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A highly sensitive assay based on new internally quenched fluorogenic peptide substrates has been developed for monitoring protease activities. These novel substrates comprise an Edans (5-(2-aminoethylamino)-1-naphthalenesulfonic acid) group at the C terminus and a Dabsyl (4-(dimethylamino)azobenzene-4′-sulfonyl chloride) fluorophore at the N terminus of the peptide chains. The Edans fluorescence increases upon peptide hydrolysis by Pseudomonas aeruginosa proteases, and this increase is directly proportional to the amount of substrate cleaved, i.e., protease activity. The substrates Dabsyl-Ala-Ala-Phe-Ala-Edans and Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans were used for testing the peptidasic activities of P. aeruginosa elastase and LasA protease, respectively. Elastase and LasA kinetic parameters were calculated and a sensitive assay was designed for the detection of P. aeruginosa proteases in bacterial supernatants. The sensitivity and the small sample requirements make the assay suitable for high-throughput screening of biological samples. Furthermore, this P. aeruginosa protease assay improves upon existing assays because it is simple, it requires only one step, and even more significantly it is enzyme specific.

In normal and pathological tissues, polymorphonuclear leukocyte proteases (elastase, cathepsin G and proteinase 3) may generate soluble peptides through limited proteolysis of elastin, the main component of mature elastic fibres. Elastin-derived peptides display diverse biological activities including cell migration, differentiation, proliferation, chemotaxis, tumor progression and up-regulation of metalloproteinases. To be biologically active, their structures must adopt a β-turn conformation which accommodates to the cell surface-located elastin binding protein. In this study, we established that human elastin exon 24-derived peptides are hydrolysed by leukocyte elastase, when the active site is fully occupied (from S5 to S′3). As shown by mass spectrometry analyses, a major cleavage site was demonstrated at a Val-Ala bond and a minor one at Gly-Val bond. For longer peptides, the hydrolysed fragments could themselves be re-hydrolysed. If the shortest fragments do not contain the GxxPG sequence known to stimulate cellular effects, some of the intermediates together with hydrolysis fragments generated by other proteases such as proteinase 3, may possess this motif.

In normal and pathological tissues, elastin-derived peptides proceed of elastin degradation by polymorphonuclear leukocyte proteases: elastase, cathepsin G and proteinase 3. They were demonstrated to have a chemotactic activity, to promote cell proliferation and protease release, . . .. To be biologically active, their structures, which reflect elastase specificity, must adopt a beta-turn conformation which accommodate to the cell surface-located elastin binding protein. In this study, we establish that human elastin exon 24-derived peptides containing at least two repeated VGVAPG sequences are hydrolyzed by the proteinase 3 (Pr3). As shown by mass spectrometry analyses, the demonstrated cleavage sites are in agreement with previously reported Pr3 substrate specificity and its lengthy substrate binding site. The characterization of the Pr3-generated products indicate that they contain at least one GXXPG sequence known to stimulate cellular effects after binding to the elastin receptor.

A series of 6-amino-2-phenyl-4H-3,1-benzoxazin-4-one aminoacyl and dipeptidyl derivatives, in which aminoacids and dipeptides are linked to the benzoxazinone moiety via an amide bond, were synthesized and tested in vitro for their inhibitory activity towards human leukocyte elastase (HLE). When compared to their values without inhibitors, the residual enzymatic activities decrease with time, indicating a time-dependent inhibition. The most potent inhibitions were obtained when Z-Arg-(Pmc), Z-Val-Phe, Z-Ala-Val or Z-Val-Ala are linked to the 6-amino group. Twenty-five new compounds were synthesized.

New N-acyl D-amino acids were isolated from Bacillus pumilus IM 1801. Their structures were determined by chemical analysis and mass spectrometry. The lipid part was identified as a mixture of fatty acids with 11, 12, 13, 15, and 16 carbon atoms in the iso, anteiso or n configuration linked by an amide bond with a D-asparagine. They exhibited surfactant properties.

HT-1080 fibrosarcoma cells express at their plasma membrane the elastin-binding protein (EBP). Occupancy of EBP by elastin fragments, tropoelastin or XGVAPG peptides was found to trigger procollagenase-1 (proMMP-1) overproduction by HT-1080 cells at the protein and enzyme levels. RT-PCR analysis indicated that elastin peptides did not modify the MMP-1 mRNA steady state levels, suggesting the involvement of a post-transcriptional mechanism. We previously reported that binding of elastin peptides to EBP induced other matrix metalloproteinases (MMP-2 and MT1-MMP) expression [20]. Since those peptides were here found to also accelerate the secretion of urokinase from HT-1080 cells, culture medium was supplemented with plasminogen together with elastin peptides at aims to induce or potentiate MMPs activation cascades. In such conditions, plasmin activity was generated and exacerbate proMMP-1 and proMMP-2 activation. As a consequence, elastin peptides and plasminogen-treated HT-1080 cells displayed a significant type I collagen matrix invasive capacity.

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections in vulnerable hosts. It may produce various virulence factors including proteases. Among them, LasA possesses both elastolytic and staphylolytic (hydrolysis of pentaglycine cross-links in the cell wall peptidoglycan) activities. To understand if its elastolytic activity results from a preference for glycine-rich substrates, we studied its ability to hydrolyse the 65 pentapeptides of human tropoelastin containing at least three glycines. As demonstrated by capillary electrophoresis (CE), 22 of these peptides were hydrolysed by LasA, generally at a single peptide bond and the catalytic ratio kcat/KM was determined for most of them. The highest value was obtained for LGGGA, 59 +/- 9 min(-1) x mmol(-1) x L. The specificity of hydrolysis was elucidated by CE, liquid secondary ion mass spectrometry and, in some cases, collision activated dissociation-mass analysis of ion kinetic energy. The preferred cleavage sites are GG and GA peptide bonds, the sequence GG(cleavage site)A being especially sensitive to hydrolysis. Both positions P2 and P'2 must be occupied for hydrolysis and the presence of an amino acid in P3 (but not in P'3) significantly increases the catalytic ratio. Considering these results, about 30 GGX sequences (X: G, A or Y) of human tropoelastin could be susceptible to LasA elastolysis.

L'elastine est un composant majeur de la matrice extracellulaire. Les peptides d'elastine (PE) derivent de sa degradation et sont presents dans le serum humain ; ils induisent des effets biologiques varies en interagissant avec des fibroblastes, des cellules phagocytaires, des lymphocytes, des cellules musculaires lisses et des cellules endotheliales, medies par les recepteurs d'elastine-laminine presents sur ces memes cellules. La voie de signalisation du recepteur elastine-laminine (REL) implique l'activation de la phospholipase C (PLC) par une proteine G sensible a la toxine de Bordetella pertussis. Le PLC induit la production d'inositol triphosphate (IP 3 ) conduisant a l'augmentation du Ca 2 + intracellulaire libre d'une part, et d'autre part du diacylglycerol (DAG); il s'ensuit la translocation de la proteine kinase C (PKC) a la membrane qui induit a son tour, la phosphorylation des proteines kinases mitogenes activees (MAPK), tel le p42/p44 MAPK. Considerant les multiples effets du REL, la connaissance des voies complexes de la signalisation aidera a sa meilleure modulation, surtout dans des conditions pathologiques comme l'atherosclerose.

The lipopeptide lichenysin (cyclo-[L-Gln1-->D-Leu2-->L-Leu3-->L-Val4--> L-Asp5-->D-Leu6-->L-Ile7-beta-OH fatty acid]) produced by Bacillus licheniformis structurally resembles surfactin from Bacillus subtilis. The main difference is the presence of a glutaminyl residue in position 1 of the peptide sequence in place of glutamic acid in surfactin. This local variation causes significant changes in the properties of the molecule compared to surfactin. Lichenysin has a higher surfactant power, the critical micellar concentration (c.m.c.) being strongly reduced from 220 to 22 microM and a much higher hemolytic activity because 100% hemolysis was observed with only 15 microM instead of 200 microM. Lichenysin is also a better chelating agent because its association constants with Ca2+ and Mg2+ are increased by a factor of 4 and 16, respectively. This effect is assigned to an increase in the accessibility of the carboxyl group to cations owing to a change in the side chain topology induced by the Glu/Gln exchange. Additionally, the propensity of the lipopeptide for extensive hydrophobic interactions, as illustrated by its low c.m.c., contributes to further stabilization of the cation and an increase in the partitioning of lichenysin into the erythrocyte membrane. Our data support the formation of a lichensyin-Ca2+ complex in a molar ratio of 2:1 instead of 1:1 with surfactin, suggesting an intermolecular salt bridge between two lichenysin molecules. Therefore, when Ca2+ ions are present in the solution, micellization occurs via a dimer assembly, with a possible long-range effect on the spatial arrangement of the micelles or other supramolecular structures. Finally, among all the surfactin peptidic variants so far known, lichenysin is the one for which the three tested activities are the most substantially improved.

With aging we assist to alterations in the vascular structure and function. One important factor in these vascular wall changes is the degradation of the elastin fibre major protein: elastin. Elastin peptides derived from the degradation are present in human sera. Elastin peptides induce on fibroblasts, phagocytic cells, lymphocytes, smooth muscle cells and endothelial cells, a variety of biological effects mediated by the elastin-laminin receptor which has been demonstrated to be present on the membrane of these cells. The transduction pathway of the ELR receptor involves the activation of phospholipase C (PLC) by a pertussis toxin sensitive G-protein. PLC induces the production of inositol trisphosphate (IP3) leading to the increase of the intracellular free calcium on one hand, and of diacylglycerol (DAG) which stimulates the translocation to the membrane of PKC leading to the phosphorylation of members of the MAPK family, such as p42/p44 MAPK. A progressive age dependent uncoupling of the elastin-laminin receptor occurs impairing its transduction pathway and which results in alteration of the calcium signaling and loss in calcium homeostasis of the cells. These alterations in the signal transduction of the elastin-laminin receptor result in modified activities of parenchymal and phagocytic cells with aging, such as free radical production and elastase release. Thus, these age-related alterations in the elastin-laminin receptor signal transduction may be involved in the atherogenesis.

We previously demonstrated that pseudolysin, a neutral metalloproteinase from thermolysin family, is a suitable catalyst for peptide bond formation through reverse proteolysis. In order to get an insight into the yield- and rate-controlling factors, we studied the kinetics and mechanism of pseudolysin-catalysed synthesis. The condensation of Z-Ala-Phe-NH2 from Z-Ala and Phe-NH2, performed in a semi-organic homogeneous system, was chosen as a reaction model. We applied the conductimetric method for measuring the initial rates of the enzymatic reaction before product precipitation. We have both optimised synthesis and conductimetric conditions. Highest velocities were obtained in the pH range 7.0-7.5. When kept below 0.15, ionic strength did not significantly alter the rate values. Kinetic studies were then performed for varied Z-Ala and Phe-NH2 concentrations at different, fixed concentrations of the alternative substrate, at pH 7.3 in 35% MeOH and for an ionic strength below 0.15. Graphical inspection and...

The Pseudomonas aeruginosa serralysin (E.C. 3.4.24.40.), which is a zinc-dependent metalloprotease from the metzincin superfamily, has quite a broad specificity, which has not yet been clearly identified. We have studied it with an original approach, using a 49-peptide library of the type Z–AXXA (amide) (X=A, L, V, F, S, R, E). The library was analyzed by LC-MS before and after enzymatic hydrolysis. A great number of hydrolyzed peptides were screened and the preferential hydrolysis was the X–X peptide bond, even if in some cases, A–X and X–A bond could be hydrolyzed. No amino acids with a ionized side chain could be found in the P 1 ′ position. The results obtained suggest that the specificity in the P n ′ position, where an hydrophobic residue was preferentially found, seems more selective that in the P n position. The P 1 position was not very specific, but, on a quantitative point of view, the enzymatic activity was particularly increased when R, F or A were in this position. The results allow us to define the P 1 ′ and P 1 residues for an optimal substrate of pseudomonal serralysin and usable for the design and the synthesis of a specific inhibitor.

Pseudomonas aeruginosa elastase was used to synthesize various N-protected dipeptide amides. The identity of the products was confirmed by FAB(+)-MS. After recrystallization, the yield of their synthesis was calculated, their purity was checked by RP-HPLC and their melting point was measured. With regard to the hydrolysis, it is well-established that the enzyme prefers hydrophobic amino acids in P'1 position and it has a wide specificity for the P1 position. This specificity was demonstrated to be quite unchanged when comparing the initial rates of peptide bond formation between different carboxyl donors (Z-aa) and nucleophiles (aa-NH2). The elastase, but not the thermolysin, was notably able to incorporate tyrosine and tryptophan in P'1 position. Furthermore, synthesis initial rates were at least 100 times faster with the elastase. To overcome the problematic condensation of some amino acids during chemical peptide synthesis, it has been previously suggested that enzymatic steps can combine with a chemical strategy. We demonstrated that the elastase readily synthesizes dipeptide derivatives containing various usual N-protecting groups. It was especially able to condense phenylalaninamide to Fmoc- and Boc-alanine. Increasing interest in peptides containing unnatural amino acids led us to try the elastase-catalyzed synthesis of Z-dipeptide amides including those amino acids in the P1 position. A synthesis was demonstrated with alphaAbu, Nle, Nva and Phg.

The V8 endoprotease from Staphylococcus aureus hydrolyzes the cyclic lipopeptide, surfactin, at 100 μM. The fragmentation lies between the two residues L-Glu1 and L-Leu2 leading to an open-chain lipopeptide. The structure of the product arising from the enzymic hydrolysis was established by chemical methods and mass spectrometry. The enzyme action has been suggested to be likely limited by the aggregation state of the lipopeptide in solution.

Immobilization of biologically active components on supports has become an important tool in biological research. The use of a spacer is often necessary to avoid steric hindrance and in order to retain biological activity. A spacer arm, exhibiting heterobifunctional character with an N -hydroxysulfosuccinimide (S-NHS) active ester linked to a maleimide moiety by an aliphatic chain can be used to link antigenic peptides to functionalized polystyrene microspheres in very selective two-step procedures. To study the kinetic parameters in aqueous buffers of such procedures, reactions of sulfosuccinimidyl 4-( N -maleimidomethyl)-1-cyclohexane carboxylate (S-SMCC) have been carried out. Alkylation of thiol groups by maleimide has been demonstrated to be a fast, specific and strongly pH dependent reaction which follows second order kinetics in the pH range 5.5–8.5. The dependence of the rate constant on the thiolate anion concentration has shown that, as with most alkylating agents, the reactive species are the thiolate anions. This alkylation was characterized by the equation v = k S− [RS − ][S-SMCC] with, at 20.5°C, k S− =8.54±0.91×10 5 l mol −1 min −1 . Hydrolysis of the active ester was shown to be the most important competing reaction and its rate constant k H has been measured. Above pH=8, hydrolysis is first order in [OH − ], and characterized by the equation k H = k OH [OH − ] with, at 20.5°C, k OH =12200±700 l mol −1 min −1 . Reaction of active esters with primary amines is strongly linked to the concentration of non-protonated amine (free amine). Of the rates investigated, the rate of aminolysis was observed to increase more rapidly than the rate of hydrolysis with increasing pH or temperature. The reaction with nitrogen nucleophiles is first order in free amine concentration and the rate constant k A remains constant whatever the excess of nucleophile: k A =34700±750 l mol −1 min −1 , at 20.5°C. All the constants concerning the reaction of the active ester are summarized in the equation: ( k obs − k H )= k A ×[amine] free . Alkylation of thiols by the maleimide group and reaction of active ester with amines have been shown to be very fast. Considering these results and the calculated constants, optimized coupling conditions and experimental procedures are proposed.

Abstract —The elastic properties of extensible tissues such as arteries and skin are mainly due to the presence of elastic fibers whose major component is the extracellular matrix protein elastin. Pathophysiological degradation of this protein leads to the generation of elastin peptides that have been identified in the circulation in the ng/mL to μg/mL range. Similar concentrations of an elastin peptide preparation (κ-elastin) were previously demonstrated to induce, among other biological actions, a dose- and endothelium-dependent vasorelaxation mediated by the elastin/laminin receptor and by endothelial NO production. To determine the elastin sequence(s) responsible for vasomotor activity and to learn more about possible signaling pathways, we have compared the action of different concentrations (10 −13 to 10 −7 mol/L) of recombinant human tropoelastin, eight synthetic elastin peptides, and a control peptide (VPVGGA) on both rat aortic ring tension and [Ca 2+ ] i of cultured human umbilical vein endothelial cells. No vasoactivity could be detected for VPVGGA and for the elastin-related sequences VGVGVA, PGVGVA, and GVGVA. Tropoelastin, VGV, PGV, and VGVAPG were found to induce an endothelium- and dose-dependent vasorelaxation and to increase endothelial [Ca 2+ ] i , whereas PVGV and VGVA produced these effects only at low concentration (10 −11 mol/L). A likely candidate for mediating the elastin peptide–related effects is the elastin/laminin receptor, since the presence of lactose strongly inhibited the vasoactivity associated with these compounds. Our results show that although the flanking amino acids modulate its activity, VGV seems to be the core sequence recognized by the elastin receptor.

Oxidized low density lipoproteins (LDL) are believed to play a central role in the events that initiate atherosclerosis. Antioxidants have been shown to decrease the oxidation of LDL, leading to the diminution of atherosclerosis. Since it is well-known that decreased levels of dehydroepiandrosterone (DHEA) are linked to the development of atherosclerosis, we studied the modulation of the oxidation of LDL by DHEA. LDL were obtained from 10 healthy subjects and oxidized by free radicals produced by γ -radiolysis of ethanol–water mixtures. The formation of conjugated dienes and thiobarbituric acid-reactive substances (TBARS), the vitamin E content, as well as the incorporation of 4-[ 14 C]DHEA in LDL and the chemotactic effect of oxidized LDL in the presence of DHEA towards monocytes, were investigated. It was found that DHEA was able to inhibit the oxidation of LDL by reducing over 90% of the conjugated dienes and TBARS formation, as well as by reducing the vitamin E disappearance and significantly decreasing the chemotactic activity towards monocytes. Our results suggest that DHEA exerts its antioxidative effect by protecting the endogenous vitamin E of LDL.

A rapid and simple capillary zone electrophoresis (CZE) method for measuring the activity of a minor protease from Pseudomonas aeruginosa is described. When glycine-containing oligopeptides were used as substrates, it was possible to separate and quantify substrate and products. Moreover oligopeptide hydrolysates were analysed by fast atom bombardment mass spectrometry providing the sites of splitting of the substrates. By comparison with CZE calibration curves constructed with reference peptides, the initial rates of hydrolysis were calculated. The method, validated for pentaglycine hydrolysis, was also used for the analysis of reaction mixtures and for monitoring the enzymic hydrolysis of various peptides in order to investigate enzyme specificity.

A spectrophotometric method was applied to assay Pseudomonas aeruginosa alkaline protease (EC 3.4.24.40) activity using N-carbobenzyloxyarginylarginyl-para-nitroanilide (Z-Arg-Arg-pNA). With the same substrate the activity of P. aeruginosa elastase (PsE) was 250-fold lower, this selectivity being even more increased in the presence of 100 μM phosphoramidon. This dipeptide derivative, which is an inhibitor of metalloproteases such as PsE was demonstrated to have no effect on alkaline protease (AP). The method was successfully applied for measurement of the enzyme activity in a culture supernatant from a clinical strain.

The biosynthesis of bacterial isoleucyl-rich surfactins was controlled by supplementation of L-isoleucine to the culture medium. Two new variants, the [Ile4,7]- and [Ile2,4,7]surfactins, were thus produced by Bacillus subtilis and their separation was achieved by reverse-phase HPLC. Amino acids of the heptapeptide moiety were analysed by chemical methods, and the lipid moiety was identified by beta-hydroxy anteiso pentadecanoic acid by combined GC/MS. Sequences were established on the basis of two-dimensional NMR data. Because conformational parameters issuing from NMR spectra suggested that the cyclic backbone fold was globally conserved in the new variants, structure-activity relationships were discussed in details on the basis of the three-dimensional model of surfactin in solution. Indeed, both variants have increased surface properties compared with that of surfactin, and this improvement is assigned to an increase of the hydrophobicity of the apolar domain favouring micellization. Furthermore, the additional Leu-to-Ile substitution at position 2 in the [Ile2,4,7]surfactin leads to a substantial increase of its affinity for calcium, when compared with that of [Ile4,7]surfactin or surfactin. This effect is assigned, from the model, to an increase in the accessibility of the acidic side chains constituting the calcium binding site. Thus, the propensities of such active lipopeptides for both hydrophobic and electrostatic interactions were improved, further substantiating that they can be rationally designed.

Electrostatic forces and hydrophobic interactions had been suggested to modify the adsorption of elastases onto insoluble fibrous elastin, which is the initial stage of elastolysis, but conflicting results had been obtained, and comparison between compounds with different structures was difficult. In order to explore these observations, we have studied the effect of six alkyltrimethylammonium bromides, with alkyl chain length ranging from six to 16 carbon atoms, on human leucocyte elastase activities, either with a synthetic substrate or with insoluble elastin. The enzymatic studies were performed either spectrophotometrically or using conductimetry, and direct binding on to elastin was conductimetrically measured. Binding of the alkyltrimethylammonium salts is increasing with alkyl chain length and we could demonstrate a cooperative binding for tetra- and hexadecyl chains. No effect of the six compounds could be evidenced on hydrolysis of a specific synthetic substrate. With insoluble elastin, elastolysis inhibition could be demonstrated for alkyl chain longer than ten carbon atoms, the effect increasing with chain length. A similar inhibition was observed with the soluble kappa-elastin, but it was less effective. The study shows that the interaction between the alkyltrimethylammonium salts and elastin plays a major role in the inhibitory potency of these molecules. As this effect is enhanced with alkyl chain length, it was concluded that hydrophobic interactions favour their binding, protecting elastin against elastase adsorption.

A gene of Pseudomonas alginovora, called aly, has been cloned in Escherichia coli using a battery of PCR techniques and sequenced. It encodes a 210-amino-acid alginate lyase (EC 4.2.2.3), Aly, in the form of a 233-amino-acid precursor. P. alginovora Aly has been overproduced in E. coli with a His-tag sequence fused at the C-terminal end under conditions in which the formation of inclusion bodies is avoided. His-tagged P. alginovora Aly has the same enzymic properties as the wild-type enzyme and has the specificity of a mannuronate lyase. It can be purified in a one-step procedure by affinity chromatography on Ni2+-nitriloacetate resin. The yield is of 5 mg of enzyme per litre of culture. The amplification factor is 12.5 compared with the level of production by wild-type P. alginovora. The six alginate lyases of known primary structure fall into three distinct classes, one of which comprises the pair P. alginovora Aly and Klebsiella pneumoniae Aly.

Elastase from Pseudomonas aeruginosa has recently been used successfully for peptide synthesis. To improve its performance we attempted to increase its catalytic stability by chemical modification. Two distinct sorts of amino group-specific modifiers, dimethyl suberimidate and cyanuric chloride-activated polyethylene glycol (PEG), gave a two-fold increase in catalytic stability at 70°C and greater degrees of stabilization at lower temperatures. Suberimidate treatment seemed to act by intramolecular crosslinking, whereas the activated PEG gave rise to an elastase-PEG conjugate. The thermal transition (T m ) for suberimidate-treated elastase was unchanged from the native value of 72°C. PEG-conjugated elastase gave anomalous T m curves; therefore, a value could not be determined. The lack of correspondence of catalytic stabilization with increased T m values is discussed.

A conductimetric method was developed for the monitoring of thermodynamically-controlled enzyme peptide synthesis. It was applied to the synthesis of Nα-benzyloxycarbonylalanylphenylalaninamide, in the presence of Pseudomonas aeruginosa elastase. A linear continuous decrease of conductance could be demonstrated in the first minutes of the reaction, allowing a precise measurement of the initial rate of synthesis. Using independent calibration graphs, the conductimetric method correlated well with liquid chromatography, the most often applied technique for enzymatic peptide synthesis monitoring. Conductimetry may therefore be considered as a promising technique for quantitative measurements of such reactions.

The effects of iturin A, at fungicidal concentrations, on yeast cells were studied by scanning electron microscopy and by transmission electron microscopy. A depression, observed in each iturin A-treated cell, was the consequence of the release of electrolytes and other cytoplasmic components. Iturin A passes through the cell wall and disrupts the plasma membrane with the formation of small vesicles and the aggregation of intramembranous particles. Moreover, iturin A passes through the plasma membrane and interacts with the nuclear membrane and probably with membranes of other cytoplasmic organelles.

International audience; When Bacillus subtilis S 499 was grown on a culture medium containing L-alanine as nitrogen source, a mixture of surfactins was obtained. Suitable chromatographic conditions allowed the separation of isoforms. Among these compounds, a new variant of surfactin was isolated and its structure was established by chemical and spectrometric methods, especially by NMR spectrometry. It contains a peptide sequence which differs from that of standard surfactin by the replacement of the L-valine residue by L-alanine residue in position 4. The folding mode of [Ala4]surfactin as deduced from NMR results was compared with that of standard surfactin and the structure/properties relationship issuing from the study of this new isoform is discussed.

A conductimetric method was applied to assay Streptococcus pyogenes pyroglutamyl peptidase (E.C. 3.4.8.11) activity using pyroglutamylalanine or pyroglutamyltyrosine. A spectrophotometric comparison using pyroglutamyl-β-naphthylamide indicated that the sensitivity was much increased when using conductimetry. The use of a C-protected dipeptide such as pyroglutamylalanineamide also increased the sensitivity. Kinetic parameters have been measured for both dipeptides, with KM values of 0.34 mM and 0.47 mM, respectively, for the alanine and tyrosine derivatives.

Surfactin is a bacterial lipopeptide containing two carboxylic groups. Its sequestering properties (surfactin binds calcium and magnesium ions) were determined in aqueous solution at pH 9.5. The calculated association constants were K = 1.5 × 105 M−1 for Ca2− and 1.9 × 104 M−1 for Mg2+. Its complexing properties were determined by the two-phase distribution system; the aqueous phase contained Ca2+ or Rb+ ions and the lipidic phase contained surfactin. At pH 9, surfactin forms a 1:1 complex with Ca2+ and a 2:1 complex with Rb+ and it shows a higher affinity for divalent cations than for monovalent cations. Surfactin is non-selective in binding monovalent cations but is slightly more effective in binding Ca2+ than Mg2+ or Ba2+. Surfactin is also a mobile carrier to transport monovalent and divalent cations across a solvent barrier and mediates the exchange between H− and inorganic cation.

Various allenenols lactones [5(E)-(2-allenylidene)-tetrahydro-2-furanones] have been synthesized by reacting 4-pentynoic acid and 2-alkynyl acetate in dimethyl sulfoxide in the presence of K 2 CO 3 , Pd(OAc) 2 as the catalyst and tri(2-furyl) phosphine as the ligand. The biological activity of these compounds as protease inhibitors has been proven.

Two Pseudomonas aeruginosa alginates were lysed by an overexpressed polymannuronate lyase AlxM B (only acting on two or more consecutive, nonacetylated mannuronate units) to prepare either mannuronate blocks (poly-M blocks) with dp∼30, or strictly alternating sequences of mannuronic and guluronic acid (poly-MG blocks) with dp>20. The poly-M blocks were obtained by lysis of a P. aeruginosa polymannuronate that has 50% O-acetylation at C-2 and C-3. The poly-MG blocks were obtained from a P. aeruginosa alginate that contained both mannuronate and guluronate residues. The polysaccharide was first deacetylated and then treated with the lyase to excise the mannuronate units from the alternating-MG blocks. Both types of blocks should have potent biological effects and should provide useful specific substrates for characterisation of other alginate lyases.

Human leukocyte elastase (HLE) has been implicated in the pathomechanism of various diseases, such as emphysema and atherosclerosis. The incidence of these diseases is increasing with aging. Therefore, it can be supposed that the HLE activity is changing with aging according to the well known age-related physiological alterations. Thus, the effects of pH, NaCl and calcium concentrations on HLE activity, separated from polymorphonculear leucocytes (PMNLs) of healthy, young (35 years) and elderly (75 years) subjects, were studied by measuring the activity with synthetic substrate and with bovine and human atherosclerotic and non-atherosclerotic aortic elastin. From our results, it may be concluded, that the elastolytic activity of HLE separated from PMNLs of elderly subjects is more sensitive to ionic strength, to pH and to the calcium concentration of the medium, than the HLE activity of young subjects. The elastolytic activity of HLE, of both young and old subjects, increases dramatically on atherosclerotic aortic elastin in the presence of calcium. These findings might explain, at least partly, the increased incidence of atherosclerosis with aging.

An enzymo-conductimetric method for the assay of ethanal is described. It is based on ethanal oxidation in presence of yeast aldehyde dehydrogenase. Experimental conditions compatible with conductimetry were optimized and kinetic parameters of the enzymatic reaction determined. Due to the low KM value of ethanal for the enzyme, an end-point assay was proposed. A linear relationship was demonstrated between experimental conductance changes and ethanal concentration up to 25 μM. Validation of the assay was made on wines by comparison with a spectrophotometric method. Both methods gave similar results, but conductimetry avoids sample treatment if solutions are coloured or turbid.

In recent years, enzymatic peptide synthesis, including synthesis of bioactive peptides and analogues, semi-synthetic proteins and bioactive peptides from recombinant precursors has been attracting increasing attention (for a review see [1, 2]). Pseudomonas aeruginosa elastase (pseudolysin), a thermolysin-like metalloproteinase has been shown to synthesize N-protected dipeptide amides with good yields and purities [3,4]. While both enzymes show some structural similarities [5] elastase but not thermolysin can incorporate tyrosine and tryptophane in P'1 position with high synthesis rates (S. Rival unpublished). Kinetic mechanisms of peptide synthesis by thermolysin have been already proposed [6–8]. In this work, mainly from conductimetric measurement of initial rates, we have studied the enzymatic mechanism of the pseudolysincatalyzed synthesis of Z-Ala–Phe–NH2 and the influence of some parameters.

Matrikines, i.e. matrix fragments with cytokine-like properties, have been ascribed a major role in regulating tumour progression. The invasive front of melanoma is characterised by intense fragmentation of dermal elastic fibres. Elastase-mediated elastolysis liberates elastin fragments, i.e. elastokines, that stimulate several aspects of melanoma progression such as to enhance melanoma cell invasion through type I collagen or increase angiogenesis. Induced-membrane-type 1 metalloprotease (MT1-MMP) expression following elastin receptor (S-Gal) occupancy by elastokines is responsible for those biological activities. Several matrix-derived peptides with a GXXPG consensus sequence adopting a type VIII beta-turn conformation were as potent as elastokines in promoting angiogenesis in a Matrigel assay, and galectin-3 also contains several similar repeats within its N-terminal domain. We propose that S-Gal might constitute a novel therapeutic target for controlling melanoma progression.

Measurement of matrix metalloproteinase (MMP) activity often remains a challenge, mainly in complex media. Two sets of methods are currently used. The first one measures the hydrolysis of natural protein substrates (labeled or not) and includes the popular zymography. These techniques which are quite sensitive, cannot generally be carried out on a continuous basis. The second one takes mainly advantage of the increase of fluorescence, which is associated to the hydrolysis of initially quenched fluorogenic peptide substrates. Quite recently, another group, which is a compromise between the other two, has been developed. It measures the hydrolysis of synthetic triple-helical peptide substrates. These different methods are described and discussed.

Publisher Summary This chapter elaborates the structural chemistry and the biological aspects of Myxococcus Xanthus metalloendopeptidase. An elastolytic activity has been evidenced by conductimetry using insoluble bovine neck ligament elastin as a substrate. This activity is totally suppressed by 10mM 1,10-phenanthroline. The molecular mass is 39,000 Da and the pI is 5.2. The sequence of the first 21 N-terminal amino acids of the enzyme is LRVNRVGSSDDIWTWANRYNL. This sequence is similar to that of an alkaline protease from Pimelobacter sp. Z-483: LIVNTVGGADDRW. For preparation, Myxococcus xanthus DK 1622 was grown in 1% casitone, 0.5% casein and 0.5% elastin media in 10 mM Tris-HCl buffer, 1mM KH 2 PO 4 , pH 7.6, 8mM MgSO 4 . Cells were collected by centrifugation at the end of the exponential-growth phase. Purification from the supernatants of cultures involved ultrafiltration, negative adsorption on DEAE-cellulose pH 7.8, ion-exchange chromatography on DEAE-cellulose and affinity chromatography on agarose-elastin at pH 6.7. Adsorbed material was eluted with 50 mM NaH 2 PO 4 , pH 7.0, containing 200 mM NaCl. The total yield was found to be extremely low (∼1%).

HT-1080 fibrosarcoma cells express at their plasma membrane the elastin-binding protein (EBP). Occupancy of EBP by elastin fragments, tropoelastin or XGVAPG peptides was found to trigger procollagenase-1 (proMMP-1) overproduction by HT-1080 cells at the protein and enzyme levels. RT-PCR analysis indicated that elastin peptides did not modify the MMP-1 mRNA steady state levels, suggesting the involvement of a post-transcriptional mechanism. We previously reported that binding of elastin peptides to EBP induced other matrix metalloproteinases (MMP-2 and MT1-MMP) expression. Since those peptides were here found to also accelerate the secretion of urokinase from HT-1080 cells, culture medium was supplemented with plasminogen together with elastin peptides at aims to induce or potentiate MMPs activation cascades. In such conditions, plasmin activity was generated and exacerbate proMMP-1 and proMMP-2 activation. As a consequence, elastin peptides and plasminogen-treated HT-1080 cells displayed a significant type I collagen matrix invasive capacity.

International audience; We have established that treatment of cultured human skin fibroblasts with tropoelastin or with heterogenic peptides, obtained after organo-alkaline or leukocyte elastase hydrolysis of insoluble elastin, induces a high expression of pro-collagenase-1 (pro-matrix metalloproteinase-1 (pro-MMP-1)). The identical effect was achieved after stimulation with a VGVAPG synthetic peptide, reflecting the elastin-derived domain known to bind to the 67-kDa elastin-binding protein. This clearly indicated involvement of this receptor in the described phenomenon. This notion was further reinforced by the fact that elastin peptides-dependent MMP-1 up-regulation has not been demonstrated in cultures preincubated with 1 mm lactose, which causes shedding of the elastin-binding protein and with pertussis toxin, which blocks the elastin-binding protein-dependent signaling pathway involving G protein, phospholipase C, and protein kinase C. Moreover, we demonstrated that diverse peptides maintaining GXXPG sequences can also induce similar cellular effects as a "principal" VGVAPG ligand of the elastin receptor. Results of our biophysical studies suggest that this peculiar consensus sequence stabilizes a type VIII beta-turn in several similar, but not identical, peptides that maintain a sufficient conformation to be recognized by the elastin receptor. We have also established that GXXPG elastin-derived peptides, in addition to pro-MMP-1, cause up-regulation of pro-matrix metalloproteinase-3 (pro-stromelysin 1). Furthermore, we found that the presence of plasmin in the culture medium activated these MMP proenzymes, leading to a consequent degradation of collagen substrate. Our results may be, therefore, relevant to pathobiology of inflammation, in which elastin-derived peptides bearing the GXXPG conformation (created after leukocyte-dependent proteolysis) bind to the elastin receptor of local fibroblasts and trigger signals leading to expression and activation of MMP-1 and MMP-3, which in turn exacerbate local connective tissue damage.

Lysis of alginates and of their saturated and unsaturated fragments was monitored by 1H NMR spectroscopy. AlxM(B) alginate lyase performs beta-elimination on the mannuronic acid (M) residues. It does not cleave the guluronic acid (G) sequences, nor the M-G or the G-M diads. In consequence, it is a true mannuronate lyase. The end product of the reaction is O-(4-deoxy-alpha-L-ery-thro-hex-4-enopyranosyl-uronic acid)-(1->(4)-O-(beta-D-mannopyranosyluronic acid)-(1->4)-O-beta-D-mannpyranuronic acid. Viscosity measurements made during degradation of a polymannuronate alginate showed that AlxM(B) behaves as an endo-enzyme. HPLC analysis of the degradation products of oligomannuronates and oligoalginates suggested that the beta-elimination requires the interaction of the enzyme with at least three sequential mannuronic acid residues. The catalytic site may possess 5 sub-sites and accommodate pentamers with different M/G ratio. Kinetic measurements showed that the specificity constant Vm/Km increased with the number of mannuronic acid residues. AlxM(B) may be reversibly inhibited by heteropolymeric blocks in a competitive manner.

The use of a peptide library of limited size, is considered to be more appropriate for studying a protease with a complex specificity, but very sensitive and efficient analytical techniques must be used. We have designed and synthesized a 49-peptide library of the type Z-AlaXXAla(amide) (X=Ala, Leu, Val, Phe, Ser, Arg, Glu) for studying the Pseudomonas aeruginosa serralysin specificity. All compounds of the peptide library could be identified by a LC-MS procedure. After hydrolysis of the library by pseudomonal serralysin, the LC-MS procedure also allowed the identification of the hydrolysis products and the different cleavage sites of the substrates.

International audience; The name surfactin refers to a bacterial cyclic lipopeptide, primarily renowned for its exceptional surfactant power since it lowers the surface tension of water from 72 mN m-1 to 27 mN m-1 at a concentration as low as 20 microM. Although surfactin was discovered about 30 years ago, there has been a revival of interest in this compound over the past decade, triggered by an increasing demand for effective biosurfactants for difficult contemporary ecological problems. This simple molecule also looks very promising as an antitumoral, antiviral and anti-Mycoplasma agent. Structural characteristics show the presence of a heptapeptide with an LLDLLDL chiral sequence linked, via a lactone bond, to a beta-hydroxy fatty acid with 13-15 C atoms. In solution, the molecule exhibits a characteristic "horse saddle" conformation that accounts for its large spectrum of biological activity, making it very attractive for both industrial applications and academic studies. Surfactin biosynthesis is catalysed non-ribosomally by the action of a large multienzyme complex consisting of four modular building blocks, called the surfactin synthetase. The biosynthetic activity involves the multicarrier thiotemplate mechanism and the enzyme is organized in structural domains that place it in the family of peptide synthetases, a class of enzymes involved in peptidic secondary-metabolite synthesis. The srfA operon, the sfp gene encoding a 4'-phosphopantetheinyltransferase and the comA regulatory gene work together for surfactin biosynthesis, while the gene encoding the acyltransferase remains to be isolated. Concerning surfactin production, there is no indication whether the genetic regulation, involving a quorum-sensing mechanism, overrides other regulation factors promoted by the fermentation conditions. Knowledge of the modular arrangement of the peptide synthetases is of the utmost relevance to combinatorial biosynthetic approaches and has been successfully used at the gene level to modify the surfactin template. Biosynthetic and genetic rationales have been described for building variants. A fine study of the structure/function relationships associated with the three-dimensional structure has led to the recognition of the specific residues required for activity. These studies will assist researchers in the selection of molecules with improved and/or refined properties useful in oil and biomedical industries.

The characterization of the specificity of alkaline protease from Pseudomonas aeruginosa has not yet been clearly defined. Some previous results suggested that its specificity was influenced more by amino acids far from the hydrolyzed peptide bond, than by amino acids in P 1 or P ′ 1 position. From other data, it was a C–(COOH)-type endoprotease where the preferential amino acid in P 1 position was an arginine residue. We have studied the hydrolysis of several biologically active peptides. Many various sites of cleavage have been characterized but no arginine in P 1 position was found, despite the presence of arginine in the peptide sequence. In fact P 1 and P ′ 1 position could be occupied by various amino acids. It seems unlikely that Pseudomonas alkaline protease may only be considered as a protease specific to arginine in P 1 position. On the other hand, we have observed that increase of the peptide chain length led to an important increase of the hydrolysis rate, suggesting an extended number of subsites.

LDL peroxidation represents one of the first event in the atherogenesis process. Inhibiting LDL oxidation may impede this process and offers a new mechanism to retard atherogenesis. 21-Aminosteroids, derived from methylprednisolone, have recently excited much interest by virtue of their ability to inhibit lipid peroxidation. The aim of our work was to investigate the effect of a novel 21-aminosteroid, U-74389G, in the LDL peroxidation initiated in a metal- and cell-free system by oxygen free radicals, .OH and O2-., generated by water gamma-radiolysis. In a concentration dependent manner, U-74389G increased the resistance of LDL to oxidation measured by the length of the lag phase, reduced the formation of conjugated dienes and thiobarbituric acid-reactive substances (TBARS), and also reduced the alpha-tocopherol disappearance by about 47% at the concentration 20 microM. U-74389G was also able to reduce the chemotactic activity of oxidized LDL towards monocytes, as well as the cholesterol accumulation in macrophages. These observations suggest that the U-74389G is a potent antioxidant by decreasing LDL peroxidation and this should be evaluated in in vivo models as a potential therapy to retard atherogenesis.

Alkaline protease from Pseudomonas aeruginosa (E.C. 3.4.24.40), also called pseudomonal serralysin, plays an important role in the infection bacterial process. The alkaline protease concentration and activity in the culture supernatants of 23 Pseudomonas aeruginosa strains from patients suffering from cystic fibrosis were determined by using an ELISA procedure and a specific enzymatic activity test. No correlation was demonstrated between the activity and the concentration of alkaline protease. This could be explained by the presence of an inactive precursor and a partial hydrolysis of the enzyme, confirmed by SDS-PAGE and Western blotting in the supernatants. The activity test is the only one allowing a quantification of the presence of an active form of alkaline protease.

Two Pseudomonas aeruginosa alginates were lysed by an overexpressed polymannuronate lyase AlxM B (only acting on two or more consecutive, nonacetylated mannuronate units) to prepare either mannuronate blocks (poly-M blocks) with dp∼30, or strictly alternating sequences of mannuronic and guluronic acid (poly-MG blocks) with dp>20. The poly-M blocks were obtained by lysis of a P. aeruginosa polymannuronate that has 50% O-acetylation at C-2 and C-3. The poly-MG blocks were obtained from a P. aeruginosa alginate that contained both mannuronate and guluronate residues. The polysaccharide was first deacetylated and then treated with the lyase to excise the mannuronate units from the alternating-MG blocks. Both types of blocks should have potent biological effects and should provide useful specific substrates for characterisation of other alginate lyases.

Pseudomonas aeruginosais a zinc metalloprotease which may be involved in many infection processes, especially in the lung. In order to evaluate the production of the enzyme in culture supernatants, we developed an assay using peptide derivatives; the conductimetric method was used for monitoring the enzymatic activities. Tetrapeptide derivatives were enzymatically synthesized by coupling Z-Ala2and X-AlaR using either thermolysin orP. aeruginosaelastase itself. In these substrates, X could be phenylalanine, tyrosine, or leucine and C-protection was performed by either an amide (NH2) or a methyl (OMe) group. Z-Ala2-Phe-AlaNH2was found to be the best substrate, giving a catalytic ratiokcat/KMof 8600 m M −1.s−1. The evaluation of the alkaline protease activity with this substrate showed that the catalytic ratio is 1000-fold lower. The sensitivity of the conductimetric method was also demonstrated with as little as 1 n M elastase (0.13 μg), being easily and accurately detected (SD, 3.8% for 10 measurements). Furthermore, the enzymatic activity was measured in a culture supernatant from a clinical strain.

An extracellular elastase, termed Myxococcus xanthus alkaline protease 1 (MAP1), has been purified from M. xanthus DK1622 culture supernatants by a combination of ion-exchange and affinity chromatographies. It consists of a single peptide chain of 39 kDa. The elastolytic activity was totally suppressed by 10 mM 1,10-phenanthroline and the enzyme may then be classified as a metalloprotease. Its pH optimum was estimated to be 8.2 with both elastin-orcein and succinyl-Ala3 p-nitroanilide as substrates. Despite its low pI (5.2), MAP1 was adsorbed on elastin at 80%, a result which privileges hydrophobic interactions between MAP1 and elastin rather than salt bridges, as for known basic elastases. About 80% of the original amidasic and elastolytic activities were conserved after a 30-min prior incubation of the enzyme at 40 degrees C; however, 70% of the amidasic activity is measured, instead of 15% for the elastolytic activity, after 30 min at 50 degrees C. Thermal denaturation at this temperature may prevent adsorption of the enzyme on elastin without any important change of the elastase structure. MAP1 readily hydrolyzes the Gly23-Phe24 bond in the oxidized insulin B chain; the peptide bonds Ala14-Leu15, Leu15-Tyr16, Phe24-Phe25, Phe25-Tyr26 are also cleaved, suggesting a primary specificity of the enzyme for hydrophobic or aromatic residues at the first amino acid towards the C-terminus from the cleavage site (P'1 position) [Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162]. This hypothesis is consistent with the fact that Ala2-Phe-Ala and Ala3-Phe-Ala are hydrolyzed even though tri-alanine to hexa-alanine oligomers are not. The evidence of an elastase with the same molecular mass and pI as MAP1 is given during fruiting body development in submerged culture of M. xanthus. The fact that aromatic amino acids have been found to be the most representative of A-signal [Kuspa, A., Plamann, L. & Kaiser, D. (1992) J. Bacteriol. 174, 3319-3326] is consistent with the hypothesis that, regarding its specificity, MAP1 is likely to play a role in development of myxobacteria.