Two glutathione transferase isoforms isolated from juvenile cysts ofTaenia crassiceps: identification, purification and characterization

2017 ◽  
Vol 92 (6) ◽  
pp. 687-695 ◽  
Author(s):  
G. Maldonado ◽  
G. Nava ◽  
A. Plancarte
Keyword(s):  
Glutathione Transferase ◽  
Taenia Solium ◽  
Single Step ◽  
Glutathione Transferases ◽  
Mass Spectrometry Analysis ◽  
Purification And Characterization ◽  
Spectrometry Analysis ◽  
Enzyme Subunits ◽  
Molecular Masses ◽  
Potential Use

AbstractWe identified and characterized the first two glutathione transferases (GSTs) isolated from juvenile cysts ofTaenia crassiceps(EC 2.5.1.18). The two glutathione transferases (TcGST1 and TcGST2) were purified in a single-step protocol using glutathione (GSH)-sepharose chromatography in combination with a GSH gradient. The specific activities of TcGST1 and TcGST2 were 26 U mg−1and 19 U mg−1, respectively, both at 25°C and pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) and GSH as substrates. TheKm(CDNB)andKcat(CDNB)values for TcGST1 and TcGST2 (0.86 μmand 62 s−1; 1.03 μmand 1.97 s−1, respectively) andKm(GSH)andKcat(GSH)values for TcGST1 and TcGST2 (0.55 μmand 11.61 s−1; 0.3 μmand 32.3 s−1, respectively) were similar to those reported for mammalian and helminth GSTs. Mass spectrometry analysis showed that eight peptides from each of the two parasite transferases were a match for gi|29825896 glutathione transferase (Taenia solium), confirming that both enzymes are GSTs. The relative molecular masses were 54,000 ± 0.9 for the native enzymes and 27,500 ± 0.5 for the enzyme subunits. Thus, TcGST1 and TcGST2 are dimeric proteins. Optimal TcGST1 and TcGST2 activities were observed at pH 8.5 in the range of 20–55°C and pH 7.5 at 35–40°C, respectively. TcGST1 and TcGST2 were inhibited by cibacron blue (CB), bromosulphophthalein (BST), rose bengal (RB), indomethacin and haematin (Hm) with 50% inhibitory concentrations (IC50) in the μmrange. TcGST1 was inhibited in a non-competitive manner by all tested inhibitors with the exception of indomethacin, which was uncompetitive. The discovery of these new GSTs facilitates the potential use ofT. crassicepsas a model to investigate multifunctional GSTs.

Label-free quantitative proteomic analysis of the YAP/TAZ interactome

2014 ◽  
Vol 306 (9) ◽  
pp. C805-C818 ◽  
Author(s):  
Priyanka Kohli ◽  
Malte P. Bartram ◽  
Sandra Habbig ◽  
Caroline Pahmeyer ◽  
Tobias Lamkemeyer ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Affinity Purification ◽  
Single Copy ◽  
Single Step ◽  
Mass Spectrometry Analysis ◽  
Single Shot ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Spectrometry Analysis

The function of an individual protein is typically defined by protein-protein interactions orchestrating the formation of large complexes critical for a wide variety of biological processes. Over the last decade the analysis of purified protein complexes by mass spectrometry became a key technique to identify protein-protein interactions. We present a fast and straightforward approach for analyses of interacting proteins combining a Flp-in single-copy cellular integration system and single-step affinity purification with single-shot mass spectrometry analysis. We applied this protocol to the analysis of the YAP and TAZ interactome. YAP and TAZ are the downstream effectors of the mammalian Hippo tumor suppressor pathway. Our study provides comprehensive interactomes for both YAP and TAZ and does not only confirm the majority of previously described interactors but, strikingly, revealed uncharacterized interaction partners that affect YAP/TAZ TEAD-dependent transcription. Among these newly identified candidates are Rassf8, thymopoetin, and the transcription factors CCAAT/enhancer-binding protein (C/EBP)β/δ and core-binding factor subunit β (Cbfb). In addition, our data allowed insights into complex stoichiometry and uncovered discrepancies between the YAP and TAZ interactomes. Taken together, the stringent approach presented here could help to significantly sharpen the understanding of protein-protein networks.

scholarly journals The N -linking glycosylation system from Actinobacillus pleuropneumoniae is required for adhesion and has potential use in glycoengineering

Open Biology ◽  
2017 ◽  
Vol 7 (1) ◽  
pp. 160212 ◽  
Author(s):  
Jon Cuccui ◽  
Vanessa S. Terra ◽  
Janine T. Bossé ◽  
Andreas Naegeli ◽  
Sherif Abouelhadid ◽  
...  
Keyword(s):  
Outer Membrane Proteins ◽  
Actinobacillus Pleuropneumoniae ◽  
Mass Spectrometry Analysis ◽  
Respiratory Pathogen ◽  
Transcriptional Unit ◽  
Biotechnological Application ◽  
Spectrometry Analysis ◽  
Porcine Pleuropneumonia ◽  
Repeat Units ◽  
Potential Use

Actinobacillus pleuropneumoniae is a mucosal respiratory pathogen causing contagious porcine pleuropneumonia. Pathogenesis studies have demonstrated a major role for the capsule, exotoxins and outer membrane proteins. Actinobacillus pleuropneumoniae can also glycosylate proteins, using a cytoplasmic N -linked glycosylating enzyme designated NGT, but its transcriptional arrangement and role in virulence remains unknown. We investigated the NGT locus and demonstrated that the putative transcriptional unit consists of rimO , ngt and a glycosyltransferase termed agt. From this information we used the A. pleuropneumoniae glycosylation locus to decorate an acceptor protein, within Escherichia coli, with a hexose polymer that reacted with an anti-dextran antibody. Mass spectrometry analysis of a truncated protein revealed that this operon could add up to 29 repeat units to the appropriate sequon. We demonstrated the importance of NGT in virulence, by creating deletion mutants and testing them in a novel respiratory cell line adhesion model. This study demonstrates the importance of the NGT glycosylation system for pathogenesis and its potential biotechnological application for glycoengineering.

scholarly journals Cloning and purification of functionally active Fas ligand interfering protein (FIP) expressed in Escherichia coli.

2008 ◽  
Vol 55 (1) ◽  
pp. 51-56 ◽  
Author(s):  
Pawel Wisniewski ◽  
Adam Master ◽  
Bozena Kaminska
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Inclusion Bodies ◽  
Structural Changes ◽  
Fas Ligand ◽  
Biological Activities ◽  
Single Step ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Soluble Fas

This report presents purification and characterization of the extracellular domain of rat Fas protein, called FIP (FasL interfering protein), expressed as inclusion bodies in Escherichia coli. FIP was extracted from the inclusion bodies, solubilized with 8 M urea, purified by a single-step immobilized metal ion (Ni(2+)) affinity chromatography and refolded. SDS/PAGE and mass spectrometry analysis of the purified protein verified its purity. Fluorescence spectrum analysis showed that the refolding procedure caused structural changes which presumably might have led to oligomerization. The purified FIP has biological activities: it binds specifically soluble Fas ligand and protects human Jurkat lymphocytes against FasL-dependent apoptosis. This efficient procedure of FIP expression in E. coli and renaturation may be useful for production of therapeutically important proteins.

scholarly journals A scalable workflow to characterize the human exposome

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xin Hu ◽  
Douglas I. Walker ◽  
Yongliang Liang ◽  
Matthew Ryan Smith ◽  
Michael L. Orr ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Mass Spectrometry ◽  
A Priori ◽  
Population Level ◽  
Sample Volume ◽  
Single Step ◽  
Environmental Chemicals ◽  
Mass Spectrometry Analysis ◽  
Environmental Chemical ◽  
Spectrometry Analysis

AbstractComplementing the genome with an understanding of the human exposome is an important challenge for contemporary science and technology. Tens of thousands of chemicals are used in commerce, yet cost for targeted environmental chemical analysis limits surveillance to a few hundred known hazards. To overcome limitations which prevent scaling to thousands of chemicals, we develop a single-step express liquid extraction and gas chromatography high-resolution mass spectrometry analysis to operationalize the human exposome. We show that the workflow supports quantification of environmental chemicals in human plasma (200 µL) and tissue (≤100 mg) samples. The method also provides high resolution, sensitivity and selectivity for exposome epidemiology of mass spectral features without a priori knowledge of chemical identity. The simplicity of the method can facilitate harmonization of environmental biomonitoring between laboratories and enable population level human exposome research with limited sample volume.

Analysis of biofluids by paper spray-MS in forensic toxicology

Bioanalysis ◽  
2020 ◽  
Vol 12 (15) ◽  
pp. 1087-1102
Author(s):  
Ana Luiza Freitas de Assis Linhares ◽  
Mauricio Yonamine
Keyword(s):  
Drugs Of Abuse ◽  
Forensic Toxicology ◽  
Ambient Ionization ◽  
Sample Volume ◽  
Mass Spectrometry Analysis ◽  
Sample Treatment ◽  
Biological Matrices ◽  
Paper Spray ◽  
Spectrometry Analysis ◽  
Potential Use

Direct ambient ionization techniques have been developed with the aim to reduce the complexity of mass spectrometry analysis by minimizing sample preparation and chromatographic separation. In this context, paper spray-MS (PS-MS) is an innovative approach that provides faster and cheaper analysis of biofluids by the addition of the sample directly to a paper. In forensic toxicology, the analytical workflow for the detection and quantification of drugs of abuse is onerous, including sample treatment, extraction and clean up, especially regarding complex biological matrices. PS-MS allows the detection of analytes of toxicological interest in blood, plasma and urine using low sample volume. This review aims to discuss the potential use, advances and challenges of PS-MS in forensic toxicology.

scholarly journals Identification and Characterization of a Novel CprA Reductive Dehalogenase Specific to Highly Chlorinated Phenols from Desulfitobacterium hafniense Strain PCP-1

2010 ◽  
Vol 76 (22) ◽  
pp. 7536-7540 ◽  
Author(s):  
Ariane Bisaillon ◽  
Réjean Beaudet ◽  
François Lépine ◽  
Eric Déziel ◽  
Richard Villemur
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mass Spectrometry Analysis ◽  
Activity Levels ◽  
Chlorinated Phenols ◽  
Quinone Oxidoreductase ◽  
Spectrometry Analysis ◽  
Reductive Dehalogenase ◽  
Molecular Masses ◽  
Desulfitobacterium Hafniense

ABSTRACT Desulfitobacterium hafniense strain PCP-1 reductively dechlorinates pentachlorophenol (PCP) to 3-chlorophenol and a variety of halogenated aromatic compounds at the ortho, meta, and para positions. Several reductive dehalogenases (RDases) are thought to be involved in this cascade of dehalogenation. We partially purified a novel RDase involved in the dechlorination of highly chlorinated phenols from strain PCP-1 cultivated in the presence of 2,4,6-trichlorophenol. The RDase was membrane associated, and the activity was sensitive to oxygen, with a half-life of 128 min upon exposure to air. The pH and temperature optima were 7.0 and 55°C, respectively. Several highly chlorinated phenols were dechlorinated at the ortho positions. The highest dechlorinating activity levels were observed with PCP, 2,3,4,5-tetrachlorophenol, and 2,3,4-trichlorophenol. 3-Chloro-4-hydroxyphenylacetate, 3-chloro-4-hydroxybenzoate, dichlorophenols, and monochlorophenols were not dechlorinated. The apparent Km value for PCP was 46.7 μM at a methyl viologen concentration of 2 mM. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activity, suggesting the involvement of a corrinoid cofactor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partially purified preparation revealed 2 bands with apparent molecular masses of 42 and 47 kDa. Mass spectrometry analysis using Mascot to search the genome sequence of D. hafniense strain DCB-2 identified the 42-kDa band as NADH-quinone oxidoreductase, subunit D, and the 47-kDa band as the putative chlorophenol RDase CprA3. This is the first report of an RDase with high affinity and high dechlorinating activity toward PCP.

scholarly journals Mass spectrometry detection of monomeric renalase in human urine

2012 ◽  
Vol 58 (5) ◽  
pp. 599-607 ◽  
Author(s):  
V.I. Fedchenko ◽  
O.A. Buneeva ◽  
A.T. Kopylov ◽  
A.A. Kaloshin ◽  
L.N. Axenova ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ammonium Sulfate ◽  
Secretory Protein ◽  
Mass Spectrometry Analysis ◽  
Mass Spectrometry Detection ◽  
Ammonium Sulfate Precipitation ◽  
Spectrometry Analysis ◽  
Molecular Masses ◽  
35 Kda Protein ◽  
Regulation Of Blood Pressure

Renalase is a recently discovered secretory protein, which is suggested to play a role (which still remains elusive) in regulation of blood pressure. Earlier it was purified from urine of healthy volunteers by means of ammonium sulfate fractionation and subsequent affinity chromatography (Xu et al. (2005) J. Clin. Invest., 115, 1275). The resultant purified preparation of renalase contained 2 proteins with molecular masses of 35 and 67-75 kDa. The authors believed that the latter represents a dimerization (aggregation) product of the 35 kDa protein. In this study we have detected relanase in urinary samples of 2 of 6 volunteers only after immunoaffinity enrichment of urinary samples subjected to ammonium sulfate precipitation. Electrophoresis of the purified preparation also demonstrated the presence of 2 proteins with molecular masses of 35 and 66 kDa, respectively. Mass spectrometry analysis of these proteins identified 35 and 66 kDa proteins as renalase and serum albumin, respectively. Thus, our results do not support suggestion on formation of renalase dimers and they indicate that urinary renalase excretion significantly varies in humans.

scholarly journals Evaluation of the chemical defense fluids of Macrotermes carbonarius and Globitermes sulphureus as possible household repellents and insecticides

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
S. Appalasamy ◽  
M. H. Alia Diyana ◽  
N. Arumugam ◽  
J. G. Boon
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Periplaneta Americana ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Controlled Environment ◽  
Spectrometry Analysis ◽  
Chromatography Mass Spectrometry ◽  
Potential Use ◽  
Macrotermes Carbonarius

AbstractThe use of chemical insecticides has had many adverse effects. This study reports a novel perspective on the application of insect-based compounds to repel and eradicate other insects in a controlled environment. In this work, defense fluid was shown to be a repellent and insecticide against termites and cockroaches and was analyzed using gas chromatography-mass spectrometry (GC–MS). Globitermes sulphureus extract at 20 mg/ml showed the highest repellency for seven days against Macrotermes gilvus and for thirty days against Periplaneta americana. In terms of toxicity, G. sulphureus extract had a low LC50 compared to M. carbonarius extract against M. gilvus. Gas chromatography–mass spectrometry analysis of the M. carbonarius extract indicated the presence of six insecticidal and two repellent compounds in the extract, whereas the G. sulphureus extract contained five insecticidal and three repellent compounds. The most obvious finding was that G. sulphureus defense fluid had higher potential as a natural repellent and termiticide than the M. carbonarius extract. Both defense fluids can play a role as alternatives in the search for new, sustainable, natural repellents and termiticides. Our results demonstrate the potential use of termite defense fluid for pest management, providing repellent and insecticidal activities comparable to those of other green repellent and termiticidal commercial products.

scholarly journals Purification and Genetic Characterization of Plantaricin NC8, a Novel Coculture-Inducible Two-Peptide Bacteriocin from Lactobacillus plantarum NC8

2003 ◽  
Vol 69 (1) ◽  
pp. 383-389 ◽  
Author(s):  
Antonio Maldonado ◽  
José Luis Ruiz-Barba ◽  
Rufino Jiménez-Díaz
Keyword(s):  
Amino Acids ◽  
Lactobacillus Plantarum ◽  
Sequence Similarity ◽  
Consensus Sequence ◽  
Mass Spectrometry Analysis ◽  
Reading Frame ◽  
Spectrometry Analysis ◽  
Complementary Action ◽  
Molecular Masses ◽  
Plantaricin Nc8

ABSTRACT A new, coculture-inducible two-peptide bacteriocin named plantaricin NC8 (PLNC8) was isolated from Lactobacillus plantarum NC8 cultures which had been induced with Lactococcus lactis MG1363 or Pediococcus pentosaceus FBB63. This bacteriocin consists of two distinct peptides, named α and β, which were separated by C2-C18 reverse-phase chromatography and whose complementary action is necessary for full plantaricin NC8 activity. N-terminal sequencing of both purified peptides showed 28 and 34 amino acids residues for PLNC8α and PLNC8β, respectively, which showed no sequence similarity to other known bacteriocins. Mass spectrometry analysis showed molecular masses of 3,587 Da (α) and 4,000 Da (β). The corresponding genes, designated plNC8A and plNC8B, were sequenced, and their nucleotide sequences revealed that both peptides are produced as bacteriocin precursors of 47 and 55 amino acids, respectively, which include N-terminal leader sequences of the double-glycine type. The mature α and β peptides contain 29 and 34 amino acids, respectively. An open reading frame, orfC, which encodes a putative immunity protein was found downstream of plNC8B and overlapping plNC8A. Upstream of the putative −35 region of plNC8B, two direct repeats of 9 bp were identified, which agrees with the consensus sequence and structure of promoters of class II bacteriocin operons whose expression is dependent on an autoinduction mechanism.

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