scholarly journals Mass spectrometric analysis of rat ovary and testis cytosolic glutathione S-transferases (GSTs): identification of a novel class-Alpha GST, rGSTA6*, in rat testis

1997 ◽  
Vol 323 (2) ◽  
pp. 503-510 ◽  
Author(s):  
Cheng-Hsilin HSIEH ◽  
Shu-Ping TSAI ◽  
Horng-I YEH ◽  
Tsuey-Chyi SHEU ◽  
Ming F. TAM
Keyword(s):  
Peptide Sequencing ◽  
Spectrometric Analysis ◽  
Sequencing Analysis ◽  
Post Translational Modifications ◽  
Rat Testis ◽  
Cytosolic Glutathione ◽  
Glutathione S Transferases ◽  
Molecular Masses ◽  
Rat Ovary ◽  
Internal Peptide

Cytosolic glutathione S-transferases (GSTs) from rat ovaries and testis were purified by a combination of GSH and S-hexylglutathione affinity chromatography. The isolated GSTs were subjected to reverse-phase HPLC, electrospray MS and N-terminal peptide sequencing analysis. The major GST isoenzymes expressed in ovaries are subunits A3, A4, M1, M2 and P1. Other isoenzymes detected are subunits A1, M3 and M6*. In rat testis, the major GST isoenzymes expressed are subunits A3, M1, M2, M3, M5* and M6*. Subunits A1, A4 and P1 are expressed in lesser amounts. We could not detect post-translational modifications of any GSTs with known cDNA sequence. The molecular masses of subunits M5* and M6*, two class-Mu GSTs that have not been cloned, were determined to be 25495 and 26538 Da respectively. An N-terminally modified protein from rat testis with molecular mass 25737 Da was isolated from the S-hexylglutathione column. Results from internal peptide sequencing analysis indicate that this is a novel class-Alpha GST that has not been previously reported. We designate this protein rGSTA6*.

scholarly journals Characterization and cloning of avian-hepatic glutathione S-transferases

1999 ◽  
Vol 343 (1) ◽  
pp. 87-93 ◽  
Author(s):  
Cheng-Hsilin HSIEH ◽  
Li-Fan LIU ◽  
Shu-Ping TSAI ◽  
Ming F. TAM
Keyword(s):  
Amino Acid ◽  
Peptide Sequencing ◽  
Cdna Sequences ◽  
Coding Regions ◽  
Liver Cdna Library ◽  
Cytosolic Glutathione ◽  
Glutathione S Transferases ◽  
Molecular Masses ◽  
Internal Peptide ◽  
Terminal Amino

Cytosolic glutathione S-transferases (GSTs) were isolated from 1-day-old Leghorn chick livers by glutathione (GSH)-affinity chromatography. After sample loading and extensive washing with 0.2 M NaCl, the column was sequentially eluted with 5 mM GSH and 1 mM S-hexylglutathione. The isolated GSTs were subjected to reverse-phase HPLC, electrospray ionization-MS, N-terminal and internal peptide sequencing analyses. The proteins recovered from the 5 mM GSH eluant were predominantly cGSTM1. A protein (cGSTM1′) with an N-terminal amino acid sequence identical to that of cGSTM1 but with the initiator methionine retained and a novel class-mu isozyme (cGSTM2*) were also recovered from this fraction. Nine class-alpha isozymes with distinctive molecular masses were identified from the 1 mM S-hexylglutathione eluant. Three of these proteins are probably variants with minor amino acid substitutions of other isozymes. Of the six remaining class-alpha isozymes, three of them have had their complete (cGSTA1 and cGSTA2) or partial (cGSTA3) cDNA sequences reported previously in the literature. A chicken liver cDNA library was screened with oligonucleotides generated from the cGSTA2 sequence as probes. Clones that encompass the complete coding regions of cGSTA3 and cGSTA4 were obtained. A clone encoding the C-terminal 187 residues of cGSTA5 was also isolated.

scholarly journals Mass spectrometric analysis of rat liver cytosolic glutathione S-transferases: modifications are limited to N-terminal processing

1995 ◽  
Vol 308 (1) ◽  
pp. 69-75 ◽  
Author(s):  
H I Yeh ◽  
C H Hsieh ◽  
L Y Wang ◽  
S P Tsai ◽  
H Y Hsu ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Female Rats ◽  
Spectrometric Analysis ◽  
Affinity Column ◽  
Female Rat ◽  
Significant Difference ◽  
Cytosolic Glutathione ◽  
Glutathione S Transferases ◽  
Molecular Masses ◽  
Rat Livers

Cytosolic glutathione S-transferases (GSTs) from rat livers were purified using an S-hexylglutathione affinity column. The GST subunits were resolved by reverse-phase HPLC and their molecular masses were determined by electrospray mass spectrometry. The major hepatic GSTs detected were subunits 1, 1′, 2, 3 and 4, with molecular mass of 25,520, 25,473, 25,188, 25,782 and 25,571 Da respectively. Subunits 6, 7 and 10 are minor components, with molecular mass of 25,551, 23,308 and 25,211 Da respectively. Alternatively, the hepatic GSTs were purified using a glutathione affinity column. Subunits 1, 1′, 2, 8 and 10 were eluted from this column with GSSG, the oxidized form of glutathione. Subunit 8 has a molecular mass of 25,553 Da. The remaining proteins on the glutathione affinity column were removed with glutathione and S-hexylglutathione. Subunits 2, 3, 4 and 6 could be detected in the eluate. We could not detect any significant difference in molecular mass between GSTs isolated from male and female rat livers. Cytosolic GSTs were isolated from livers of buthionine sulphoximine-treated female rats for MS analysis. The molecular masses obtained were identical to those determined for the controls.

scholarly journals Rat kidney glutathione S-transferase 1 subunits have C-terminal truncations

1996 ◽  
Vol 314 (3) ◽  
pp. 1017-1025 ◽  
Author(s):  
Horng-I. YEH ◽  
Jing-Yu LEE ◽  
Shu-Ping TSAI ◽  
Cheng-Hsilin HSIEH ◽  
Ming F. TAM
Keyword(s):  
Rat Liver ◽  
Peptide Mapping ◽  
Rat Kidney ◽  
Cdna Sequencing ◽  
Sequencing Data ◽  
Glutathione S Transferase ◽  
Reverse Phase Hplc ◽  
Cytosolic Glutathione ◽  
Glutathione S Transferases ◽  
Molecular Masses

Cytosolic glutathione S-transferases (GSTs) from rat kidneys were purified by a combination of glutathione and S-hexylglutathione affinity columns. The isolated GSTs were subjected to reverse-phase HPLC and electrospray MS analysis. The major GST isoenzymes expressed in kidney are subunits 1, 2, 7 and 8. GST 1´, 3, and 4 are expressed in minor amounts. GST 10 is barely detectable in the male kidney cytosol. The molecular masses of these rat kidney GST subunits were determined by MS. The values obtained for subunits 1´, 2, 3, 4, 7, 8 and 10 are identical with those obtained for rat liver GSTs. Rat kidney GST 1 consists of three polypeptides, with molecular masses of 25517, 25372 and 24982 Da. Results from peptide mapping, MS and amino-acid-sequencing analyses indicate that the major components were generated by deleting the C-terminal phenylalanine (24982 Da) and the C-terminal IFKF tetrapeptide (25372 Da) from the GST 1 subunit, respectively. The 1-chloro-2,4-dinitrobenzene-conjugating and peroxidase activities of kidney GST 1 are substantially lower than for its counterpart from rat liver. In addition, rat kidney GST 1 has an arginine and a valine residue at positions 151 and 207 respectively. The results are in contradiction with the SWISS-PROT and GenBank rat liver GST 1 cDNA-sequencing data, which give a lysine and a methionine at the corresponding positions. Further analyses indicate that rat liver GST 1 also has C-terminal phenylalanine deletion, and an arginine and a valine residue at positions 151 and 207 respectively. However, the C-terminal-tetrapeptide-deleted form was not observed for rat liver GST 1.

Purification and characterization of a glutathione S-transferase Omega in pig: evidence for two distinct organ-specific transcripts

2001 ◽  
Vol 358 (1) ◽  
pp. 257-262 ◽  
Author(s):  
Patrick ROUIMI ◽  
Patricia ANGLADE ◽  
Anne BENZEKRI ◽  
Philippe COSTET ◽  
Laurent DEBRAUWER ◽  
...  
Keyword(s):  
Peptide Sequencing ◽  
Cross Reactivity ◽  
Glutathione S Transferase ◽  
Gene Encoding ◽  
Simple Method ◽  
Organ Specific ◽  
Cytosolic Glutathione ◽  
A Subunit ◽  
Internal Peptide

A cytosolic glutathione S-transferase (GST, EC 2.5.1.18) from the recently characterized Omega class [GSTO; Board et al. 2000, J. Biol. Chem. 275, 24798–24806] has been identified in pig organs. It was found widely distributed in the different tissues investigated and especially abundant in liver and muscle. The hepatic enzyme has been purified to homogeneity by using its selective affinity for S-hexylglutathione over GSH, thus providing a simple method to isolate mammalian GSTO. The dimeric protein has a subunit molecular mass of 27328Da as measured by electrospray ionization MS. Internal peptide sequencing and complete cDNA sequencing revealed strong similarities with its human recombinant orthologue and two rodent GST-like proteins with the ability to catalyse the GSH-dependent reduction of dehydroascorbate. Additional similarities, including the presence of a specific N-terminal extension and of immunological cross-reactivity, support the results. Moreover, this gene encoding GSTO generates two organ-specific transcripts, suggesting transcriptional mechanisms with a significance that is as yet uncharacterized.

scholarly journals Novel functional association of rat testicular membrane-associated cytosolic glutathione S transferases and cyclooxygenase in vitro

2005 ◽  
Vol 7 (2) ◽  
pp. 171-178 ◽  
Author(s):  
S. Neeraja ◽  
B. Ramakrishna ◽  
A. S. Sreenath ◽  
G. V. Reddy ◽  
P. R. K. Reddy ◽  
...  
Keyword(s):  
Functional Association ◽  
Cytosolic Glutathione ◽  
Glutathione S Transferases

Cytosolic glutathione S-transferases of Oesophagostomum dentatum

Parasitology ◽  
2008 ◽  
Vol 135 (10) ◽  
pp. 1215-1223 ◽  
Author(s):  
A. JOACHIM ◽  
B. RUTTKOWSKI
Keyword(s):  
Amino Acids ◽  
Pcr Primers ◽  
Mrna Levels ◽  
Fourth Stage ◽  
Glutathione S Transferase ◽  
Cdna Sequences ◽  
Oesophagostomum Dentatum ◽  
Cytosolic Glutathione ◽  
Glutathione S Transferases

SUMMARYOesophagostomum dentatum stages were investigated for glutathione S-transferase (GST) expression at the protein and mRNA levels. GST activity was detected in all stages (infectious and parasitic stages including third- and fourth-stage larvae of different ages as well as males and females) and could be dose-dependently inhibited with sulfobromophthalein (SBP). Addition of SBP to in vitro larval cultures reversibly inhibited development from third- to fourth-stage larvae. Two glutathione-affinity purified proteins (23 and 25 kDa) were detected in lysates of exsheathed third-stage larvae by SDS-PAGE. PCR-primers were designed based on peptide sequences and conserved GST sequences of other nematodes for complete cDNA sequences (621 and 624 nt) of 2 isoforms, Od-GST1 and Od-GST2, with 72% nucleotide similarity and 75% for the deduced proteins. Genomic sequences consisted of 7 exons and 6 introns spanning 1296 bp for Od-GST1 and 1579 and 1606 bp for Od-GST2. Quantitative real-time-PCR revealed considerably elevated levels of Od-GST1 in the early parasitic stages and slightly reduced levels of Od-GST2 in male worms. Both Od-GSTs were most similar to GST of Ancylostoma caninum (nucleotides: 73 and 70%; amino acids: 80 and 73%). The first three exons (75 amino acids) corresponded to a synthetic prostaglandin D2 synthase (53% similarity). O. dentatum GSTs might be involved in intrinsic metabolic pathways which could play a role both in nematode physiology and in host-parasite interactions.

scholarly journals Triatoma infestansApyrases Belong to the 5′-Nucleotidase Family

2004 ◽  
Vol 279 (19) ◽  
pp. 19607-19613 ◽  
Author(s):  
Eric Faudry ◽  
Silene P. Lozzi ◽  
Jaime M. Santana ◽  
Marian D'Souza-Ault ◽  
Sylvie Kieffer ◽  
...  
Keyword(s):  
Blood Platelets ◽  
Gel Filtration ◽  
Peptide Sequencing ◽  
Triatoma Infestans ◽  
Nucleoside Triphosphate ◽  
Blood Sucking ◽  
Tandem Mass Spectroscopy ◽  
Molecular Masses ◽  
Sequence Similarities ◽  
Human Blood Platelets

Apyrases are nucleoside triphosphate-diphosphohydrolases (EC 3.6.1.5) present in a variety of organisms. The apyrase activity found in the saliva of hematophagous insects is correlated with the prevention of ADP-induced platelet aggregation of the host during blood sucking. Purification of apyrase activity from the saliva of the triatomine bugTriatoma infestanswas achieved by affinity chromatography on oligo(dT)-cellulose and gel filtration chromatography. The isolated fraction includes fiveN-glycosylated polypeptides of 88, 82, 79, 68 and 67 kDa apparent molecular masses. The isolated apyrase mixture completely inhibited aggregation of human blood platelets. Labeling with the ATP substrate analogue 5′-p-fluorosulfonylbenzoyladenosine showed that the five species have ATP-binding characteristic of functional apyrases. Furthermore, tandem mass spectroscopy peptide sequencing showed that the five species share sequence similarities with the apyrase fromAedes aegyptiand with 5′-nucleotidases from other species. The complete cDNA of the 79-kDa enzyme was cloned, and its sequence confirmed that it encodes for an apyrase belonging to the 5′-nucleotidase family. The gene multiplication leading to the unusual salivary apyrase diversity inT. infestanscould represent an important mechanism amplifying the enzyme expression during the insect evolution to hematophagy, in addition to an escape from the host immune response, thus enhancing acquisition of a meal by this triatomine vector of Chagas' disease.

scholarly journals Involvement of Sialoadhesin in Entry of Porcine Reproductive and Respiratory Syndrome Virus into Porcine Alveolar Macrophages

2003 ◽  
Vol 77 (15) ◽  
pp. 8207-8215 ◽  
Author(s):  
Nathalie Vanderheijden ◽  
Peter L. Delputte ◽  
Herman W. Favoreel ◽  
Joël Vandekerckhove ◽  
Jozef Van Damme ◽  
...  
Keyword(s):  
Amino Acid ◽  
Viral Entry ◽  
Alveolar Macrophages ◽  
Amino Acid Identity ◽  
Peptide Sequencing ◽  
Respiratory Syndrome Virus ◽  
Vesicle Membrane ◽  
Virus Particles ◽  
Internal Peptide ◽  
Peptide Data

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) shows a very restricted tropism for cells of the monocyte/macrophage lineage. It enters cells via receptor-mediated endocytosis. A monoclonal antibody (MAb) that is able to block PRRSV infection of porcine alveolar macrophages (PAM) and that recognizes a 210-kDa protein (p210) was described previously (MAb41D3) (X. Duan, H. Nauwynck, H. Favoreel, and M. Pensaert, J. Virol. 72:4520-4523, 1998). In the present study, the p210 protein was purified from PAM by immunoaffinity using MAb41D3 and was subjected to internal peptide sequencing after tryptic digestion. Amino acid sequence identities ranging from 56 to 91% with mouse sialoadhesin, a macrophage-restricted receptor, were obtained with four p210 peptides. Using these peptide data, the full p210 cDNA sequence (5,193 bp) was subsequently determined. It shared 69 and 78% amino acid identity, respectively, with mouse and human sialoadhesins. Swine (PK-15) cells resistant to viral entry were transfected with the cloned p210 cDNA and inoculated with European or American PRRSV strains. Internalized virus particles were detected only in PK-15 cells expressing the recombinant sialoadhesin, demonstrating that this glycoprotein mediated uptake of both types of strains. However, nucleocapsid disintegration, like that observed in infected Marc-145 cells as a result of virus uncoating after fusion of the virus with the endocytic vesicle membrane, was not observed, suggesting a block in the fusion process. The ability of porcine sialoadhesin to mediate endocytosis was demonstrated by specific internalization of MAb41D3 into PAM. Altogether, these results show that sialoadhesin is involved in the entry process of PRRSV in PAM.

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