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PubMed1982

Proline transport by synaptosomal membrane vesicles isolated from rat brain: energetics and inhibition by free fatty acids.

Synaptosomal membrane vesicles have been employed to study the energetics of proline transport and the inhibition of proline transport by unsaturated free fatty acids. Active uptake of proline into synaptosomal membrane vesicles requires extravesicular Na+ and is primarily driven by a Na+ gradient created by diluting K+-loaded vesicles into Na+-containing buffers. Uptake of proline under these conditions is enhanced up to 2-fold by a valinomycin-induced diffusion potential (interior negative). Proline transport is reduced in the absence of external C1- or internal K+. Strong (40-90%) inhibition of proline uptake occurs upon collapse of the Na+ gradient by ionophores such as gramicidin D or activation of the action potential Na+ channel by veratridine or Tityus serrulatus venom. Less (15-25%) inhibition is obtained with the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, which also prevents the stimulation of proline uptake by the valinomycin-induced diffusion potential. Unsaturated free fatty acids inhibit proline uptake. The inhibition is greatest for arachidonic acid and was somewhat less for oleic acid. The saturated fatty acids palmitic and stearic have little or no inhibitory capacity. Endogenous unsaturated free fatty acids may exert similar inhibitory effects on the reuptake systems for neuroactive amino acids and thus modulate their action in the central nervous system.

Animals↗
PubMed1997

Aspartate 55 in the Na+/proline permease of Escherichia coli is essential for Na+-coupled proline uptake.

Four acidic residues in the N-terminal domain of Na+/proline permease of Escherichia coli (Asp33, Asp34, and Asp55 in putative loop 2, Glu75 in putative transmembrane domain II) were individually replaced with neutral or charged amino acid residues. Replacement of Glu75, the only residue in the permease presumed to be in the middle of a transmembrane domain, Asp33, or Asp34 had little or no influence on the kinetics of Na+-coupled proline transport. In contrast, removal of the carboxylate at position 55 (Asp55 --> Asn or Asp55 --> Cys permease) impaired proline uptake completely while lengthening of the side chain at this position by one methylene group (Asp55 --> Glu permease) allowed transport at a reduced initial rate. Importantly, all permease molecules were present in the membrane at concentrations comparable to the wild-type protein. Kinetic analysis of Na+-coupled proline transport catalyzed by Asp55 --> Glu permease revealed a 5-fold increase of the K(m) for proline and a 30-fold decrease of the V(max) compared to wild-type. Remarkably, replacement of Asp55 by Glu led to a 50-fold decrease of the apparent affinity of the permease for Na+. Furthermore, replacement of Asp55 with Cys or Asn blocked proline-induced Na+ uptake whereas significant Na+ transport was observed with Asp55 --> Glu permease. In addition, transport of proline down its concentration gradient was not detectable with deenergized cells containing Asp55 --> Glu permease at low Na+ concentrations. However, downhill transport activity was observed in the presence of high Na+ concentrations. Replacement of Asp55 with Asn or Cys impaired downhill transport under all conditions tested. The observations demonstrate that a carboxylate at position 55 of proline permease is essential for Na+-coupled proline transport. It is suggested that Asp55 may be involved in binding of the coupling ion.

Amino Acid Sequence↗
PubMed1998

A conserved aspartate residue, Asp187, is important for Na+-dependent proline binding and transport by the Na+/proline transporter of Escherichia coli.

Asp187 in the Na+/proline transporter of Escherichia coli (PutP) is conserved within the Na+/solute cotransporter family. Information on the role of this residue has been gained by amino acid substitution analysis. PutP with Glu, Asn, or Cys in place of Asp187 catalyzed Na+-coupled proline uptake at 75%, 25%, and 1.5%, respectively, of the Vmax of PutP-wild-type while the apparent Km for proline was only slightly altered. Importantly, acetylation or amidoacetylation of an engineered transporter containing a single Cys at position 187 stimulated proline uptake. Strikingly, PutP-D187C exhibited high-affinity proline binding even at very low Na+ concentrations (2 microM) while proline binding to PutP-wild-type, -D187E, and -D187N was strictly dependent on the Na+ concentration. The apparent independence of proline binding from the Na+ concentration can at least partially be attributed to an enhanced Na+ affinity of PutP-D187C. In addition, reaction of PutP containing a single Cys at position 187 with N-ethylmaleimide was inhibited by Na+ but not by Li+ or proline. The results indicate that electrostatic interactions of the amino acid side chain at position 187 in PutP with other parts of the transporter and/or the coupling ion are crucial for active proline transport. It is suggested that Asp187 is located close to the pathway of the coupling ion through the membrane and may be involved in the release of Na+ on the cytoplasmic side of the membrane.

Amino Acid Sequence↗
PubMed1999

The 2.1 A structure of a cysteine protease with proline specificity from ginger rhizome, Zingiber officinale.

A cysteine protease from ginger rhizome (GP-II) cleaves peptides and proteins with proline at the P(2) position. The unusual specificity for proline makes GP-II an attractive tool for protein sequencing and identification of stably folded domains in proteins. The enzyme is a 221 amino acid glycoprotein possessing two N-linked oligosaccharide chains (8% glycosylated by weight) at Asn99 and Asn156. The availability of the sequence of these glycosyl chains afforded the opportunity to observe their structure and impact on protein conformation. The three-dimensional structure of GP-II has been determined by X-ray crystallography to a resolution of 2.1 A (overall R-factor = 0.214, free R = 0.248). The overall structure of GP-II is similar to that of the homologous cysteine proteases papain, actinidin, and glycyl endopeptidase, folding into two distinct domains of roughly equal size which are divided by a cleft. The observed N-linked glycosyl chains (half the total carbohydrate sequence) participate in both crystallographic and noncrystallographic contacts, tethering the proteins together via hydrogen bonds to the carbohydrate residues without intervening ordered water molecules. The putative S(2) binding pocket (the proline recognition site) was identified by superposition of the GP-II structure with structures of four previously determined papain-inhibitor complexes. The particular enzymic amino acids forming the S(2) pocket of GP-II (Trp, Met, and Ala) are similar to those found in the proline binding pockets of the unrelated enzymes alpha-lytic protease and cyclophilin. However, there is no conserved three-dimensional arrangement of these residues between the three enzymes (i.e., no proline binding motif). Thus, the particular amino acids found at S(2) are consistent with a binding pocket for a moiety with the steric characteristics and charge distribution of proline. Size exclusion is also a mechanism for selectivity compared to the S(2) binding pocket of papain. The S(2) binding pocket of GP-II greatly restricts the size of the side chain which could be bound because of the occurrence of a tryptophan in place of the corresponding tyrosine in papain. In light of the nature of the binding pocket, the specificity of GP-II for proline over other small nonpolar amino acids may be attributed to a direct effect of proline on the substrate peptide backbone conformation.

Binding Sites↗
PubMed1990

Effect of prostaglandin E2 on proline uptake and protein synthesis by cultured human mesangial cells.

PGE2 production by glomeruli is increased in a variety of glomerular diseases. Potentially, this process may affect mesangial cell protein synthesis and mesangial cell growth. Thus studies have been undertaken, using cultured human mesangial cells, to assess the effects of PGE2 on proline uptake, protein synthesis and cell proliferation. In the presence of 140 mM NaCl, incubation of mesangial cells with 0.01 to 1 microM PGE2 for 72 hours resulted in a marked decrease of 14C proline uptake, but did not modify 14C leucine uptake. Substitution of choline to sodium inhibited 14C proline uptake by 85% which became independent of PGE2, indicating that this PG specifically altered sodium-dependent proline uptake. Inhibition of this component reached 35 to 50% with 1 microM PGE2. The inhibitory effect of PGE2 on sodium-dependent proline uptake required a lag time of 48 hours, and was suppressed by ouabain, an inhibitor of Na+, K+ ATPase activity. PGE2 did not modify the Vmax of the transport system (1.007 vs. 1.023 nmol/mg/min) but increased (P less than 0.01) its Km (1.179 vs. 0.823 mM). 8-bromo-cyclic AMP also inhibited sodium-independent proline uptake, and PGE2 markedly increased cyclic AMP production. Taken together, these results suggested that PGE2 acted via cyclic AMP stimulation. PGE2 under identical conditions (1 microM, 72 hr incubation) produced a decrease in collagen synthesis estimated by the relative rate of collagen production after incubation of mesangial cells with 14C proline (percentage of 14C radioactivity in collagenase-sensitive proteins over total proteins). PGE2 also diminished the intracellular free proline pool. More generally, PGE2 inhibited cell proliferation and cell total proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Division↗
PubMed1991

Interleukin-1 beta-induced changes in the kinetic constants of L-proline uptake in human skin fibroblasts.

The effects of interleukin-1 beta (IL-1) on L-proline uptake in human skin fibroblasts were investigated. Exposure of the fibroblasts to IL-1 (5, 10 or 50 pg/ml) for 2 h did not change L-proline uptake. In contrast, inhibition was observed after 6 h of IL-1 treatment, and only 60% of the control uptake remained after incubation for 24 h with 10 pg of IL-1/ml. IL-1 depressed the activity of both transfer systems; the low-affinity system inhibited by alpha-(methylamino)isobutyric acid (Me-AIB), corresponding to system A, and a high-affinity transfer system which is unaffected by Me-AIB. The inhibitory effect increased as the L-proline concentration decreased. To determine whether IL-1-induced prostaglandin release influences proline uptake, indomethacin (14 microM) was added as a cyclo-oxygenase inhibitor. Indomethacin itself decreased L-proline uptake but to a lesser extent than did IL-1. When IL-1 was tested in the presence of indomethacin, the inhibition of L-proline uptake was still observed, with values between those obtained with each substance in isolation. This suggests that the inhibitory effect of IL-1 on proline uptake by skin fibroblast does not only involve the prostaglandins that accumulate in the medium, but no firm conclusion can be drawn, due to the fact that the inhibition by the two agents was not statistically independent. Kinetic analyses for 1 min combined with inhibition experiments showed that IL-1 induced a decrease in the Km and Vmax, values of the high-affinity transport system, whereas it increased the Km of system A. Therefore the two systems of proline uptake in skin fibroblasts are probably inhibited by IL-1 via different mechanisms.

Biological Transport↗
PubMed1996

Purification and initial characterization of proline 4-hydroxylase from Streptomyces griseoviridus P8648: a 2-oxoacid, ferrous-dependent dioxygenase involved in etamycin biosynthesis.

Proline 4-hydroxylase is a 2-oxoacid, ferrous-ion-dependent dioxygenase involved in the biosynthesis of the secondary metabolite etamycin. The purification, in low yield, of proline 4-hydroxylase from Streptomyces griseoviridus P8648 to near, apparent homogeneity and its initial characterization are reported. In most respects proline 4-hydroxylase is a typical member of the 2-oxoacid-dependent dioxygenase family. It is monomeric (M(r) approx. 38,000) (by gel filtration on Superdex-G75) and has typically strict requirements for ferrous ion and 2-oxoglutarate. The enzyme was inhibited by aromatic analogues of 2-oxoglutarate. L-Proline-uncoupled turnover of 2-oxoglutarate to succinate and CO2 was observed. The addition of L-ascorbate did not stimulate L-proline-coupled turnover of 2-oxoglutarate, but did stimulate L-proline-uncoupled turnover. L-Ascorbate caused a time-dependent inhibition of L-proline hydroxylation. The enzyme was completely inactivated by preincubation with diethyl pyrocarbonate under histidine-modifying conditions. This inactivation could be partially prevented by the inclusion of L-proline and 2-oxoglutarate in the preincubation mixture, suggesting the presence of histidine residue(s) at the active site.

Anti-Bacterial Agents↗
PubMed1996

Multiple cis-trans conformers of the prolactin receptor proline-rich motif (PRM) peptide detected by reverse-phase HPLC, CD and NMR spectroscopy.

An eight-amino-acid synthetic peptide (IIe1-Phe2-Pro3-Pro4-Val5-Pro6-Gly7-Pro8) corresponding to the conserved proline-rich motif (PRM) of the intracellular domain of the prolactin receptor (PRL-R) was studied by one- and two-dimensional (1D and 2D) proton NMR spectroscopy in water and DMSO in order to characterize its conformational dynamics. The purified PRL-R PRM peptide eluted as two partially resolved peaks in equilibrium on reverse-phase HPLC (RP-HPLC) at 20 degrees C with a ratio of 60:40. At 30 degrees C, the two peaks coalesced into a single peak The two RP-HPLC peaks correspond to two peptide conformers resulting from the slow cis-trans isomerization of one of the four proline amide bonds. Although the peptide has only three amide (NH) protons, its ID NMR spectrum in water contains approximately 15 discernible NH region peaks, providing evidence for multiple conformers. The amide resonances were assigned on the basis of 2D-COSY spectra, chemical shift values resonance splitting patterns and temperature coefficients. The cis:trans ratio for each proline in water, calculated from integrated intensities and/or peak heights of the appropriate resonances, were Phe2-Pro3 (35:65), Pro3-Pro4 (40:60), Val5-Pro6 (70:30), and Gly7-Pro8 (30:70). Temperature studies (25-70 degrees C) were used to semi-quantitatively estimate the rates of isomerization for the different prolines. In water, Pro8 undergoes rapid isomerization; Pro3 isomerizes at an intermediate rate; while Pro4 and Pro6 both appear to isomerize very slowly since no coalescence of amide resonances was observed. In DMSO, only Pro4 displayed slow isomerization. Slow kinetics combined with a similar 60:40 ratio of conformers determined by RP-HPLC and NMR suggests that isomerization of the Pro3-Pro4 bond generates the two RP-HPLC peaks. Both proximal and distal proline isomerization effects were observed in NMR experiments. All of the 16 theoretical (24 = 16) proline configurations appear to exist in equilibrium in water The predominant (19%) conformation, trans3-trans4-cis6-trans8, may reflect the configuration of the PRM prolines in the native PRL-R. Isomerization of Pro6 from cis to trans generates an interaction between the peptide N-and C-termini, suggesting an overall pseudo-cyclic conformation. This all-trans proline configuration may play an important biochemical role in the function of cytokine/haematopoietin receptors. A model is proposed which suggests that isomerization of the PRM by an immunophilin such as the FK 506-binding protein (FKBP) serves as an on-off switch for cytokine receptor activation.

Amides↗
PubMed1975

Role of epithelial architecture and intracellular metabolism in proline uptake and transtubular reclamation in PRO/re mouse kidney.

The homozygous PRO/Re mouse has less than 1 percent of the very high proline oxidase activity that characterizes normal kidney cortex. In PRO/Re mouse the endogenous proline concentration is eight times normal in plasma and four times normal in kidney cortex cell, but 50 times normal in urine. The integrity of the membrane transport systems for proline uptake at the antiluminal surface of absorbing epithelium is retained in PRO/Re kidney, as determined by the slice method. Clearance studies in vivo under steady-state conditions indicate that the integrity of the luminal uptake system shared by glycine and proline, and serving proline absorption, is also intact. The exaggerated renal clearance of proline in PRO/Re mice (50 times normal) is explained when its raised intracellular concentration, caused by impaired proline oxidation, is considered. Backflux into urine flowing down the nephron will occur under these conditions, thus impairing net reclamation of proline in PRO/Re kidney. The findings reveal that membrane transport and intracellular metabolism of a substrate are, indeed, independent functions, but that metabolism of a substance can influence its transcellular transport.

Animals↗
PubMed2004

Phospholipase D is a negative regulator of proline biosynthesis in Arabidopsis thaliana.

Accumulation of proline has been observed in a large number of plant species in response to drought and salt stresses, suggesting a key role of this amino acid in plant stress adaptation. Upstream components of the proline biosynthesis signal transduction pathways are still poorly defined. We provide experimental evidence that phospholipase D (PLD) is involved in the regulation of proline metabolism in Arabidopsis thaliana. The application of primary butyl alcohols, which divert part of PLD-derived phosphatidic acid by transphosphatidylation, stimulated proline biosynthesis even without hyperosmotic constraints. Moreover, application of primary butyl alcohols enhanced the proline responsiveness of seedlings to mild hyperosmotic stress. These data indicate that some PLDs are negative regulators of proline biosynthesis and that plants present a higher proline responsiveness to hyperosmotic stress when this regulator is abolished. We clearly demonstrate that PLD signaling for proline biosynthesis is similar to RD29A gene expression and different from the abscisic acid-dependent RAB18 gene expression. Our data reveal that PLDs play positive and negative roles in hyperosmotic stress signal transduction in plants, contributing to a precise regulation of ion homeostasis and plant salt tolerance.

Abscisic Acid↗
PubMed1994

Production of proline-rich proteins by the parotid glands of rats is enhanced by feeding diets containing tannins from faba beans (Vicia faba L.).

Feeding a casein-based diet with either 400 g/kg of tannin-containing faba bean hulls (Vicia faba L.) (1.41% condensed tannins) or 60 g/kg of a tannin-rich hull extract of faba beans (1.99% condensed tannins) to rats over a period of 7 d resulted in a 2.6 and 1.5 fold increase in weight of the parotid glands, respectively, (P < 0.05) and a corresponding 5.5 and 3.7 fold increase in the level of proline-rich proteins in the glands (P < 0.05). In a dose-response experiment, increasing the level of tannin-rich hull extract in the diet (0.0, 3.8, 7.5, 15.0, 30.0 and 60.0 g/kg) resulted in a linear increase in both the relative size of parotid glands (R2 = 0.90; P < 0.05) and the quantity of proline-rich proteins in the glands (R2 = 0.89; P < 0.05). The apparent digestibility of total (R2 = 0.97) and individual amino acids (R2 varied from 0.27 to 0.99) decreased linearly (P < 0.05). The quantity of proline-rich proteins in the cecum of rats was estimated from the decrease in digestibility of proline, glycine and glutamic acid. The estimated secretions of proline-rich proteins, when calculated on the basis of the three respective amino acids, were 3.5, 3.5 and 3.9 mg of proline-rich proteins per 10 mg of additional hull extract (21.8% condensed tannins). The results indicate that tannins from faba beans stimulate the parotid glands to increase the secretion of proline-rich proteins in rats. The proline-rich proteins then interact with dietary condensed tannins to reduce their antinutritional effects.

Amino Acids↗
PubMed2000

Prokink: a protocol for numerical evaluation of helix distortions by proline.

Proline residues are known to perturb the structure of helices by introducing a kink between the segments preceding and following the proline residue. The distortion of the helical structure results from the avoided steric clash between the ring of the proline at position (i) and the backbone carbonyl at position (i - 4), as well as the elimination of helix backbone H-bonds for the carbonyls at positions (i - 3) and (i - 4). Both the departure from the ideal helical pattern and the reduction in H-bond stabilization contribute to the observed flexibility of a proline-containing alpha-helix. The special local flexibility of the proline kink can confer an important role on the proline-containing helix in the conformational changes related to the function of the protein. As a useful tool in determining and evaluating the role of proline-induced flexibility and distortions in protein function, we present here a protocol to quantify the geometry of the distortion introduced in helices by prolines both as a time-averaged value and for individual 'snapshots' along a molecular dynamics simulation.

Algorithms↗
PubMed1992

Proline residues in transmembrane helices of channel and transport proteins: a molecular modelling study.

Proline residues are commonly found in putative transbilayer helices of many integral membrane proteins which act as transporters, channels and receptors. Intramembranous prolines are often conserved between homologous proteins. It has been suggested that such intrahelical prolines provide liganding sites for cations via exposure of the backbone carbonyl oxygen atoms of residues i-3 and i-4 (relative to the proline). Molecular modelling studies have been carried out to evaluate this proposal. Bundles of parallel proline-kinked helices are considered as simplified models of ion channels. The energetics of K+ ion-helix bundle interactions are explored. It is shown that carbonyl oxygens exposed by the proline-induced kink and at the C-terminus of the helices may provide cation-liganding sites. 'Hybrid' bundles of antiparallel helices, only some of which contain proline residues, are considered as models of transport proteins. Again, proline-exposed carbonyl oxygens are shown to be capable of liganding cations. The roles of alpha-helix dipoles and of the geometry of helix packing are considered in relation to cation-bundle interactions. Implications with respect to modelling of ion channel and transport proteins are discussed.

Binding Sites↗
PubMed2005

Effects of luminal deoxynivalenol and L-proline on electrophysiological parameters in the jejunums of laying hens.

Most amino acids are cotransported with sodium. Deoxynivalenol (DON) decreases glucose absorption in the chicken small intestine in vivo and in vitro, and this effect is apparently mediated by the inhibition of the sodium D-glucose cotransporter. DON could selectively modulate the activities of other intestinal transporters. In order to assess this hypothesis, a study was conducted to characterize the in vitro effects of DON in the presence of mucosal amino acids, using L-proline as a model, on the electrophysiological parameters in the jejunums of laying hens. L-Proline (mucosal concentration of 1 mmol/L) was added to a stripped proximal part of jejunum sheets mounted in Ussing chambers in Ringer buffer, and the electrical properties were measured. The transmural potential difference (PD) was nearly constant between the treatments. The tissue resistance (Rt) was higher (P < 0.05) in the tissues exposed to DON compared with basal values and the values after addition of L-proline. Addition of L-proline on the luminal side of the isolated mucosa increased (P < 0.05) the short circuit-current (Isc), and it decreased (P < 0.05) after addition of DON, indicating that the proline-induced Isc was altered by DON. The addition of proline after incubation of the tissues with DON had no effect (P > 0.05) on PD or Rt. Proline did not increase the Isc under these conditions. DON decreased (P < 0.1) the Isc after addition of proline, indicating that DON inhibited the Na+-amino acid co-transport. We concluded from the present study that the amino acid cotransporter activity appears to be highly sensitive to DON suppression.

Animals↗
PubMed1984

Active transport of proline by Coxiella burnetii.

The obligate intracellular rickettsia, Coxiella burnetii, was shown to possess an energy dependent proline transport system which displayed a high degree of specificity and was highly dependent on pH. Transport was maximal at pH 3.0 to 4.5, a pH range approximately that of the host cell phagolysosome where the agent replicates. Transport was inhibited by the uncouplers carbonyl cyanide m-chlorophenylhydrazone and dinitrophenol, but not by sodium arsenite. In the presence of glutamate, a preferred energy source, proline uptake was enhanced more than two-fold. This enhancement of proline uptake was greatly decreased in the presence of sodium arsenite. The addition of glutamate decreased the apparent Km for proline transport from 45 microM to 15 microM, with the Vmax increasing from 3.6 pmol s-1 (mg dry wt)-1 to 4.8 pmol s-1 (mg dry wt)-1. Two proline analogues, furoic acid and azetidine-2-carboxylic acid, were effective inhibitors of proline transport. D-Proline, 4-hydroxyproline, glycine and proline amide inhibited transport minimally, while no inhibition was seen with succinate, pyruvate or glutamate.

2,4-Dinitrophenol↗
PubMed1999

Positional preference of proline in alpha-helices.

Conformational free energy calculations have been carried out for proline-containing alanine-based pentadecapeptides with the sequence Ac-(Ala)n-Pro-(Ala)m-NHMe, where n + m = 14, to figure out the positional preference of proline in alpha-helices. The relative free energy of each peptide was calculated by subtracting the free energy of the extended conformation from that of the alpha-helical one, which is used here as a measure of preference. The highest propensity is found for the peptide with proline at the N-terminus (i.e., Ncap + 1 position), and the next propensities are found at Ncap, N' (Ncap - 1), and C' (Ccap + 1) positions. These computed results are reasonably consistent with the positional propensities estimated from X-ray structures of proteins. The breaking in hydrogen bonds around proline is found to play a role in destabilizing alpha-helical conformations, which, however, provides the favored hydration of the corresponding N-H and C=O groups. The highest preference of proline at the beginning of alpha-helix appears to be due to the favored electrostatic and nonbonded energies between two residues preceding proline and the intrinsic stability of alpha-helical conformation of the proline residue itself as well as no disturbance in hydrogen bonds of alpha-helix by proline. The average free energy change for the substitution of Ala by Pro in a alpha-helix is computed to be 4.6 kcal/mol, which is in good agreement with the experimental value of approximately 4 kcal/mol estimated for an oligopeptide dimer and proteins of barnase and T4 lysozyme.

Peptides↗
PubMed2006

Transgenic Medicago truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced tolerance to osmotic stress.

Legume root nodule nitrogen-fixing activity is severely affected by osmotic stress. Proline accumulation has been shown to induce tolerance to salt stress, and transgenic plants over-expressing Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), which accumulates high levels of proline, display enhanced osmotolerance. Here, we transformed the model legume Medicago truncatula with the P5CS gene from Vigna aconitifolia, and nodule activity was evaluated under osmotic stress in transgenic plants that showed high proline accumulation levels. Nitrogen fixation was significantly less affected by salt treatment compared to wild-type (WT) plants. To our knowledge, this is the first time that transgenic legumes have been produced that display nitrogen-fixing activity with enhanced tolerance to osmotic stress. We studied the expression of M. truncatula proline-related endogenous genes M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 1 (MtP5CS1), M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 2 (MtP5CS2), M. truncatula ornithine delta-aminotransferase (MtOAT), M. truncatula proline dehydrogenase (MtProDH) and a proline transporter gene in both WT and transgenic plants. Our results indicate that proline metabolism is finely regulated in response to osmotic stress in an organ-specific manner. The transgenic model allowed us to analyse some of the biochemical and molecular mechanisms that are activated in the nodule in response to high salt conditions, and to ascertain the essential role of proline in the maintenance of nitrogen-fixing activity under osmotic stress.

Adaptation, Physiological↗
PubMed1983

The location of redox-sensitive groups in the carrier protein of proline at the outer and inner surface of the membrane in Escherichia coli.

Evidence is presented in this report for the presence of two sets of dithiols associated with proline transport activity in Escherichia coli. One set is located at the outer surface, the other at the inner surface of the cytoplasmic membrane. Treatment of right-side-out membrane vesicles from E. coli ML 308-225 with the membrane-impermeable oxidant ferricyanide resulted in inhibition of L-proline uptake without having significant effect on the magnitude of the delta approximately mu H+. Subsequent addition of reducing agents restored proline transport activity. The membrane-impermeable SH-reagent glutathione hexane maleimide inhibited proline transport in right-side-out membrane vesicles irreversibly. Pretreatment of the vesicles with ferricyanide protected the carrier against inactivation by glutathione hexane maleimide. Electron transfer in the respiratory chain of right-side-out vesicles led to the generation of a delta approximately mu H+, interior negative and alkaline, and the conversion of a disulphide to a dithiol in the proline carrier as is shown by the increased inhibition of proline transport by the membrane impermeable dithiol reagent 4-(2-arsonophenyl)azo-3-hydroxy-2,7-naphthalene disulphonic acid (thorin). The inhibition exerted by thorin was completely reversed by dithiothreitol. Pretreatment of the vesicles with thorin protected against glutathione hexane maleimide inhibition, indicating that both reagents react with the same group. Treatment of inside-out membrane vesicles with ferricyanide inactivated the proline transport system reversibly. The oxidizing effect of ferricyanide in inside-out vesicles resulted in protection against inhibition by glutathione hexane maleimide. Imposition in these vesicles of a delta approximately mu H+, interior positive and acid, also protected the proline carrier against glutathione hexane maleimide inactivation, indicating that a dithiol is converted to a disulphide upon energization.

Arsenicals↗