Comparative enzymatic studies of human renin acting on pure natural or synthetic substrates.

Some of the essential structural requirements for the enzymatic reaction of pure human renin acting on pure human and rat angiotensinogen and on their synthetic tetradecapeptide substrates were investigated. The five carboxy terminal amino acids of synthetic tetradecapeptides played a significant role in substrate recognition and/or hydrolysis by human renin. Kinetic constants Km, Kcat and kcat/Km of the various human renin assays were different according to the substrate used. The presence of either an asparagine or a threonine residue in the S'4 renin subsite did not affect significantly the kinetic constant values. A tyrosine residue, rather than a histidine residue, in the S'3 renin subsite gave the best synthetic substrate studied. When tyrosine residue was present in the S'2 renin subsite an important decrease in kcat was observed. Human angiotensinogen was hydrolysed by human renin with lower Km and kcat values than those measured with human and porcine synthetic substrates, suggesting that the 3-dimensional structure of human angiotensinogen plays a key role in the hydrolysis. This finding was supported by assays performed with rat angiotensinogen, which was cleared by human renin with the same kcat value as rat tetradecapeptide, but with a 49-fold lower Km. Between human and rat angiotensinogen a kcat/Km value of only 2-fold higher has been found in the renin assay using human substrate.

Crystallization and preliminary X-ray study of porcine trypsin, free and complexed with Ecballium elaterium trypsin inhibitor, a member of the squash inhibitors family.

Porcine trypsin has been crystallized either free or complexed with synthetic Ecballium elaterium trypsin inhibitor II, a 28-residue peptide with three disulfide bridges. The crystals diffract beyond 2.0 A. Crystals are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 77.32 A, b = 53.81 A, c = 46.91 A, for the free trypsin, and a = 62.25 A, b = 62.27 A, c = 84.66 A for the complex with E. elaterium trypsin inhibitor II.

Contraction induced by glicentin on smooth muscle cells from the human colon is abolished by exendin (9-39).

UNLABELLED: Glicentin and glucagon-like peptide-1 (7-36) amide (GLP-1) are gut hormones released during digestion. Glicentin and GLP-1 slow down gastric emptying and glicentin can switch off the duodenojejunal fed motor pattern. The effect of glicentin on the motor activity of colon has never been reported in humans. Our aim was to determine if circular smooth muscle cells (SMC) from the human colon are target cells for glicentin or GLP-1, and if their motility is dependent upon these digestive hormones. METHODS: Twenty-two resections were performed on patients operated for colon adenocarcinoma. The SMC were isolated from colonic circular muscle layer and cell contraction was assessed. RESULTS: Glicentin caused a dose-related contraction of SMC, when GLP-1 determined a contraction of weak amplitude. Exendin-(9-39), described as a GLP-1 receptor antagonist, inhibited contraction due to glicentin or GLP-1. In contrast, on antral SMC from rabbit, GLP-1 exerts neither relaxation nor contraction; however, exendin-(9-39) dose dependently reduced the contractile activity of glicentin [glicentin EC(50) = 5 pM, exendin-(9-39) pA(2) = -9.36]. CONCLUSIONS: The circular muscle from the human colon is a target tissue for glicentin and GLP-1. Whereas glicentin is a long-life digestive hormone which would contribute to segmental contraction, the biological activity of GLP-1 remains unknown on this tissue. On the digestive smooth muscle, exendin-(9-39) behaved as an antagonist for two members of the glucagon-receptor family, GLP-1 and glicentin.

New monoclonal antibodies as probes for human cardiac troponin I: epitopic analysis with synthetic peptides.

Forty monoclonal antibodies (MAbs) specific for human cardiac troponin I (TnI) were selected to develop a new alternative for specific biological diagnosis of acute myocardial infarction. Using an immunoenzymatic sandwich assay, these MAbs were employed in the mapping of human cardiac TnI and showed six different epitopes. Parts of the TnI peptide sequences were synthesised; the sequences were chosen from the published sequences of mammalian TnI. Immunological assays showed that 8 out of 40 MAbs recognised a RAYATEPHAK (P2) N-terminus cardiac-specific sequence of human TnI. The information obtained from epitopic mapping of TnI and the properties of the peptides allowed pairs of MAbs to be selected for the development of a future specific TnI assay.

Characterization and 2D NMR study of the stable [9-21, 15-27] 2 disulfide intermediate in the folding of the 3 disulfide trypsin inhibitor EETI II.

The three disulfide Ecballium elaterium trypsin inhibitor II (EETI II) reduction with dithiothreitol (DTT) and reoxidation of the fully reduced derivative have been examined. A common stable intermediate has been observed for both processes. Isolation and sequencing of carboxymethylated material showed that the intermediate lacks the [2-19] bridge. The NMR study showed a very strong structural conservation as compared to the native EETI II, suggesting that the bridges are the [9-21] and [15-27] native ones. The differences occurred in sections 2-7 (containing the free cysteine 2 and the Arg 4-Ile 5 active site) and 19-21 (containing the second free cysteine). Distance geometry calculations and restrained molecular dynamics refinements were also in favor of a [9-21, 15-27] arrangement and resulted in a well-conserved (7-28) segment.

Glicentin and oxyntomodulin modulate both the phosphoinositide and cyclic adenosine monophosphate signaling pathways in gastric myocytes.

We have investigated the transduction pathways mediating the contractile effect of two glucagon-containing peptides, glicentin (GLIC) and oxyntomodulin (OXM), on smooth muscle cells isolated from rabbit antrum. Low concentrations of GLIC induced a biphasic and rapid (first phase at 5-8 sec) Ins(1,4,5)P3 production. By comparison, higher concentrations of OXM or OXM(19-37) were required to obtain biphasic time-courses of Ins(1,4,5)P3 production. In a Ca2+ free medium, the first phase of Ins(1,4,5)P3 production induced by GLIC or OXM was maintained, while the second phase disappeared. In saponin-permeabilized cells, all three peptides induced cell contraction with similar efficacies and potencies. Exogenous Ins(1,4,5)P3 mimicked the contractile effect of the peptides and heparin, which inhibits the Ins(1,4,5)P3 binding to its receptor, prevented contraction stimulated by each effector. We conclude that a Ca2+ mobilization from the intracellular stores is essential in the contractile effects of GLIC and OXM. Using the fluo-3 probe, a [Ca2+]i increase was observed in the presence of GLIC, OXM, or OXM(19-37). The three peptides reduced by 30-40% the cAMP content of cells stimulated by forskolin. This effect was pertussis toxin sensitive as demonstrated with OXM(19-37). Our data constitute important clues for the existence in smooth muscle cells of receptor(s) specific for the GLIC/OXM hormones, coupled via G protein(s) to both Ca2+ and cAMP pathways.

Purification from human plasma of a tetrapeptide that potentiates insulin-like growth factor-I activity in chick embryo cartilage.

Human plasma has been shown to contain a low molecular weight factor that potentiates human IGF-I stimulation of glycosaminoglycan synthesis in chick embryo cartilage. The peptide was purified and characterized by Edman degradation and electrospray mass spectrometry. The primary structure determined was: Trp-Gly-His-Glu. A homologous synthetic peptide similarly promoted matrix biosynthesis in cartilage exposed to IGF-I.

Immunological detection of prohormone convertases in two different proglucagon processing cell lines.

The distribution of the prohormone convertases, PC1/3, PC2 and PC5/6, was determined by immunoblotting in two cell lines. In alpha TC1-6 cells, the proglucagon processing occurred according to the pancreatic A-cell type. In STC-1 cells, proglucagon was processed in a manner reminiscent of the intestinal L-cell type. PC1/3 was undetectable in both proglucagon processing cell lines whereas PC2 displayed a strong immunostaining in the alpha TC1-6 cells and was barely detectable in the STC-1 cells. PC5/6 was detected as a 70 kDa protein in both cell lines. These results suggest a possible role of PC2 in the processing of proglucagon into glucagon in the A-cells, whereas in L-cells it would require still undetermined endoproteases.

Miniglucagon production from glucagon: an extracellular processing of a hormone used as a prohormone.

Glucagon is secondarily processed into its C-terminal (19-29) fragment, referred to as 'miniglucagon', which modulates the glucagon action. This extracellular processing, occurring at the level of of the glucagon target cells, is due to the presence at the cell surface of a new 100-kDa processing enzyme with characteristics of both thiol- and metalloprotease.

Molecular recognition between serine proteases and new bioactive microproteins with a knotted structure.

Microproteins with proteinase inhibitory activity, 28 to 30 amino acids long, with 3 disulfide bridges have been isolated from Ecballium elaterium seeds. A peptide (EETI II) was isolated and behaved as a powerful trypsin inhibitor (Kd = 10(-11) to 10(-12) M). It was sequenced, chemically synthesized and the 3-D structure determined by 2-D 1H NMR. The information gained in the process enabled us to synthesize modified derivatives with inhibitory activity towards pancreatic elastase, chymotrypsin and human leucocyte elastase (Kd = 10(-7) to 10(-9) respectively). The most striking characteristic that appeared during the synthetic approach was the unfailing ability of the 28 amino acid peptides to refold and correctly close the 3 disulfide bridges, giving in each case an active compound. These disulfide bridges are assembled in a particular knotted structure, shared by few other bioactive peptides and called the 'knottin' structure. Molecular modeling of the peptide and a comparison with the other active molecules with similar topology allowed the synthesis of a chimaeric peptide, bearing 1 active site against a seryl-protease (trypsin), and 1 against a metallo-protease (carboxypeptidase A). The bis-headed peptide was able to inhibit both enzymes separately and concomitantly.

(1)H NMR Study of the solution structure of sarafotoxin-S6b.

Sarafotoxin-S6b has been synthesized and studied by (1)H NMR in 50 50 acetonitrile/water mixture. All spin systems were identified and assigned with the aid of 2D experiments. On the basis of these data, a 3D structure of sarafotoxin is proposed and compared to that of [Nle(7)]endothelin obtained in the same conditions. From this study, it appeared that sarafotoxin-S6b and [Nle(7)]endothelin roughly share the same 3D structure, the main differences being located in the 4-7 loop bearing the sequence variation.

Miniglucagon: a local regulator of islet physiology.

Miniglucagon, or glucagon-[19-29], is partially processed from glucagon in its target tissues where it modulates the glucagon action. In the islets of Langerhans, the glucagon-producing A cells contain miniglucagon at a significant level (2-5% of the glucagon content). We studied a possible control of insulin release by miniglucagon using as a model the MIN6 cell line. Miniglucagon, in the 10(-14) to 10(-9) M range, inhibited insulin release induced by glucose, glucagon, tGLP-1, or glibenclamide by 85-100% with an IC50 close to 1 pM. While no change in the cyclic AMP content was noted, Ca2+ influx was reduced in parallel with the inhibition of insulin release. Use of pharmacological modulators of L-type voltage-sensitive Ca2+ channels and bacterial toxins indicates that miniglucagon blocks insulin release by closing this type of channel via a pertussis toxin-sensitive G protein. Miniglucagon is a novel, possibly physiologically relevant, local regulator of islet function.

Oxyntomodulin and glicentin are potent inhibitors of the fed motility pattern in small intestine.

Glicentin (GLIC) and oxyntomodulin (OXM or GLIC 33-69) are gut hormones which regulate digestion. They are known to reduce digestive secretions and to delay gastric emptying. Their biological activities on intestinal motility are still unknown. The effect of a systemic GLIC or OXM increase was investigated in rats on the food intake, the postprandial myoelectrical activity of small intestine and the orocaecal transit. An OXM or GLIC i.v. infusion was applied during the 5 min preceding food onset and during the first 15 min of food intake. This determined a three- to fourfold increase of the preprandial OXM-GLIC level. The OXM or GLIC plasma increase did not modify food intake. OXM infusion slowed down gastric emptying when the stomach contained 3/4 of the ingested food (before T 3 h). The quantity of food delivered in jejunum was subsequently smaller (P < 0.05). In the small intestine, the duration of postprandial myoelectrical activity (50-60 min g(-1) of ingested food) was reduced by 70% (P < 0.001) on duodenum or jejunum and by 54% (P < 0.01) on ileum in OXM-treated rats. An interdigestive motility profile was settled and an acceleration of both gastric emptying and transit rate was thereafter evidenced (after T 3 h). GLIC also reduced the duration of the postprandial myoelectrical activity on duodenum and jejunum (65 and 63% respectively, P < 0.05), but was not as efficient as OXM on ileum. In pathological states such as acute adult gastroenteritis, OXM and GLIC exhibit a two- to fivefold increase in their plasma concentrations. The present findings suggest that OXM and GLIC could, in that disease, contribute to exclude pathogens, due to their joined action on gut motility.

Oxyntomodulin inhibits pancreatic secretion through the nervous system in rats.

Glicentin (GLIC), oxyntomodulin (OXM), and peptide YY (PYY) released in blood by ileocolonic L-cells after meals may inhibit pancreatic secretion. Whereas OXM interacts with glucagon and tGLP-1 receptors, OXM 19-37, a biologically active fragment, does not. The purpose of this study was to measure the effect of OXM, OXM 19-37, GLIC, tGLP-1, and PYY on pancreatic secretion stimulated by 2 deoxyglucose (2DG), electrical stimulation of the vagus nerves (VES), acetylcholine and cholecystokinin octapeptide (CCK8) in anesthetized rats. The effect of OXM was also studied in dispersed pancreatic acini. Plasma oxyntomodulin-like immunoreactivity (OLI) was measured by radioimmunoassay after the exogenous infusion of OXM and after an intraduodenal meal. OXM 19-37, infused at doses mimicking postprandial plasma levels of OLI, decreased pancreatic secretion stimulated by 2DG, VES, or CCK8. Similar effects were found with OXM and GLIC. OXM 19-37 did not change the pancreatic stimulation induced by acetylcholine in vivo, or CCK-induced amylase release in isolated acini. Vagotomy completely suppressed the inhibitory effect of OXM 19-37 on CCK8-stimulated pancreatic secretion. PYY inhibited the effect of 2DG, but not that of CCK8, whereas tGLP-1, even in pharmacologic doses, had no effect on stimulated pancreatic secretion. OXM, OXM 19-37, but not tGLP-1, inhibit pancreatic secretion at physiologic doses, through a vagal neural indirect mechanism, different from that used by PYY, and probably through a GLIC-related peptide-specific receptor.

Comparison of binding characteristics of endothelin receptors on subpopulations of astrocytes.

Astrocytes in primary culture originating from different brain areas of the mouse embryo (striatum, cerebral cortex and mesencephalon) were compared for their [125I]-Endothelin-1 (ET-1) binding characteristics, in terms of affinity, binding capacity and specificity. Our results indicate that astrocytes from mesencephalon express about twice as many receptors as astrocytes from striatum or cortex (149,000 +/- 9700 vs 63,700 +/- 5600 and 81,900 +/- 5300, respectively), with similar affinities. Specificity patterns for the various peptides of the endothelins/sarafotoxins family (ET-1, -2, -3; SRTXa, b, c) are comparable in the three subpopulations of astrocytes: ET-1, -2 and SRTXb exhibit higher affinities than SRTXa and SRTXc. In addition, ET-3 and SRTXc seem to discriminate between different subsets of [125I]-ET-1 binding sites in the three subpopulations.

Use of carboethoxysulfenyl chloride for disulfide bond formation.

The use of carboethoxysulfenyl chloride for disulfide bond formation and concomitant cyclization of five peptides was investigated. Even though cyclic peptides were obtained very rapidly and in good yields when cyclization was performed in aqueous media at different pHs (4 to 7), the final crude peptides were found to contain closely related impurities which, in the case of somatostatin and pressinoic acid, were not generated by air oxidation. This observation may limit the use of carboethoxysulfenyl chloride to those cases where other methods of disulfide bond formation prove inadequate.

Folding of the squash trypsin inhibitor EETI II. Evidence of native and non-native local structural preferences in a linear analogue.

A peptide, corresponding to the entire sequence of the squash trypsin inhibitor EETI II (Ecballium elaterium trypsin inhibitor) in which the six cysteines, engaged in three disulphide bridges in native EETI II, have been replaced by six serines, has been synthesised. CD, Fourier-transform infrared spectroscopy (FTIR) and 1H-NMR studies of this peptide revealed that some secondary structures present in native EETI II are still populated in the absence of disulphide bonds. Native-like secondary structures were observed for segments 10-15 (helix), 16-19 and 22-25 (reverse turns) but no native tertiary interaction was detected. However, a non-native local interaction between the aromatic ring of Phe26 and the amide group of Gly28 was observed. It is hypothesised that the 10-15, 16-19 and 22-25 native-like local conformations could play a major role in the early folding of EETI II.

Solid phase synthesis of a trypsin inhibitor isolated from the Cucurbitaceae Ecballium elaterium.

The synthesis of a 28-residue peptide isolated from Ecballium elaterium of the Cucurbitaceae family which strongly inhibits trypsin activities (Ka = 8.10(-11)M), using BOP as the coupling reagent in a solid phase procedure is presented. This micro protein contains three disulfide bridges in its sequence and was obtained after oxidation of the six half-cystine residues either by air or with the use of carboethoxysulfenyl chloride. After purification by semi-preparative HPLC, the synthetic product was shown by trypsin inhibition tests to be identical with the trypsin inhibitor EETI II isolated from Ecballium elaterium.

1H 2D NMR and distance geometry study of the folding of Ecballium elaterium trypsin inhibitor, a member of the squash inhibitors family.

The solution conformation of synthetic Ecballium elaterium trypsin inhibitor II, a 28-residue peptide with 3 disulfide bridges, has been studied by 1H 2D NMR measurements. Secondary structure elements were determined: a miniantiparallel beta-sheet Met 7-Cys 9 and Gly 25-Cys 27, a beta-hairpin 20-28 with beta-turn 22-25, and two tight turns Asp 12-Cys 15 and Leu 16-Cys 19. A set of interproton distance restraints deduced from two-dimensional nuclear Overhauser enhancement spectra and 13 phi backbone torsion angles restraints were used as the basis of three-dimensional structure computations including disulfide bridges arrangement by using distance geometry calculations. Computations for the 15 possible S-S linkage combinations lead to the proposal of the array 2-19, 9-21, 15-27 as the most probable structure for EETI II.

Min-21 and min-23, the smallest peptides that fold like a cystine-stabilized beta-sheet motif: design, solution structure, and thermal stability.

Small disulfide-rich proteins provide examples of simple and stable scaffolds for design purposes. The cystine-stabilized beta-sheet (CSB) motif is one such elementary structural motif and is found in many protein families with no evolutionary relationships. In this paper, we present NMR structural studies and stability measurements of two short peptides of 21 and 23 residues that correspond to the isolated CSB motif taken from a 28-residue squash trypsin inhibitor. The two peptides contain two disulfide bridges instead of three for the parent protein, but were shown to fold in a native-like fashion, indicating that the CSB motif can be considered an autonomous folding unit. The 23-residue peptide was truncated at the N-terminus. It has a well-defined conformation close to that of the parent squash inhibitor, and although less stable than the native protein, it still exhibits a high T(m) of about 100 degrees C. We suggest that this peptide is a very good starting building block for engineering new bioactive molecules by grafting different active or recognition sites onto it. The 21-residue peptide was further shortened by removing two residues in the loop connecting the second and third cysteines. This peptide exhibited a less well-defined conformation and is less stable by about 1 kcal mol(-)(1), but it might be useful if a higher flexibility is desired. The lower stability of the 21-residue peptide is supposed to result from inadequate lengths of segments connecting the first three cysteines, thus providing new insights into the structural determinants of the CSB motif.