelial cells has attracted considerable interest in investigations into how biologically active peptides express diverse physiological functions in the body. It has been postulated that the tripeptide, Gly-Pro-Hyp, which is frequently found in collagen sequences, exhibits bioactivity. However, the mechanism of uptake of dietary di- and tripeptides by intestinal epithelial cells is not well understood. In this study, we used porcine brush-border membrane (BBM) vesicles to assess Gly-Pro-Hyp uptake, because these vesicles can structurally and functionally mimic in vivo conditions of human intestinal apical membranes. The present study demonstrated the time-dependent degradation of this tripeptide into the free-form Gly and a dipeptide, Pro-Hyp, on the apical side of the BBM vesicles. In parallel with the hydrolysis of the tripeptide, the dipeptide Pro-Hyp was identified in the BBM intravesicular space environment. We found that the transcellular transport of Pro-Hyp across the BBM was inhibited by the addition of a competitive substrate (Gly-Pro) for peptide transporter (PEPT1) and was pH-dependent. These results indicate that Gly-Pro-Hyp can be partially hydrolyzed by the brush-border membrane-bound aminopeptidase N to remove Gly, and that the resulting Pro-Hyp is, in part, transported into the small intestinal epithelial cells via the H+-coupled PEPT1. Gly-Pro-Hyp cannot cross the epithelial apical membrane in an intact form, and Pro-Hyp is highly resistant to hydrolysis by intestinal mucosal apical proteases.     

Uncoupled ATPase activity and heat production by the sarcoplasmic reticulum Ca2+-ATPase. Regulation by ADP

Sarcoplasmic reticulum vesicles of rabbit skeletal muscle accumulate Ca2+ at the expense of ATP hydrolysis. The heat released during the hydrolysis of each ATP molecule varies depending on whether or not a Ca2+ gradient is formed across the vesicle membrane. After Ca2+ accumulation, a part of the Ca2+-ATPase activity is not coupled with Ca2+ transport (Yu, X., and Inesi, G. (1995) J. Biol. Chem. 270, 4361-4367). I now show that both the heat produced during substrate hydrolysis and the uncoupled ATPase activity vary depending on the ADP/ATP ratio in the medium. With a low ratio, the Ca2+ transport is exothermic, and the formation of the gradient increases the amount of heat produced during the hydrolysis of each ATP molecule cleaved. With a high ADP/ATP ratio, the Ca2+ transport is endothermic, and formation of a gradient increased the amount of heat absorbed from the medium. Heat is absorbed from the medium when the Ca2+ efflux is coupled with the synthesis of ATP (5.7 kcal/mol of ATP). When there is no ATP synthesis, the Ca2+ efflux is exothermic (14-16 kcal/Ca2+ mol). It is concluded that in the presence of a low ADP concentration the uncoupled ATPase activity is the dominant route of heat production. With a high ADP/ATP ratio, the uncoupled ATPase activity is abolished, and the Ca2+ transport is endothermic. The possible correlation of these findings with thermogenesis and anoxia is discussed.     

Studies on transport of amino acids from peptides by rat small intestine in vitro. Synthesis, properties and uptake of a photosensitive tetrapeptide

By comparison with what is known of disaccharides transport, it has been suggested that intestinal aminopeptidase N could, hydrolyze, on the surface of the microvillus membrane, oligopeptides longer than tripeptides and itself subserve the translocation function for the amino acids released from these peptides. This article describes the synthesis of the tritiated azido-tetrapeptides p-azido[3H]phenylalanyl-alanyl-glycyl-glycine containing L or D-alanine. The synthesized products possess a function which displays all the characteristics of an aryl-azide. The photosensitive tetrapeptide formed with LAla-Gly-Gly is as good a substrate for porcine and rat aminopeptidases N as unmodified peptides while the tetrapeptide formed with DLa-Gly-Gly is not hydrolyzed at all. In addition a pattern of stepwise hydrolysis could be demonstrated and aminopeptidase N is the only exopeptidase present in the mucosal cells capable of utilizing the modified tetrapeptide as substrate. Uptake assays performed on everted rings of jejunum with the azido-tetrapeptide as substrate have shown that: (a) the azido-tetrapeptide is not transported intact but must be hydrolyzed first; (b) p-azido-phenylalanine is not released in the external medium and therefore its observed uptake is not from the bulk medium and (c) the azido-D-tetrapeptide is only accumulated by passive diffusion. These observations suggest the presence on the brush border membrane of an aminopeptidase-related transport system.     

Monitoring enzyme synthesis as a means of studying peptide transport and utilization in Escherichia coli.

A new method has been developed for measuring peptide transport in aminoacid auxotrophs of Escherichia coli by following induction of beta-galactosidase. Appearance of the enzyme was determined after addition of inducer and peptides to amino-acid starved bacteria. For a given number of lysine equivalents, the rate and the extent of enzyme synthesis were the same for lysine and lysyl peptides; similar results were found for glycine and glycl peptides. Saturation constants for peptide transport were determined from the exogenous peptide concentration that gave half maximal rates of enzyme synthesis. The saturation constants, studies with mutants defective in peptide transport, and detection of competition between peptides for uptake, all endorsed earlier conclusions from growth tests about the structural specificities for peptide transport. The new method is quicker, more sensitive and more informative than growth tests.     

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.     

Differences in coupling of energy to glycine and phenylalanine transport in aerobically grown Escherichia coli.

Differences exist in the coupling of energy to transport of glycine and phenylalanine in aerobically grown cells of Escherichia coli. Energy derived from respiration, although involved in both uptake systems, is not employed identically as shown by kinetic effects of cyanide and anoxia and by temperature dependencies. Additional evidence for aerobic differences was provided by the effects of azide which greatly decreased initial rates of uptake of glycine but not phenylalanine. The effect on glycine uptake was not due to uncoupling of oxidative phosphorylation or to a decrease in respiration rate. Evidence for anaerobic differences was provided by the addition of either glucose or ferricyanide to cell suspensions containing glycerol, thereby maintaining anoxic uptake of phenylalanine, but not glycine, at the aerobic level. Ferricyanide stimulation required a functional Ca, Mg-adenosine 5'-triphosphatase and involved cell metabolism. Ferricyanide was also found to produce differential stimulation of other amino acid transport systems; tyrosine, tryptophan and leucine uptakes were stimulated whereas those for alanine, proline, threonine, and glutamine were relatively unaffected.     

Peptide utilization in Escherichia coli: Studies with peptides containing β-alanyl residues

A glycine auxotroph of Escherichia coli can utilize glycine oligopeptides as a source of its required amino acid. Glycylglycyl-β-alanine and β-alanylglycylglycine are both readily hydrolysed by intracellular peptidases, but only the former supports growth of the glycine auxotroph. Glycylglycyl-β-alanine is not nutritionally active towards a glycine mutant that is unable to transport oligopeptides. The nutritional responses to these β-alanine peptides are interpreted in terms of the structural requirements of the oligopeptide transport system, for which an α-peptide bond is required but the C-terminal α-carboxyl group is not essential. Dipeptides of β-alanine are generally poor sources of amino acids for auxotrophs of E. coli, although β-alanylhistidine (carnosine) is as effective as the free amino acid in supporting growth of a histidine auxotroph; this observation does not accord with the structural requirements established for dipeptide transport in general, and may indicate a separate uptake process. The results are related to the occurrence of β-alanyl peptides in the normal environment of enteric bacteria, and to the known ability of the intestine to transport carnosine.     

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.