Amino acid sequence and molecular modelling of glycoprotein IIb-IIIa and fibronectin receptor iso-antagonists from Trimeresurus elegans venom.

Low-molecular-mass Arg-Gly-Asp (RGD)-containing polypeptides were isolated from the venom of Trimeresurus elegans by a simple two-step procedure consisting of membrane filtration and reverse-phase HPLC. A combination of electrospray MS, fast-atom bombardment MS and Edman degradation allowed us to ascertain the presence in the venom of different isoforms and to determine their primary structures. The amino acid sequences resembled the structure of elegantin, the only disintegrin previously reported from the T.

Transglutaminase from rat coagulating gland secretion. Post-translational modifications and activation by phosphatidic acids.

Structural and biochemical characteristics of transglutaminase purified by a rapid chromatographic procedure from the rat coagulating gland (anterior prostate) secretion are reported. Fast atom bombardment mapping and automated Edman degradation experiments allowed us to verify that at least 85% of the entire transglutaminase amino acid sequence is identical to that derived from the cDNA of the major androgen-dependent rat prostate protein called DP1. The enzyme was found NH2 terminally blocked and largely post-translationally modified, since the presence of N-linked oligosaccharides, as well as of complex lipidic structures, was observed.

Glutathione-dependent pathways of refolding of RNase T1 by oxidation and disulfide isomerization: catalysis by protein disulfide isomerase.

Protein folding, associated with oxidation and isomerization of disulfide bonds, was studied using reduced and denatured RNase T1 (rd-RNase T1) and mixed disulfide between glutathione and reduced RNase T1 (GS-RNase T1) as starting materials. Folding was initiated by addition of free glutathione (GSH + GSSG) and was monitored by electrospray mass spectrometry (ES-MS) time-course analysis. This permitted both the identification and quantitation of the population of intermediates present during the refolding process.

Assignment of protein disulphides by a computer method using mass spectrometric data.

We designed a computer program for the assignment of protein disulphides using mass spectrometric data. All the theoretical linear peptides containing from one to three cysteines are generated on the basis of the protein sequence. Their combination ways are determined in order to create all the possible disulphide-bridged fragments containing from two to six cysteines and to calculate their molecular weight.

Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: the case of Minibody.

A strategy that combines limited proteolysis experiments and mass spectrometric analysis of the fragments generated has been developed to probe protease-accessible sites on the protein surface. This integrated approach has been employed to investigate the tertiary structure of the Minibody, a de novo designed 64-residue protein consisting of a beta-sheet scaffold based on the heavy-chain variable-domain structure of a mouse immunoglobulin and containing two segments corresponding to the hypervariable H1 and H2 regions. The low solubility of the protein prevented a detailed characterization by NMR and/or X-ray.

Structure of fuscopeptins, phytotoxic metabolites of Pseudomonas fuscovaginae.

The structure of the fuscopeptins, bioactive lipodepsipeptides produced in culture by the gramineae pathogen Pseudomonas fuscovaginae, has been determined. The combined use of FAB mass spectroscopy NMR spectroscopy and chemical and enzymatic procedures allowed one to define a peptide moiety corresponding to Z-Dhb-D-Pro-L-Leu-D-Ala-D-Ala-D-Ala-D-Ala-D-Val-Gly-D-Ala-D-Val-D-Ala-D- Val-Z-Dhb-Da-Thr-L-Ala-L-Dab-D-Dab-L-Phe with the terminal carboxyl group closing a macrocyclic ring on the hydroxyl group of the allothreonine residue. The N-terminus is in turn acylated by 3-hydroxyoctanoate in fuscopeptin A and 3-hydroxydecanoate in fuscopeptin B.

The gene, protein and glycan structures of laccase from Pleurotus ostreatus.

A member of the laccase multigene family in Pleurotus ostreatus has been cloned and sequenced. The gene structure has been determined by comparison with the corresponding cDNA, synthesized by reverse transcription/PCR amplification. The gene encode a laccase isoenzyme of 533 amino acids which has already been purified and characterized [Palmieri, G.

Identification of disulphide bonds in the refolding of bovine pancreatic RNase A.

Background: Comprehension of the rules that govern the folding process is still far from satisfactory, though it is nevertheless clear that all the information required to define the folding is encoded in the amino acid sequence. In proteins that contain disulphide bonds, folding is associated with disulphide bond formation. Protein species with different numbers of disulphides tend to accumulate during the process; these species can be trapped in a stable form, by quenching any remaining free SH groups, and then characterized in order to identify the disulphide bonds formed.

Neurokinin receptors could be differentiated by their capacity to respond to the transglutaminase-synthesized gamma-(glutamyl5)spermine derivative of substance P.

Four different gamma-(glutamyl5)amine derivatives of substance P (SP) were synthesized in vitro in the presence of purified guinea pig liver transglutaminase and Ca2+. The 1,3-diaminopropane, spermidine, spermine (Spm), and monodansylcadaverine adducts of the neuropeptide were purified by HPLC on a reversed-phase column and characterized by fast atom bombardment mass spectrometry. The gamma-(glutamyl5)Spm derivative of SP (Spm-SP) was found to be able, like the parent neuropeptide, to provoke rabbit aorta relaxation, to decrease rat arterial blood pressure, and to inhibit collagen-induced platelet aggregation.

Structural analysis of saposin C and B. Complete localization of disulfide bridges.

Saposins A, B, C, and D are a group of homologous glycoproteins derived from a single precursor, prosaposin, and apparently involved in the stimulation of the enzymatic degradation of sphingolipids in lysosomes. All saposins have six cysteine residues at similar positions. In the present study we have investigated the disulfide structure of saposins B and C using advanced mass spectrometric procedures.

Amino acid sequence and disulphide-bridge pattern of three gamma-thionins from Sorghum bicolor.

The complete primary structure of a new alpha-amylase inhibitor from Sorghum bicolor belonging to the gamma-thionin family has been determined and the amino acid sequences of two components of the family already elucidated have been corrected by combining the classical Edman degradation with advanced mass spectrometric procedures. The same integrated approach allowed us to define the pattern of the disulphide bridges in the three isoinhibitors. The arrangement of the cysteine pairing was determined as Cys3-Cys47, Cys14-Cys34, Cys20-Cys41 and Cys24-Cys43.

[The predictive value for major arrhythmic events of ventricular arrhythmias, particularly nonsustained ventricular tachycardias, in the subacute phase of a fibrinolyzed infarct. An analysis of GISSI-2 data. Gruppo Italiano per lo Studio della Streptochinasi nell'Infarto Miocardico].

Background: The relationship between ventricular arrhythmias (VA) in the subacute phase of a myocardial infarction (MI) and subsequent major arrhythmic events, i.e. sustained ventricular tachycardia (SVT) and sudden death (SD), is well known.

Novel bioactive lipodepsipeptides from Pseudomonas syringae: the pseudomycins.

The covalent structure and most of the stereochemistry of the pseudomycins, bioactive metabolites of a transposon-generated mutant of a Pseudomonas syringae wild-type strain proposed for the biological control of Dutch elm disease, have been determined. While two pseudomycins are identical to the known syringopeptins 25-A and 25-B, pseudomycins A, B, C, C' are new lipodepsinonapeptides. For all of these the peptide moiety corresponds to L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp(3-OH) -L-Thr (4-Cl) with the terminal carboxyl group closing a macrocyclic ring on the OH group of the N-terminal Ser.

Biological activities of CNBr fragments of a major protein secreted from the rat seminal vesicle epithelium.

Two fragments of SV-IV, one of the major proteins secreted from the rat seminal vesicle epithelium, were produced in vitro by protein cleavage with CNBr at level of the single methionine residue (Met-70) occurring in its polypeptide chain. After their purification by reversed-phase chromatography, SV-IV/A (1-70 fragment) and SV-IV/B (71-90 fragment) were assayed as transglutaminase substrates, and their anti-inflammatory, anti-thrombotic and immunosuppressive properties were evaluated in comparison with native SV-IV. Both fragments retained the SV-IV ability to act as transglutaminase substrates in vitro; fast atom bombardment mass spectrometry analyses of the reaction products pointed to Gln-9 and Gln-86 as acyl donor sites, and to Lys-59, -79 and -80 as acyl acceptor sites.

Analysis of RNase A refolding intermediates by electrospray/mass spectrometry.

Electrospray/mass spectrometry (ES/MS) was extensively used to obtain information on disulphide-containing intermediates formed during refolding of bovine pancreatic ribonuclease A. The analysis showed the existence of an equilibrated population of disulphide bonded intermediates, and indicates that intermediates containing two intramolecular S-S are predominant until late stages of the refolding process. Mixed disulphides with exogenous glutathione were also detected, supporting previous evidence of conformational restrictions on the ability of RNase A to form intramolecular disulphides.

Hb F-Sassari: a novel G gamma variant with a threonine residue at position gamma 75, characterized by mass spectrometric techniques.

The cord blood sample of a Caucasian newborn contained about 40% of an abnormal fetal hemoglobin. The mutated gamma chain was isolated using reversed phase high performance liquid chromatography and characterized by means of electrospray and fast atom bombardment mass spectrometric techniques as a G gamma-globin variant with an Ile-->Thr substitution at position gamma 75. The variant chain shows the same structure as the previously described Hb F-Charlotte that was demonstrated to be an A gamma variant with an Ile-->Thr substitution at position gamma 75 and an additional Ala-->Gly substitution at gamma 136.

Post-translational modifications in aspartate aminotransferase from Sulfolobus solfataricus. Detection of N-epsilon-methyllysines by mass spectrometry.

Advanced mass spectrometric procedures have been extensively used to provide an accurate structural characterization of aspartate aminotransferase from Sulfolobus solfataricus. The amino acid sequence of this enzyme had previously been deduced from the DNA sequence. The accurate molecular mass of the protein, determined using electrospray mass spectrometry, demonstrated that the amino acid sequence deduced was correct and ruled out the possible presence of large covalent modifications which had been postulated to fit the much higher molecular mass obtained from previous SDS/PAGE experiments.

Production and structural characterization of amino terminally histidine tagged human oncostatin M in E. coli.

Oncostatin M is a cytokine that acts as a growth regulator on a wide variety of cells and has diverse biological activities including acute phase protein induction, LDL receptor up-regulation and cell-specific gene expression. In order to gather information about the Onc M structure, we established a protocol for large scale production and single step purification of this functional cytokine from bacterial cells. The cDNA of human Onc M was cloned by RT-PCR from total RNA of PMA induced U937 cells.

Relevance of chlorine-substituent for the antifungal activity of syringomycin and syringotoxin, metabolites of the phytopathogenic bacterium Pseudomonas syringae pv. syringae.

Structural analogues of syringomycin and syringotoxin were produced by fermentation, characterized by FAB-MS and amino acid analysis and compared to the parent compounds in the antibiosis test against Rhodotorula pilimanae. The C-terminal residue was shown to be important for the activity.

[D-Leu2]deltorphin, a 17 amino acid opioid peptide from the skin of the Brazilian hylid frog, Phyllomedusa burmeisteri.

A novel 17 amino acid peptide, having a D-leucine in position 2 of its sequence, has been isolated from methanol extracts of the skin of the Brazilian frog, Phyllomedusa burmeisteri. The sequence of the peptide is: Tyr-D-Leu-Phe-Ala-Asp-Val-Ser-Thr-Ile-Gly-Asp-Phe-Phe-His-Ser-Ile-NH2. It displays a poor affinity for delta-opioid binding sites, both in the periphery and in the central nervous system.

Sheep haemoglobin I or beta B13(A10)Gly-->Ser: an example of a CpG mutation in vertebrates. Characterization using FAB-mass spectrometry and amino acid sequencing.

1. The amino acid substitution which characterizes the haemoglobin I variant from sheep has been ascertained using a combination of Fast Atom Bombardment mass spectrometry and protein sequencing. 2.

Hb O-Arab [beta 121(GH4)Glu-->Lys]: association with DNA polymorphisms of African ancestry in two Mediterranean families.

Hb O-Arab [beta 121(GH4)Glu-->Lys] was detected in two Mediterranean families, one from Southern Italy and the other from Albania. The GAA-->AAA mutation at codon 121 was characterized by DNA sequencing. The mutant genes were associated with the same beta-globin gene framework variant and with the rare Hpa I/3' beta polymorphic restriction site generating a 7.

Electrospray mass spectrometric analysis of river buffalo (Bubalus bubalis) hemoglobins. Re-examination of alpha 1 and alpha 3 globin chain sequences.

1. The accurate molecular weight of the globin chains from river buffalo hemoglobin components has been determined by means of electrospray mass spectrometry. 2.