Transport of L-proline,L-proline-containing peptides and related drugs at mammalian epithelial cell membranes

Summary. Membrane transport of L-proline has received considerable attention in basic and pharmaceutical research recently. Of the most recently cloned members of the solute carrier family, two are “proline transporters”. The amino acid transporter PAT1, expressed in intestine, kidney, brain and other organs, mediates the uptake of proline and derivatives in a pH gradient-dependent manner. The Na⁺-dependent proline transporter SIT1, cloned in 2005, exhibits the properties of the long-sought classical IMINO system. Proline-containing peptides are of interest for several reasons. Many biologically important peptide sequences contain highly conserved proline residues. Xaa-Pro peptides are very often resistant to enzymatic hydrolysis and display, in contrast to Pro-Xaa peptides, a high affinity to the H⁺/peptide cotransporter PEPT1 which is expressed in intestinal, renal, lung and biliary duct epithelial cells. Furthermore, several orally available drugs are recognized by PEPT1 as Xaa-Pro analogues due to their sterical resemblance to small peptides.     

Synthesis, tandem MS- and NMR-based characterization, and quantification of the carbon 13-labeled advanced glycation endproduct, 6-N-carboxymethyllysine

Summary. 6-N-carboxymethyllysine (CML), generated by the glycation and/or oxidation of lysine residues, has been measured in biological materials and food products using techniques such as ELISA, HPLC with fluorescence detection and mass spectrometry methods. Only limited information has been reported regarding the preparation of standards labeled with either deuterium, ¹³C or ¹⁵N atoms to be used as internal standards. In the present paper, a synthesis of carbon-13 labeled CML is described using l,2-¹³C₂-glyoxylic acid and 2-N-acetyllysine as starting materials. The resulting labeled 2-N-acetyl-CML was purified by HPLC-UV as a dibutyl ester. After a deprotection step, the yield was evaluated to be 53% when the reaction was conducted 17 h at 37°C. CML was extensively studied by ¹H- and ¹³C-NMR and the fragments observed in the collision induced dissociation (CID) spectrum were also assigned. Finally, the standards of CML and carbon-13 labeled CML were accurately quantified based on ¹H-NMR and tandem MS using lysine as an internal reference.     

Alterations in neutrophil (PMN) free intracellular alpha-keto acid profiles and immune functions induced by L-alanyl-L-glutamine, arginine or taurine

Summary. The objective of this study was to determine the dose as well as duration of exposure-dependent effects of L-alanyl-L-glutamine, arginine or taurine on polymorphonuclear neutrophil (PMN) free α-keto acid profiles and, in a parallel study, on PMN immune functions. Exogenous L-alanyl-L-glutamine significantly increased PMN α-ketoglutarate, pyruvate PMN superoxide anion (O₂⁻) generation, hydrogen peroxide (H₂O₂) formation and released myeloperoxidase (MPO) activity. Arginine also led to significant increases in α-ketoglutarate, pyruvate, MPO release and H₂O₂ generation. Formation of O₂⁻ on the other hand was decreased by arginine. Incubation with taurine resulted in lower intracellular pyruvate and α-ketobutyrate levels, decreased O₂⁻ and H₂O₂ formation and a concomitant significantly increased MPO activity. We therefore believe that considerable changes in PMN free-α-keto-acid profiles, induced for example by L-alanyl-L-glutamine, arginine or taurine, may be one of the determinants in cell nutrition that considerably modulates the immunological competence of PMN.     

Relationship between NO synthesis, arginine transport, and intracellular arginine levels in vascular smooth muscle cells

Summary. The present study was designed to evaluate the relevance of arginine transport in nitric oxide (NO) synthesis in vascular smooth muscle cells. For this purpose, NO synthesis and arginine transport (system B^0,+ and y⁺) were evaluated in cells treated with IL-1β or angiotensin II (Ang II). In addition, the effects of 5 mM lysine and glutamine, competitive inhibitors of systems y⁺ and B^0,+ respectively, were examined. L-arginine transport was estimated with ³H-labelled arginine and NO was determined with the Griess reagent. These studies were done in control conditions, arginine-starved cells, and in cells incubated in media containing 10 mM arginine. Our data indicate that induction of NO biosynthesis by IL-1β depends on external arginine when cells are arginine-depleted for 24 hours. The concentration of arginine producing half maximal activation of NO synthesis in arginine-depleted cells ([arginine]_i < 10 μM) was 41.1 ± 18 μM. By contrast, in normal culture conditions, NO synthesis occurred independently of arginine transport. Neither 5 mM lysine or glutamine which abolished arginine transport through systems y⁺ and B^0,+, respectively, reduced nitrite release in cells incubated in normal media. This suggests that the relevance of arginine uptake to NO synthesis depends on the status of intracellular arginine pools. Intracellular arginine concentrations were not affected by the stimulation of NO production using IL-1β or its inhibition using Ang II, but were markedly reduced by arginine starvation for 48 h. Aspartate levels were also reduced by arginine-depletion, but were not affected in cells incubated with 10 mM arginine. By contrast, glutamate levels were reduced in arginine-starved cells and were increased in cells incubated in arginine-supplemented medium. Ornithine levels were markedly increased by arginine supplementation. Altogether, these findings indicate that NO synthesis is normally independent of membrane transport. However in arginine-depleted cells, membrane transport is essential for NO synthesis. It is concluded that arginine transport is required for the long-term maintenance of intracellular arginine pools. Received February 7, 1999; Accepted June 21, 1999     

The peptides of α-aminosuberic acid I New intermediates for synthetic studies

Summary. The paper describes the synthesis of α-aminosuberic acid derivatives suitable for the synthesis of peptides. These include Z-, Boc- and Fmoc-protection on the α-amino group, benzyl ester, Boc-hydrazide and Z-hydrazide as well as the free carboxylic function in the side chain, and methyl ester, benzyl ester or free α-carboxylic group. Their use is demonstrated on the synthesis of the respective derivatives of Asu-Val-Leu. The enzyme catalyzed reaction was succesfully used both as a route to L-Asu from the D,L-compound as well as for the direct synthesis of the optically active tripeptide derivative from the Z-D,L-Asu-OH. Received February 5, 1999     

Recombinant horseradish peroxidase isoenzyme C: the effect of distal haem cavity mutations (His42→Leu and Arg38→Leu) on compound I formation and substrate binding

 Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the role of two key distal haem cavity residues, histidine 42 and arginine 38, in the formation of compound I and in substrate binding. The role of these residues as general acid-base catalysts, originally proposed for cytochrome c peroxidase by Poulos and Kraut in 1980 was assessed for HRPC. Replacement of histidine 42 by leucine [(H42L)HRPC*] decreased the apparent bimolecular rate constant for the reaction with hydrogen peroxide by five orders of magnitude (k ₁ = 1.4×10² M^–1s^–1) compared with both native-glycosylated and recombinant forms of HRPC (k ₁ = 1.7×10⁷ M^–1s^–1). The first-order rate constant for the heterolytic cleavage of the oxygen-oxygen bond to form compound I was estimated to be four orders of magnitude slower for this variant. Replacement of arginine 38 by leucine [(R38L)HRPC*] decreased the observed pseudo-first-order rate constant for the reaction with hydrogen peroxide by three orders of magnitude (k ₁ = 1.1×10⁴ M^–1s^–1), while the observed rate constant of oxygen bond scission was decreased sixfold (k ₂ = 142 s^–1). These rate constants are consistent with arginine 38 having two roles in catalysing compound I formation: firstly, promotion of proton transfer to the imidazole group of histidine 42 to facilitate peroxide anion binding to the haem, and secondly, stabilisation of the transition state for the heterolytic cleavage of the oxygen-oxygen bond. These roles for arginine 38 explain, in part, why dioxygen-binding globins, which do not have an arginine in the distal cavity, are poor peroxidases. Binding studies of benzhydroxamic acid to (H42L)HRPC* and (R38L)HRPC* indicate that both histidine 42 and arginine 38 are involved in the modulation of substrate affinity. Received: 21 July 1995 / Accepted: 27 November 1995     

Chemical Modification of Insulin by <Emphasis Type="Italic">N</Emphasis>-phosphorylation

In this paper, the reactions of bovine insulin and small peptides, such as actin binding domain of thymosin β₄ and Growth Hormone Releasing Factor (GRF 1–29 amino acids) with diisopropyloxyphosphite (DIPPH) and dimethyloxyphosphite (DMPH) were studied by modified Todd reaction. The MALDI-TOF or ESI-MS results showed that lysine, histidine and arginine residues in insulin could be phosphorylated under the water/ethanol system. The N,N,N-diisopropyloxyphosphorylated insulin analogues were characterized using MALDI-TOF and ³¹P NMR. These insulin analogues with different phosphorylation degree were separated and identified through LC-ESI-MS. In addition, circular dichroism (CD) spectra showed that the conformation of N,N,N-dimethyloxyphosphorylated insulin were only changed a little, whereas, that of N,N,N-diisopropyloxyphosphorylated insulin was changed completely.     

Use of 15N reverse gradient two-dimensional nuclear magnetic resonance spectroscopy to follow metabolic activity in Nicotiana plumbaginifolia cell-suspension cultures

Nitrogen metabolism was monitored in suspension cultured cells of Nicotiana plumbaginifolia Viv. using nuclear magnetic resonance (NMR) spectroscopy following the feeding of (¹⁵NH₄)₂SO₄ and K¹⁵NO₃. By using two-dimensional ¹⁵N-¹H NMR with heteronuclear single-quantum-coherence spectroscopy and heteronuclear multiple-bond-coherence spectroscopy sequences, an enhanced resolution of the incorporation of ¹⁵N label into a range of compounds could be detected. Thus, in addition to the amino acids normally observed in one-dimensional ¹⁵N NMR (glutamine, aspartate, alanine), several other amino acids could be resolved, notably serine, glycine and proline. Furthermore, it was found that the peak normally assigned to the non-protein amino-acid γ-aminobutyric acid in the one-dimensional ¹⁵N NMR spectrum was resolved into a several components. A peak of N-acetylated compounds was resolved, probably composed of the intermediates in arginine biosynthesis, N-acetylglutamate and N-acetylornithine and, possibly, the intermediate of putrescine degradation into γ-aminobutyric acid, N-acetylputrescine. The occurrence of ¹⁵N-label in agmatine and the low detection of labelled putrescine indicate that crucial intermediates of the pathway from glutamate to polyamines and/or the tobacco alkaloids could be monitored. For the first time, labelling of the peptide glutathione and of the nucleotide uridine could be seen. Received: 29 March 1999 / Accepted: 15 July 1999     

Free amino acid and dipeptide changes in the body fluids from Alzheimer’s disease subjects

Summary. Our aim was to determine changes in free amino acid (FAA) and dipeptide (DP) concentrations in probable Alzheimer’s disease (pAD) subjects compared with control (CT) subjects using liquid chromatography and electrospray ionization tandem mass spectrometry (LCMS²). We recruited gender- and age-matched study participants based on neurological and neuropsychological assessments. We measured FAAs and DPs in cerebrospinal fluid (CSF), plasma and urine using LCMS² with selected reaction monitoring (SRM). Imidazole-containing FAAs (histidine, methyl-histidine), catecholamines (L-DOPA and dopamine), citrulline, ornithine, glycine and antioxidant DPs (carnosine and anserine) accounted for the major changes between CT and pAD. Carnosine levels were significantly lower in pAD (328.4 ± 91.31 nmol/dl) than in CT plasma (654.23 ± 100.61 nmol/dl). In contrast, L-DOPA levels were higher in pAD (1400.84 ± 253.68) than CT (513.10 ± 121.61 nmol/dl) plasma. These data underscore the importance of FAA and DP metabolism in the pathogenesis of AD. Since our data show changes in antioxidants, neurotransmitters and their precursors, or FAA associated with urea metabolism in pAD compared with CT, we propose that manipulation of these metabolic pathways may be important in preventing AD progression.     

Residue 116 determines the C-terminal anchor residue of HLA-B*3501 and -B*5101 binding peptides but does not explain the general affinity difference

 HLA-B^*3501 and -B^*5101 molecules, which belong to the HLA-B5 cross-reactive group, bind peptides carrying similar anchor residues at P2 and the C-terminus, but differences are observed in the preference for a Tyr residue at the C-terminus and the affinity of peptides. A recent study of HLA-B^*3501 crystal structure suggested that residue 116 on the floor of the F-pocket determines a preference for anchor residues at the C-terminus. In order to evaluate the role of the residue 116 in the peptide binding to both HLA-B^*3501 and HLA-B^*5101 molecules, we generated HLA-B^*3501 mutant molecules carrying Tyr at residue 116 (B^*3501–116Y) and tested the binding of a panel of nonamer peptides to the B^*3501–116Y molecules by a stabilization assay with RMA-S transfectants expressing the mutant molecules. The substitution of Tyr for Ser at residue 116 markedly reduced the affinity of nonamer peptides carrying Tyr at P9, while it enhanced that of nonamer peptides carrying Ile and Leu at P9. On the other hand, the affinity of peptides carrying aliphatic hydrophobic residues at P9 to B^*3501–116Y molecules was much higher than that to HLA-B^*3501 and HLA-B^*5101 molecules. These results indicate that residue 116 is critical for the structural difference of the F-pocket between HLA-B^*3501 and HLA-B^*5101 which determines the C-terminal anchor residues, while leaving other residues which differ between HLA-B^*3501 and HLA-B^*5101 may be responsible for the low peptide binding property of the latter. Received: 18 April 1997 / Revised: 18 September 1997     

Identification of an oxidation product of aminoethylcysteine ketimine dimer

Summary. In continuation of our previous work dedicated to the detection of the oxidation products of aminoethylcysteine ketimine dimer by oxygen reactive species, we give here data for the identification of the α, β unsaturated sulfoxide as the main product of interaction of the dimer with H₂O₂. Identification has been done on the basis of mass spectrometry and NMR analyses of the product isolated by preparative chromatography. Received March 24, 1998, Accepted October 20, 1998     

Structure, dynamics and mapping of membrane-binding residues of micelle-bound antimicrobial peptides by natural abundance C NMR spectroscopy

Worldwide bacterial resistance to traditional antibiotics has drawn much research attention to naturally occurring antimicrobial peptides (AMPs) owing to their potential as alternative antimicrobials. Structural studies of AMPs are essential for an in-depth understanding of their activity, mechanism of action, and in guiding peptide design. Two-dimensional solution proton NMR spectroscopy has been the major tool. In this article, we describe the applications of natural abundance ¹³C NMR spectroscopy that provides complementary information to 2D ¹H NMR. The correlation of ¹³Cα secondary shifts with both 3D structure and heteronuclear ¹⁵N NOE values indicates that natural abundance carbon chemical shifts are useful probes for backbone structure and dynamics of membrane peptides. Using human LL-37-derived peptides (GF-17, KR-12, and RI-10), as well as amphibian antimicrobial and anticancer peptide aurein 1.2 and its analog LLAA, as models, we show that the cross peak intensity plots of 2D ¹H-¹³Cα HSQC spectra versus residue number present a wave-like pattern (HSQC wave) where key hydrophobic residues of micelle-bound peptides are located in the troughs with weaker intensities, probably due to fast exchange between the free and bound forms. In all the cases, the identification of aromatic phenylalanines as a key membrane-binding residue is consistent with previous intermolecular Phe-lipid NOE observations. Furthermore, mutation of one of the key hydrophobic residues of KR-12 to Ala significantly reduced the antibacterial activity of the peptide mutants. These results illustrate that natural abundance heteronuclear-correlated NMR spectroscopy can be utilized to probe backbone structure and dynamics, and perhaps to map key membrane-binding residues of peptides in complex with micelles. ¹H-¹³Cα HSQC wave, along with other NMR waves such as dipolar wave and chemical shift wave, offers novel insights into peptide-membrane interactions from different angles.     

Identification of Important Amino Acid Residues of the Na+-Ca2+ Exchanger Inhibitory Peptide,XIP

The Na⁺-Ca²⁺ exchanger plays an important role in cardiac contractility by moving Ca²⁺ across the plasma membrane during excitation-contraction coupling. A 20 amino acid peptide, XIP, synthesized to mimic a region of the exchanger, inhibits exchange activity. We identify here amino acid residues important for inhibitory function. Effects of modified peptides on Na⁺-Ca²⁺ exchange activity were determined. Exchange activity was assessed as ⁴⁵Ca²⁺ uptake into Na⁺-loaded cardiac sarcolemmal vesicles. We find that the entire length of XIP is important for maximal potency, though the major inhibitory components are between residues 5 and 16. Basic and aromatic residues are most important for the inhibitory function of XIP. Substitutions of arginine 12 and arginine 14 with alanine or glutamine dramatically decrease the potency of XIP, suggesting that these residues play a key role in possible charge-charge interactions. Substitutions of other basic residues with alanines or glutamines had less effect on the potency of XIP. All aromatic residues participate in binding with the exchanger, probably via hydrophobic interactions as indicated by tryptophan fluorescence. A tyrosine is required at position 6 for maximal inhibition and phenylalanine 5 and tyrosine 8 can only be replaced by other aromatic residues. Tyrosine 10 and tyrosine 13 can be replaced with other bulky residues. A specific conformation of XIP, with structural constrains provided by all parts of the molecule, is required for optimal inhibitory function. Received: 19 September 1996/Revised: 20 November 1996     

A method for simple identification of signature peptides derived from polyUb-K48 and K63 by MALDI-TOF MS and chemically assisted MS/MS fragmentation

Summary. A simple method is described to identify signature peptides derived from polyubiquitin (polyUb) chains. The method is based on MALDI-TOF MS/MS analysis after chemically assisted fragmentation, and works on peptides isolated from polyacrylamide gels. PolyUb chains branched at K48 and K63 were chosen as models for Ub-protein conjugates. They were resolved by SDS-PAGE, and their tryptic peptides (in-gel-trypsinolysis) derivatized with 3-sulfopropinic acid NHSester to obtain chemically assisted fragmentation during the MS/MS analysis. PolyUb-K63 produced a single peptide identified as ⁵⁵TLSDYNIQK⁶³ (GG)ESTLHLVLR⁷². PolyUb-K48 produced two branched signature peptides identified as ⁴³LIFAGK⁴⁸(GG)QLEDGR⁵⁴ and ⁴³LIFAGK⁴⁸(LRGG)QLEDGR⁵⁴. The recovery of signature peptide with LRGG as branched chain underscores the need to take limited proteolysis into account in the search for detection of ubiquitinated peptides in proteomics studies. In conclusion, a simple method has been described allowing the identification of signature peptides, which are diagnostic markers of the majority of polyUb-conjugated proteins. In principle, the method should be applicable also for other more rare signature peptides.     

Geranylhydroquinone 3′′-hydroxylase, a cytochrome P-450 monooxygenase from Lithospermum erythrorhizon cell suspension cultures

Geranylhydroquinone 3′′-hydroxylase, which is likely to be involved in shikonin and dihydroechinofuran biosynthesis, was identified in cell suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae). The enzyme hydroxylates the isoprenoid side chain of geranylhydroquinone (GHQ), a known precursor of shikonin. Proton/proton correlation spectroscopic and proton/proton long-range correlation spectroscopic studies confirmed that hydroxylation takes place specifically at position 3′′, i.e. at the methyl group involved in the cyclization reaction. The enzyme is membrane-bound and was found in the microsomal fraction. It requires NADPH and molecular oxygen as cofactors, and is inhibited by cytochrome P-450 inhibitors such as cytochrome c and CO. The inhibitory effect of CO is reversed by illumination. These data suggest that the enzyme is a cytochrome P-450-dependent monooxygenase. The optimum pH of GHQ 3′′-hydroxylase is 7.4, and the apparent K _m value for GHQ is 1.5 μM. The reaction velocity obtained with 3-geranyl-4-hydroxybenzoic acid was more than 100 times lower than that obtained with geranylhydroquinone. Received: 20 March 1999 / Accepted: 20 July 1999     

Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures

Maize (Zea mays L.) cell cultures incorporated radioactivity from [¹⁴C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5–20 min, the CoA pool had lost its ¹⁴C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a pulse–chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age. In young (1–3 d) cultures, polysaccharide-bound [¹⁴C]feruloyl- and [¹⁴C]diferuloyl residues were both detectable within 1 min of [¹⁴C]cinnamate feeding. Thus, feruloyl residues were dimerised <1 min after their attachment to polysaccharides. For at least the first 2.3 h after [¹⁴C]cinnamate feeding, polysaccharide-bound [¹⁴C]diferuloyl residues remained almost constant at ≈7% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides <1 min after the biosynthesis of the latter, and >10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H₂O₂ (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [¹⁴C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H₂O₂ induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H₂O₂-limited. In both 2- and 4-d-old cultures, polysaccharide-bound ¹⁴C-trimers and larger coupling products exceeded [¹⁴C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed. Received: 19 January 2000 / Accepted: 13 April 2000     

Chemical modification of canavanine with p-nitrophenylglyoxal. Factors influencing the chemistry and reactivity of alpha-dicarbonyl-guanidino reactions

The role of structural features and deprotonation of guanidino derivatives on chemical reactions with p-nitrophenylglyoxal has been investigated. Canavanine, an arginine analog, reacts to form a yellow product, which absorbs maximally at 350 nm (epsilon = 6500) and at 278 nm (epsilon = 14 500). Elemental analysis, fast atom bombardment mass spectral analysis, n.m.r. and i.r. studies suggest that the product is a 5-(p-nitrophenyl)4-oxo-2 imidazoline derivative of canalaline. Kinetic studies show that the second order rate constant for the reaction increases with increasing pH in the range of pH 7-11.0. It is concluded that the pH dependence of the reaction can be explained by general base catalysis and not simply by a deprotonation of the guanidinoxy side chain. The reaction of arginine, polyarginine, and other derivatives differs markedly from that of canavanine. The results suggest that change in the tautomeric equilibria between the imino and amino forms of the guanidino group may partly account for differences in reaction of canavanine and arginine and the reactions of specific arginyl residues in proteins.     

<Emphasis Type="Italic">N</Emphasis>-Methylation of <Emphasis Type="Italic">N</Emphasis><Superscript>α</Superscript>-Acetylated,Fully C<Superscript>α</Superscript>-Ethylated,Linear Peptides

“Mono-N-methyl scan” is a rational approach for the optimization of the peptide biological properties. N-Methylation of the –CONH– functionality is also a useful tool for discriminating solvent exposed from intramolecularly H-bonded secondary amide groups in peptides. We are currently extending this reaction to linear peptides based on C^α-tetrasubstituted α-amino acids. Following our study on the synthesis and conformation of the mono-N-methylated peptides from C^α-methylated residues, in this work we investigated the N-methylation reaction on homo-peptides to the pentamer level from the C^α-ethylated residue C^α,α-diethylglycine. Under the classical experimental conditions used, exclusively mono-N-methylation (on the N-terminal, acetylated residue) takes place, as unambiguously shown by mass spectrometry, 2D-NMR, and X-ray diffraction techniques. This backbone modification does not seem to involve any significant change in the peptide conformation in the crystalline state. Dedicated to the memory of Prof. Miroslav T. Leplawy (Technical University of Łodz, Poland), who performed the first synthesis of the extremely sterically demanding C^α,α-diethylglycine peptides.     

Free radical scavenging potential of L-proline: evidence from in vitro assays

Summary. An assessment of the potential of proline to scavenge free radicals was made in a couple of in vitro assay systems, namely graft co-polymerization and autooxidation of pyrogallol. Both these assays are essentially dependent upon free radical mechanisms. Graft co-polymerization involved a ceric (Ce⁴⁺) ion- or γ-radiation-induced grafting of methyl acrylate (MA) onto a cellulose backbone. The degree of grafting, measured gravimetrically, was taken as a measure of free radical generation. The γ-radiation-dependent grafting was far greater than that due to Ce⁴⁺ ions. Inclusion of proline in the assay, irrespective of the initiator used, led to suppression of grafting in a concentration-dependent manner indicating the ability of proline to scavenge free radicals. The γ-radiation-dependent grafting was also suppressed by hydroquinone and glutathione but not by ascorbate, glycine and spermine. In contrast to graft co-polymerization, proline did not inhibit the autooxidation of pyrogallol, a reaction involving superoxide radical generation. A subset of data constitutes an evidence for the ability of proline to scavenge free radicals in vitro. It is implied by extension that free proline, known to accumulate in plant tissues during abiotic stresses, would contribute to scavenging of surplus free radicals produced under a variety of abiotic stresses. Authors’ address: Shanti S. Sharma, Department of Biosciences, Himachal Pradesh University, Shimla 171 005, India     

Almost all about citrulline in mammals

Summary. Citrulline (Cit, C₆H₁₃N₃O₃), which is a ubiquitous amino acid in mammals, is strongly related to arginine. Citrulline metabolism in mammals is divided into two fields: free citrulline and citrullinated proteins. Free citrulline metabolism involves three key enzymes: NO synthase (NOS) and ornithine carbamoyltransferase (OCT) which produce citrulline, and argininosuccinate synthetase (ASS) that converts it into argininosuccinate. The tissue distribution of these enzymes distinguishes three “orthogonal” metabolic pathways for citrulline. Firstly, in the liver, citrulline is locally synthesized by OCT and metabolized by ASS for urea production. Secondly, in most of the tissues producing NO, citrulline is recycled into arginine via ASS to increase arginine availability for NO production. Thirdly, citrulline is synthesized in the gut from glutamine (with OCT), released into the blood and converted back into arginine in the kidneys (by ASS); in this pathway, circulating citrulline is in fact a masked form of arginine to avoid liver captation. Each of these pathways has related pathologies and, even more interestingly, citrulline could potentially be used to monitor or treat some of these pathologies. Citrulline has long been administered in the treatment of inherited urea cycle disorders, and recent studies suggest that citrulline may be used to control the production of NO. Recently, citrulline was demonstrated as a potentially useful marker of short bowel function in a wide range of pathologies. One of the most promising research directions deals with the administration of citrulline as a more efficient alternative to arginine, especially against underlying splanchnic sequestration of amino acids. Protein citrullination results from post-translational modification of arginine; that occurs mainly in keratinization-related proteins and myelins, and insufficiencies in this citrullination occur in some auto-immune diseases such as rheumatoid arthritis, psoriasis or multiple sclerosis.