The Experts below are selected from a list of 195 Experts worldwide ranked by ideXlab platform
Pierre E. Bougis - One of the best experts on this subject based on the ideXlab platform.
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The scorpion toxin Bot IX is a potent member of the α-like family and has a unique N-Terminal Sequence extension
FEBS Letters, 2016Co-Authors: Marie-france Martin-eauclaire, Juan Salvatierra, Frank Bosmans, Pierre E. BougisAbstract:We report the detailed chemical, immunological and pharmacological characterization of the α-toxin Bot IX from the Moroccan scorpion Buthus occitanus tunetanus venom. Bot IX, which consists of 70 amino acids, is a highly atypical toxin. It carries a unique N-Terminal Sequence extension and is highly lethal in mice. Voltage clamp recordings on oocytes expressing rat Nav1.2 or insect BgNav1 reveal that, similar to other α-like toxins, Bot IX inhibits fast inactivation of both variants. Moreover, Bot IX belongs to the same structural/immunological group as the α-like toxin Bot I. Remarkably, radioiodinated Bot IX competes efficiently with the classical α-toxin AaH II from Androctonus australis, and displays one of the highest affinities for Nav channels.
Marie-france Martin-eauclaire - One of the best experts on this subject based on the ideXlab platform.
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The scorpion toxin Bot IX is a potent member of the α-like family and has a unique N-Terminal Sequence extension
FEBS Letters, 2016Co-Authors: Marie-france Martin-eauclaire, Juan Salvatierra, Frank Bosmans, Pierre E. BougisAbstract:We report the detailed chemical, immunological and pharmacological characterization of the α-toxin Bot IX from the Moroccan scorpion Buthus occitanus tunetanus venom. Bot IX, which consists of 70 amino acids, is a highly atypical toxin. It carries a unique N-Terminal Sequence extension and is highly lethal in mice. Voltage clamp recordings on oocytes expressing rat Nav1.2 or insect BgNav1 reveal that, similar to other α-like toxins, Bot IX inhibits fast inactivation of both variants. Moreover, Bot IX belongs to the same structural/immunological group as the α-like toxin Bot I. Remarkably, radioiodinated Bot IX competes efficiently with the classical α-toxin AaH II from Androctonus australis, and displays one of the highest affinities for Nav channels.
John E. Shively - One of the best experts on this subject based on the ideXlab platform.
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Automated carboxy-Terminal Sequence analysis of polypeptides containing C-Terminal proline.
Analytical biochemistry, 1995Co-Authors: Jerome M. Bailey, G. Issai, John E. ShivelyAbstract:Abstract Proteins and peptides can be Sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on automation of the thiocyanate chemistry using diphenyl phosphoroisothiocyanatidate (DPP-ITC) and pyridine to derivatize the C-Terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C-Terminal amino acid (Bailey, J. M., Nikfarjam, F., Shenoy, N. S., and Shively, J. E. (1992) Protein Sci. 1, 1622-1633). A major limitation of this approach was the inability to derivatize C-Terminal proline. We now describe chemistry based on the DPP-ITC/pyridine reaction which is capable of derivatizing C-Terminal proline to a thiohydantoin. The reaction of DPP-ITC/pyridine with C-Terminal proline rapidly forms an acyl isothiocyanate which is capable of forming a quaternary amine containing thiohydantoin. Unlike formation of peptidylthiohydantoins with the other 19 commonly occurring amino acids in which cyclization to a thiohydantoin is concomitant with loss of a proton from the amide nitrogen, proline has no amide proton and as a result the newly formed proline thiohydantoin contains an unprotonated ring nitrogen. This cyclic structure if left unprotonated will regenerate C-Terminal proline during the cleavage reaction. However, if protonated by the addition of acid, the proline thiohydantoin ring is stabilized and can be readily hydrolyzed to proline thiohydantoin and a shortened peptide by the addition of water vapor or alternatively by sodium or potassium trimethylsilanolate, the reagent normally used for the cleavage reaction. By introducing vapor-phase trifluoroacetic acid (TFA) for the protonation reaction and water vapor for the hydrolysis reaction we have been able to automate the chemistry required for derivatization of C-Terminal proline. Since the TFA/water steps have no effect on peptidylthiohydantoins formed from the other 19 amino acids, the additional steps required for proline were readily integrated into the automated sequencing program, providing for the first time an automated sequencing program which permits the C-Terminal Sequence analysis of all 20 of the commonly occurring amino acids. Automated programs are described for the C-Terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene and larger polypeptides noncovalently applied to Zitex (porous Teflon).
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C-Terminal Sequence analysis of polypeptides containing C-Terminal proline
Techniques in Protein Chemistry, 1995Co-Authors: Jerome M. Bailey, G. Issai, John E. ShivelyAbstract:Publisher Summary The use of diphenylphosphoroisothiocyanatidate (DPP-ITC) and pyridine combines the activation and derivatization steps, and has permitted application of the C-Terminal chemistry to a wide variety of protein samples with cycle times similar to those employed for N-Terminal Sequence analysis. This chapter describes the development of chemistry based on the DPP-ITC/pyridine reaction, which permits the efficient derivatization and hydrolysis of peptidyl C-Terminal proline to a thiohydantoin and discusses the integration of this chemistry into an automated method for the C-Terminal Sequence analysis of polypeptides containing C-Terminal proline. The chapter discusses automated chemistry, which is capable of the C-Terminal Sequence analysis of polypeptides containing CTerminal proline. The use of DPP-ITC/pyridine for derivatization permits the direct formation of an acylisothiocyanate at the C-terminus without the need for oxazolinone formation. Once an acylisothiocyanate is formed, it can cyclize to a quaternary amine, containing thiohydantoin. The quaternary amine, containing proline thiohydantoin can be readily cleaved with water vapor, or alternatively with the silanolate salt normally used for the cleavage reaction.
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A Chemical Method for the C-Terminal Sequence Analysis of Proteins
Methods, 1994Co-Authors: Jerome M. Bailey, John E. ShivelyAbstract:Abstract During the past several years there have been increased interest and substantial progress toward the development of a routine automated method for the C-Terminal Sequence analysis of peptides and proteins. Such a procedure would complement current methodology for N-Terminal Sequence determination and would be especially valuable for the Sequence determination of proteins with blocked N-termini and for the detection of post-translational processing at the C-terminus of natural and recombinant proteins. Of the chemical methods proposed, the method based on the derivatization of the carboxy-Terminal amino acid to a thiohydantoin has been the most widely studied and appears to offer the most promise. Recent work from several laboratories has solved many of the limitations of the thiocyanate approach. This work, coupled with the availability of modern analytical instrumentation, has made the prospects for a routine automated method for C-Terminal Sequence analysis feasible. This article discusses some of the most recent advances in the chemistry, describes detailed procedures for automation of the chemistry on our new compact protein Sequencer and the prototypic G1009A Hewlett-Packard C-Terminal protein sequencing system, and presents several examples of the application of this automated approach to the C-Terminal sequencing of peptide and protein samples.
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Compact protein Sequencer for the C-Terminal Sequence analysis of peptides and proteins.
Analytical biochemistry, 1993Co-Authors: J.m. Bailey, M. Rusnak, John E. ShivelyAbstract:We describe the construction of a compact protein Sequencer designed specifically for the C-Terminal Sequence analysis of peptides and proteins. This Sequencer has a vertical flow path and is equipped with a continuous flow reactor (CFR). The flow paths for the various reagents and solvents have been minimized. A unique feature of this instrument is the design of a quadrate valve (quad valve) which permits the delivery of four solvents or reagents to the conversion flask (CF). Combination of two of these quad valves in series permits the delivery of eight solvents and reagents to the CFR. The CF contains three inputs from the top, one for transfer of the contents of the CFR, one which is used as a vent, and one for input of solvents or reagents from the CF quad valve. The CF drains from the bottom, connecting to a switching valve which allows delivery either to a waste bottle or to an on-line HPLC. Another unique feature of this instrument is the design of an optical flow detector which permits injection of approximately 90% of the contents of the CF for HPLC analysis. The overall size of the instrument (11 w x 16.5 h x 23.5 d in.) is smaller than commercially available instruments for protein sequencing and represents the first time an instrument has been constructed specifically for C-Terminal Sequence analysis. The utility of this instrument is demonstrated with the C-Terminal Sequence analysis of protein samples noncovalently applied to Zitex strips and with a peptide covalently attached to carboxylic acid-modified polyethylene film.
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Automated carboxy-Terminal Sequence analysis of peptides.
Protein science : a publication of the Protein Society, 1992Co-Authors: Jerome M. Bailey, Narmada R. Shenoy, Michael Ronk, John E. ShivelyAbstract:Proteins and peptides can be Sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on solution phase conditions for formation of the peptidylthiohydantoins with trimethylsilylisothiocyanate (TMS-ITC) and for hydrolysis of these peptidylthiohydantoins into an amino acid thiohydantoin derivative and a new shortened peptide capable of continued degradation (Bailey, J. M. & Shively, J. E., 1990, Biochemistry 29, 3145-3156). The current study is a continuation of this work and describes the construction of an instrument for automated C-Terminal sequencing, the application of the thiocyanate chemistry to peptides covalently coupled to a novel polyethylene solid support (Shenoy, N. R., Bailey, J. M., & Shively, J. E., 1992, Protein Sci. I, 58-67), the use of sodium trimethylsilanolate as a novel reagent for the specific cleavage of the derivatized C-Terminal amino acid, and the development of methodology to Sequence through the difficult amino acid, aspartate. Automated programs are described for the C-Terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene. The chemistry involves activation with acetic anhydride, derivatization with TMS-ITC, and cleavage of the derivatized C-Terminal amino acid with sodium trimethylsilanolate. The thiohydantoin amino acid is identified by on-line high performance liquid chromatography using a Phenomenex Ultracarb 5 ODS(30) column and a triethylamine/phosphoric acid buffer system containing pentanesulfonic acid. The generality of our automated C-Terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids were found to Sequence in high yield (90% or greater) except for asparagine and aspartate, which could be only partially removed, and proline, which was found not be capable of derivatization. In spite of these current limitations, the methodology should be a valuable new tool for the C-Terminal Sequence analysis of peptides.
Juan Salvatierra - One of the best experts on this subject based on the ideXlab platform.
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The scorpion toxin Bot IX is a potent member of the α-like family and has a unique N-Terminal Sequence extension
FEBS Letters, 2016Co-Authors: Marie-france Martin-eauclaire, Juan Salvatierra, Frank Bosmans, Pierre E. BougisAbstract:We report the detailed chemical, immunological and pharmacological characterization of the α-toxin Bot IX from the Moroccan scorpion Buthus occitanus tunetanus venom. Bot IX, which consists of 70 amino acids, is a highly atypical toxin. It carries a unique N-Terminal Sequence extension and is highly lethal in mice. Voltage clamp recordings on oocytes expressing rat Nav1.2 or insect BgNav1 reveal that, similar to other α-like toxins, Bot IX inhibits fast inactivation of both variants. Moreover, Bot IX belongs to the same structural/immunological group as the α-like toxin Bot I. Remarkably, radioiodinated Bot IX competes efficiently with the classical α-toxin AaH II from Androctonus australis, and displays one of the highest affinities for Nav channels.
Frank Bosmans - One of the best experts on this subject based on the ideXlab platform.
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The scorpion toxin Bot IX is a potent member of the α-like family and has a unique N-Terminal Sequence extension
FEBS Letters, 2016Co-Authors: Marie-france Martin-eauclaire, Juan Salvatierra, Frank Bosmans, Pierre E. BougisAbstract:We report the detailed chemical, immunological and pharmacological characterization of the α-toxin Bot IX from the Moroccan scorpion Buthus occitanus tunetanus venom. Bot IX, which consists of 70 amino acids, is a highly atypical toxin. It carries a unique N-Terminal Sequence extension and is highly lethal in mice. Voltage clamp recordings on oocytes expressing rat Nav1.2 or insect BgNav1 reveal that, similar to other α-like toxins, Bot IX inhibits fast inactivation of both variants. Moreover, Bot IX belongs to the same structural/immunological group as the α-like toxin Bot I. Remarkably, radioiodinated Bot IX competes efficiently with the classical α-toxin AaH II from Androctonus australis, and displays one of the highest affinities for Nav channels.
