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Sample records for nucleotidyl transferase subunits

  1. A single RNA-dependent RNA polymerase assembles with mutually exclusive nucleotidyl transferase subunits to direct different pathways of small RNA biogenesis.

    PubMed

    Lee, Suzanne Rebecca; Talsky, Kristin Benjamin; Collins, Kathleen

    2009-07-01

    Members of the conserved family of eukaryotic RNA-dependent RNA polymerases (Rdrs) synthesize double-stranded RNA (dsRNA) intermediates in diverse pathways of small RNA (sRNA) biogenesis and RNA-mediated silencing. Rdr-dependent pathways of sRNA production are poorly characterized relative to Rdr-independent pathways, and the Rdr enzymes themselves are poorly characterized relative to their viral RNA-dependent RNA polymerase counterparts. We previously described a physical and functional coupling of the Tetrahymena thermophila Rdr, Rdr1, and a Dicer enzyme, Dcr2, in the production of approximately 24-nucleotide (nt) sRNA in vitro. Here we characterize the endogenous complexes that harbor Rdr1, termed RDRCs. Distinct RDRCs assemble to contain Rdr1 and subsets of the total of four tightly Rdr1-associated proteins. Of particular interest are two RDRC subunits, Rdn1 and Rdn2, which possess noncanonical ribonucleotidyl transferase motifs. We show that the two Rdn proteins are uridine-specific polymerases of separate RDRCs. Two additional RDRC subunits, Rdf1 and Rdf2, are present only in RDRCs containing Rdn1. Rdr1 catalytic activity is retained in RDRCs purified from cell extracts lacking any of the nonessential RDRC subunits (Rdn2, Rdf1, Rdf2) or if the RDRC harbors a catalytically inactive Rdn. However, specific disruption of each RDRC imposes distinct loss-of-function consequences at the cellular level and has a differential impact on the accumulation of specific 23-24-nt sRNA sequences in vivo. The biochemical and biological phenotypes of RDRC subunit disruption reveal a previously unanticipated complexity of Rdr-dependent sRNA biogenesis in vivo. PMID:19451546

  2. Nucleotidyl transferase assisted DNA labeling with different click chemistries

    PubMed Central

    Winz, Marie-Luise; Linder, Eva Christina; André, Timon; Becker, Juliane; Jäschke, Andres

    2015-01-01

    Here, we present a simple, modular and efficient strategy that allows the 3′-terminal labeling of DNA, regardless of whether it has been chemically or enzymatically synthesized or isolated from natural sources. We first incorporate a range of modified nucleotides at the 3′-terminus, using terminal deoxynucleotidyl transferase. In the second step, we convert the incorporated nucleotides, using either of four highly efficient click chemistry-type reactions, namely copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, Staudinger ligation or Diels-Alder reaction with inverse electron demand. Moreover, we create internal modifications, making use of either ligation or primer extension, after the nucleotidyl transferase step, prior to the click reaction. We further study the influence of linker variants on the reactivity of azides in different click reactions. We find that different click reactions exhibit distinct substrate preferences, a fact that is often overlooked, but should be considered when labeling oligonucleotides or other biomolecules with click chemistry. Finally, our findings allowed us to extend our previously published RNA labeling strategy to the use of a different copper-free click chemistry, namely the Staudinger ligation. PMID:26013812

  3. Distinct and cooperative activities of HESO1 and URT1 nucleotidyl transferases in microRNA turnover in Arabidopsis.

    PubMed

    Tu, Bin; Liu, Li; Xu, Chi; Zhai, Jixian; Li, Shengben; Lopez, Miguel A; Zhao, Yuanyuan; Yu, Yu; Ramachandran, Vanitharani; Ren, Guodong; Yu, Bin; Li, Shigui; Meyers, Blake C; Mo, Beixin; Chen, Xuemei

    2015-04-01

    3' uridylation is increasingly recognized as a conserved RNA modification process associated with RNA turnover in eukaryotes. 2'-O-methylation on the 3' terminal ribose protects micro(mi)RNAs from 3' truncation and 3' uridylation in Arabidopsis. Previously, we identified HESO1 as the nucleotidyl transferase that uridylates most unmethylated miRNAs in vivo, but substantial 3' tailing of miRNAs still remains in heso1 loss-of-function mutants. In this study, we found that among nine other potential nucleotidyl transferases, UTP:RNA uridylyltransferase 1 (URT1) is the single most predominant nucleotidyl transferase that tails miRNAs. URT1 and HESO1 prefer substrates with different 3' end nucleotides in vitro and act cooperatively to tail different forms of the same miRNAs in vivo. Moreover, both HESO1 and URT1 exhibit nucleotidyl transferase activity on AGO1-bound miRNAs. Although these enzymes are able to add long tails to AGO1-bound miRNAs, the tailed miRNAs remain associated with AGO1. Moreover, tailing of AGO1-bound miRNA165/6 drastically reduces the slicing activity of AGO1-miR165/6, suggesting that tailing reduces miRNA activity. However, monouridylation of miR171a by URT1 endows the miRNA the ability to trigger the biogenesis of secondary siRNAs. Therefore, 3' tailing could affect the activities of miRNAs in addition to leading to miRNA degradation. PMID:25928405

  4. Synergistic and independent actions of multiple terminal nucleotidyl transferases in the 3' tailing of small RNAs in Arabidopsis.

    PubMed

    Wang, Xiaoyan; Zhang, Shuxin; Dou, Yongchao; Zhang, Chi; Chen, Xuemei; Yu, Bin; Ren, Guodong

    2015-04-01

    All types of small RNAs in plants, piwi-interacting RNAs (piRNAs) in animals and a subset of siRNAs in Drosophila and C. elegans are subject to HEN1 mediated 3' terminal 2'-O-methylation. This modification plays a pivotal role in protecting small RNAs from 3' uridylation, trimming and degradation. In Arabidopsis, HESO1 is a major enzyme that uridylates small RNAs to trigger their degradation. However, U-tail is still present in null hen1 heso1 mutants, suggesting the existence of (an) enzymatic activities redundant with HESO1. Here, we report that UTP: RNA uridylyltransferase (URT1) is a functional paralog of HESO1. URT1 interacts with AGO1 and plays a predominant role in miRNA uridylation when HESO1 is absent. Uridylation of miRNA is globally abolished in a hen1 heso1 urt1 triple mutant, accompanied by an extensive increase of 3'-to-5' trimming. In contrast, disruption of URT1 appears not to affect the heterochromatic siRNA uridylation. This indicates the involvement of additional nucleotidyl transferases in the siRNA pathway. Analysis of miRNA tailings in the hen1 heso1 urt1 triple mutant also reveals the existence of previously unknown enzymatic activities that can add non-uridine nucleotides. Importantly, we show HESO1 may also act redundantly with URT1 in miRNA uridylation when HEN1 is fully competent. Taken together, our data not only reveal a synergistic action of HESO1 and URT1 in the 3' uridylation of miRNAs, but also independent activities of multiple terminal nucleotidyl transferases in the 3' tailing of small RNAs and an antagonistic relationship between uridylation and trimming. Our results may provide further insight into the mechanisms of small RNA 3' end modification and stability control. PMID:25928341

  5. Genomic cloning and characterization of the rat glutathione S-transferase-A3-subunit gene.

    PubMed

    Fotouhi-Ardakani, N; Batist, G

    1999-05-01

    The rat glutathione S-transferase-A3-subunit (GSTA3) gene is a member of the class Alpha GSTs, which we have previously reported to be overexpressed in anti-cancer-drug-resistant cells. In this study, we report the isolation and characterization of the entire rat GSTA3 (rGST Yc1) subunit gene. The rat GSTA3 subunit gene is approximately 15 kb in length and consists of seven exons interrupted by introns of different lengths. Exon 1, with a length of 219 bp, contains only the 5'-untranslated region of the gene. Each exon-intron splicing junction exhibited the consensus sequence for a mammalian splice site. The transcription start site and exon 1 of rat GSTA3 were characterized by a combination of primer extension and rapid amplification of the cDNA ends. Position +1 was identified 219 bp upstream of the first exon-intron splicing junction. The proximal promoter region of the rat GSTA3 subunit gene does not contain typical TATA or CAAT boxes. A computer-based search for potential transcription-factor binding sites revealed the existence of a number of motifs such as anti-oxidant-responsive element, ras-response element, activator protein-1, nuclear factor-kappaB, cAMP-response-element-binding protein, Barbie box and E box. The functional activity of the regulatory region of the rat GSTA3 subunit gene was shown by its ability to drive the expression of a chloramphenicol acetyltransferase reporter gene in rat mammary carcinoma cells, and its activity was greater in melphalan-resistant cells known to have transcriptional activation of this gene by previous studies. The structure of the gene, with a large intron upstream of the translation-initiation site, may explain why the isolation of this promoter has been so elusive. This information will provide the opportunity to examine the involvement of the rat GSTA3 subunit gene in drug resistance and carcinogenesis. PMID:10215608

  6. Genomic cloning and characterization of the rat glutathione S-transferase-A3-subunit gene.

    PubMed Central

    Fotouhi-Ardakani, N; Batist, G

    1999-01-01

    The rat glutathione S-transferase-A3-subunit (GSTA3) gene is a member of the class Alpha GSTs, which we have previously reported to be overexpressed in anti-cancer-drug-resistant cells. In this study, we report the isolation and characterization of the entire rat GSTA3 (rGST Yc1) subunit gene. The rat GSTA3 subunit gene is approximately 15 kb in length and consists of seven exons interrupted by introns of different lengths. Exon 1, with a length of 219 bp, contains only the 5'-untranslated region of the gene. Each exon-intron splicing junction exhibited the consensus sequence for a mammalian splice site. The transcription start site and exon 1 of rat GSTA3 were characterized by a combination of primer extension and rapid amplification of the cDNA ends. Position +1 was identified 219 bp upstream of the first exon-intron splicing junction. The proximal promoter region of the rat GSTA3 subunit gene does not contain typical TATA or CAAT boxes. A computer-based search for potential transcription-factor binding sites revealed the existence of a number of motifs such as anti-oxidant-responsive element, ras-response element, activator protein-1, nuclear factor-kappaB, cAMP-response-element-binding protein, Barbie box and E box. The functional activity of the regulatory region of the rat GSTA3 subunit gene was shown by its ability to drive the expression of a chloramphenicol acetyltransferase reporter gene in rat mammary carcinoma cells, and its activity was greater in melphalan-resistant cells known to have transcriptional activation of this gene by previous studies. The structure of the gene, with a large intron upstream of the translation-initiation site, may explain why the isolation of this promoter has been so elusive. This information will provide the opportunity to examine the involvement of the rat GSTA3 subunit gene in drug resistance and carcinogenesis. PMID:10215608

  7. X-ray Crystal Structures Elucidate the Nucleotidyl Transfer Reaction of Transcript Initiation Using Two Nucleotides

    SciTech Connect

    M Gleghorn; E Davydova; R Basu; L Rothman-Denes; K Murakami

    2011-12-31

    We have determined the X-ray crystal structures of the pre- and postcatalytic forms of the initiation complex of bacteriophage N4 RNA polymerase that provide the complete set of atomic images depicting the process of transcript initiation by a single-subunit RNA polymerase. As observed during T7 RNA polymerase transcript elongation, substrate loading for the initiation process also drives a conformational change of the O helix, but only the correct base pairing between the +2 substrate and DNA base is able to complete the O-helix conformational transition. Substrate binding also facilitates catalytic metal binding that leads to alignment of the reactive groups of substrates for the nucleotidyl transfer reaction. Although all nucleic acid polymerases use two divalent metals for catalysis, they differ in the requirements and the timing of binding of each metal. In the case of bacteriophage RNA polymerase, we propose that catalytic metal binding is the last step before the nucleotidyl transfer reaction.

  8. Rab geranylgeranyl transferase β subunit is essential for male fertility and tip growth in Arabidopsis

    PubMed Central

    Gutkowska, Malgorzata; Wnuk, Marta; Nowakowska, Julita; Lichocka, Malgorzata; Stronkowski, Michal M.; Swiezewska, Ewa

    2015-01-01

    Rab proteins, key players in vesicular transport in all eukaryotic cells, are post-translationally modified by lipid moieties. Two geranylgeranyl groups are attached to the Rab protein by the heterodimeric enzyme Rab geranylgeranyl transferase (RGT) αβ. Partial impairment in this enzyme activity in Arabidopsis, by disruption of the AtRGTB1 gene, is known to influence plant stature and disturb gravitropic and light responses. Here it is shown that mutations in each of the RGTB genes cause a tip growth defect, visible as root hair and pollen tube deformations. Moreover, FM 1–43 styryl dye endocytosis and recycling are affected in the mutant root hairs. Finally, it is demonstrated that the double mutant, with both AtRGTB genes disrupted, is non-viable due to absolute male sterility. Doubly mutated pollen is shrunken, has an abnormal exine structure, and shows strong disorganization of internal membranes, particularly of the endoplasmic reticulum system. PMID:25316062

  9. Solution Structure of Alg13: The Sugar Donor Subunit of a Yeast N-Acetylglucosamine Transferase

    PubMed Central

    Wang, Xu; Weldeghorghis, Thomas; Zhang, Guofeng; Imperiali, Barbara; Prestegard, James H.

    2008-01-01

    Summary The solution structure of Alg13, the glycosyl-donor binding domain of an important bipartite glycosyltransferase in the yeast S. cerevisiae, is presented. This glycosyl transferase is unusual in that it is only active in the presence of a binding partner, Alg14. Alg13 is found to adopt a unique topology amongst glycosyltransferases. Rather than the conventional Rossmann fold found in all GT-B type enzymes, the N-terminal half of the protein is a Rossmann-like fold with a mixed parallel and anti-parallel β sheet. The Rossmann fold of the C-terminal half of Alg13 is conserved. However, while conventional GT-B type enzymes usually possess three helices at the C-terminus, only two helices are present in Alg13. Titration of Alg13 with both UDP-GlcNAc, the native glycosyl donor, and a paramagnetic mimic, UDP-TEMPO, shows that the interaction of Alg13 with the sugar donor is primarily through the residues in the C-terminal half of the protein. PMID:18547528

  10. Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site.

    PubMed

    Wongsantichon, Jantana; Robinson, Robert C; Ketterman, Albert J

    2015-01-01

    Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance. We report a new Epsilon class protein crystal structure from Drosophila melanogaster for the glutathione transferase DmGSTE6. The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order. This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme. PMID:26487708

  11. Epsilon glutathione transferases possess a unique class-conserved subunit interface motif that directly interacts with glutathione in the active site

    PubMed Central

    Wongsantichon, Jantana; Robinson, Robert C.; Ketterman, Albert J.

    2015-01-01

    Epsilon class glutathione transferases (GSTs) have been shown to contribute significantly to insecticide resistance. We report a new Epsilon class protein crystal structure from Drosophila melanogaster for the glutathione transferase DmGSTE6. The structure reveals a novel Epsilon clasp motif that is conserved across hundreds of millions of years of evolution of the insect Diptera order. This histidine-serine motif lies in the subunit interface and appears to contribute to quaternary stability as well as directly connecting the two glutathiones in the active sites of this dimeric enzyme. PMID:26487708

  12. Loss of the oligosaccharyl transferase subunit TUSC3 promotes proliferation and migration of ovarian cancer cells.

    PubMed

    Vaňhara, Petr; Horak, Peter; Pils, Dietmar; Anees, Mariam; Petz, Michaela; Gregor, Wolfgang; Zeillinger, Robert; Krainer, Michael

    2013-04-01

    Consequences of deregulated protein N-glycosylation on cancer pathogenesis are poorly understood. TUSC3 is a gene with a putative function in N-glycosylation, located on the short arm of chromosome 8. This is a chromosomal region of frequent genetic loss in ovarian cancer. We established recently that the expression of TUSC3 is epigenetically decreased in epithelial ovarian cancer compared to benign controls and provides prognostic information on patient survival. Therefore, we analyzed the consequences of silenced TUSC3 expression on proliferation, invasion and migration of ovarian cell lines. In addition, we performed subcellular fractionation, co-immunofluorescence and co-immunoprecipitation experiments to establish the molecular localization of TUSC3 in ovarian cancer cells. We demonstrated that TUSC3 is localized in the endoplasmic reticulum as a subunit of the oligosaccharyltransferase complex and is capable of modulation of glycosylation patterning of ovarian cancer cells. Most importantly, silencing of TUSC3 enhances proliferation and migration of ovarian cancer cells in vitro. Our observations suggest a role for N-glycosylating events in ovarian cancer pathogenesis in general, and identify TUSC3 as a tumor suppressor gene in ovarian cancer in particular. PMID:23404293

  13. Subunit diversity and tissue distribution of human glutathione S-transferases: interpretations based on electrospray ionization-MS and peptide sequence-specific antisera.

    PubMed Central

    Rowe, J D; Nieves, E; Listowsky, I

    1997-01-01

    Uncertainties about the composition and identities of glutathione S-transferases (GSTs) in human tissue have impeded studies on their biological functions. A rigorous protocol has therefore been developed to characterize the human proteins. Cytosolic GST subunits were resolved by reverse-phase HPLC methods, individual components were assigned to Alpha, Mu and Pi classes on the basis of their immunoreactivities, and peptide-sequence-specific antisera were used to distinguish among five different Mu-class subunits (GSTM1-GSTM5). Each subunit type was characterized and identified unambiguously by electrospray ionization-MS. Acetylation of N-terminal residues in the GSTA1, GSTA2, GSTM3 and GSTM4 subunits were the only natural post-translational modifications detected. The unique structure of GSTM3, with N- and C-terminal peptide extensions predicted from cDNA sequences, was confirmed. Only testis and brain were rich sources of GSTM3 subunits. Subunit profiles were distinct and characteristic of the particular tissue type, and this tissue specificity in GST expression was evident even in organs from different individuals. For instance, livers had relatively simple GST compositions, consisting of a preponderance of Alpha-class subunits and GSTM1 (when present). By contrast, representation of most subunit types was a characteristic feature of testis, which had the highest levels of GSTs. GSTM4 and GSTM5 subunits, here identified for the first time in human tissue extracts, were minor components, with GSTM5 found only in brain, lung and testis. Specimens devoid of GSTM1 subunits, particularly those from null-genotype individuals, were readily discerned at the protein level. Liver was the only rich source of the GSTM1 subunit (although it also constituted a major fraction of adrenal GSTs), and so the functional consequences of the GSTM1 gene deletion are likely to vary in extrahepatic tissues. PMID:9230131

  14. Regulation of glutathione S-transferase Ya subunit gene expression: identification of a unique xenobiotic-responsive element controlling inducible expression by planar aromatic compounds.

    PubMed Central

    Rushmore, T H; King, R G; Paulson, K E; Pickett, C B

    1990-01-01

    We have identified a region in the 5' flanking sequence of the glutathione S-transferase (RX:glutathione R-transferase, EC 2.5.1.18) Ya subunit gene that contains a unique xenobiotic-responsive element (XRE). The regulatory region spans nucleotides -722 to -682 of the 5' flanking sequence and is responsible for part of the basal level as well as inducible expression of the Ya subunit gene by planar aromatic compounds such as beta-naphthoflavone (beta-NF) and 3-methyl-cholanthrene. The DNA sequence of this region (beta-NF-responsive element) is distinct from the DNA sequence of the XRE found in the cytochrome P-450 IA1 gene. In addition to the region containing the beta-NF-responsive element, two other regulatory regions of the Ya subunit gene have been identified. One region spans nucleotides -867 to -857 and has a DNA sequence with identity to the hepatocyte nuclear factor 1 recognition motif found in several liver-specific genes. The second region spans nucleotides -908 to -899 and contains a DNA sequence with identity to the XRE found in the cytochrome P-450 IA1 gene. The XRE sequence also contributes to part of the responsiveness of the Ya subunit gene to planar aromatic compounds. Our data suggest that regulation of gene expression by planar aromatic compounds can be mediated by a DNA sequence that is distinct from the XRE sequence. Images PMID:2160079

  15. Structural basis of substrate specificity of human oligosaccharyl transferase subunit N33/Tusc3 and its role in regulating protein N-glycosylation.

    PubMed

    Mohorko, Elisabeth; Owen, Robin L; Malojčić, Goran; Brozzo, Maurice S; Aebi, Markus; Glockshuber, Rudi

    2014-04-01

    N-linked glycosylation of proteins in the endoplasmic reticulum (ER) is essential in eukaryotes and catalyzed by oligosaccharyl transferase (OST). Human OST is a hetero-oligomer of seven subunits. The subunit N33/Tusc3 is a tumor suppressor candidate, and defects in the subunit N33/Tusc3 are linked with nonsyndromic mental retardation. Here, we show that N33/Tusc3 possesses a membrane-anchored N-terminal thioredoxin domain located in the ER lumen that may form transient mixed disulfide complexes with OST substrates. X-ray structures of complexes between N33/Tusc3 and two different peptides as model substrates reveal a defined peptide-binding groove adjacent to the active site that can accommodate peptides in opposite orientations. Structural and biochemical data show that N33/Tusc3 prefers peptides bearing a hydrophobic residue two residues away from the cysteine forming the mixed disulfide with N33/Tusc3. Our results support a model in which N33/Tusc3 increases glycosylation efficiency for a subset of human glycoproteins by slowing glycoprotein folding. PMID:24685145

  16. Evidence that glutathione S-transferases B1B1 and B2B2 are the products of separate genes and that their expression in human liver is subject to inter-individual variation. Molecular relationships between the B1 and B2 subunits and other Alpha class glutathione S-transferases.

    PubMed Central

    Hayes, J D; Kerr, L A; Cronshaw, A D

    1989-01-01

    The Alpha class glutathione S-transferases (GSTs) in human liver are composed of polypeptides of Mr 25,900. These enzymes are dimeric, and two immunochemically distinct subunits, B1 and B2, have been described that combine to form GSTs B1B1, B1B2 and B2B2 [Stockman, Beckett & Hayes (1985) Biochem. J. 227, 457-465]. Gradient affinity elution from GSH-Sepharose has been used to resolve the three Alpha class GSTs, and this method has been applied to demonstrate marked inter-individual differences in the hepatic content of GSTs B1B1, B1B2 and B2B2. The B1 and B2 subunits can be resolved by reverse-phase h.p.l.c., and their elution positions suggest that they are equivalent to the alpha chi and alpha y h.p.l.c. peaks described by Ketterer and his colleagues [Ostlund Farrants, Meyer, Coles, Southan, Aitken, Johnson & Ketterer (1987) Biochem. J. 245, 423-428]. The B1 and B2 subunits have now been cleaved with CNBr and the fragments subjected to automated amino acid sequence analysis. The sequence data show that B1 and B2 subunits do not arise from post-translational modification, as had been previously believed for the hepatic Alpha class GSTs, but are instead the products of separate genes; B1 and B2 subunits were found to contain different amino acid residues at positions 88, 110, 111, 112, 116, 124 and 127. The relationship between the B1 and B2 subunits and the cloned GTH1 and GTH2 cDNA sequences [Rhoads, Zarlengo & Tu (1987) Biochem. Biophys. Res. Commun. 145, 474-481] is discussed. PMID:2604726

  17. Molecular cloning and heterologous expression of a cDNA encoding a mouse glutathione S-transferase Yc subunit possessing high catalytic activity for aflatoxin B1-8,9-epoxide.

    PubMed Central

    Hayes, J D; Judah, D J; Neal, G E; Nguyen, T

    1992-01-01

    Resistance to the carcinogenic effects of aflatoxin B1 (AFB1) in the mouse is due to the constitutive expression of an Alpha-class glutathione S-transferase (GST), YcYc, with high detoxification activity towards AFB1-8,9-epoxide. A cDNA clone (pmusGST Yc) for a murine GST Yc polypeptide has been isolated. Sequencing has shown the cDNA insert of pmusGST Yc to be 922 bp in length, with an open reading frame of 663 bp that encodes a polypeptide of M(r) 25358. The primary structure of the murine GST Yc subunit predicted by pmusGST Yc is in complete agreement with the partial amino acid sequence of the aflatoxin-metabolizing mouse liver GST described previously [McLellan, Kerr, Cronshaw & Hayes (1991) Biochem. J. 276, 461-469]. A plasmid, termed pKK-musGST Yc, which permits the expression of the murine Yc subunit in Escherichia coli, has been constructed. The murine GST expressed in E. coli was purified and found to be catalytically active towards several GST substrates, including AFB1-8,9-epoxide. This enzyme was also found to possess electrophoretic and immunochemical properties closely similar to those of the GST Yc subunit from mouse liver. However, the GST synthesized in E. coli and the constitutive mouse liver Alpha-class GST exhibited small differences in their chromatographic behaviour during reverse-phase h.p.l.c. Automated Edman degradation revealed alanine to be the N-terminal amino acid in the GST Yc subunit expressed in E. coli, whereas the enzyme in mouse liver possesses a blocked N-terminus. Although sequencing showed that the purified Yc subunit from E. coli lacked the initiator methionine, the amino acid sequence obtained over the first eleven N-terminal residues agreed with that predicted from the cDNA clone, pmusGST Yc. Comparison of the deduced amino acid sequence of the mouse Yc polypeptide with the primary structures of the rat Alpha-class GST enzymes revealed that it is more closely related to the ethoxyquin-induced rat liver Yc2 subunit than to

  18. Discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the RNA polymerase-containing protein of all nidoviruses

    PubMed Central

    Lehmann, Kathleen C.; Gulyaeva, Anastasia; Zevenhoven-Dobbe, Jessika C.; Janssen, George M. C.; Ruben, Mark; Overkleeft, Hermen S.; van Veelen, Peter A.; Samborskiy, Dmitry V.; Kravchenko, Alexander A.; Leontovich, Andrey M.; Sidorov, Igor A.; Snijder, Eric J.; Posthuma, Clara C.; Gorbalenya, Alexander E.

    2015-01-01

    RNA viruses encode an RNA-dependent RNA polymerase (RdRp) that catalyzes the synthesis of their RNA(s). In the case of positive-stranded RNA viruses belonging to the order Nidovirales, the RdRp resides in a replicase subunit that is unusually large. Bioinformatics analysis of this non-structural protein has now revealed a nidoviral signature domain (genetic marker) that is N-terminally adjacent to the RdRp and has no apparent homologs elsewhere. Based on its conservation profile, this domain is proposed to have nucleotidylation activity. We used recombinant non-structural protein 9 of the arterivirus equine arteritis virus (EAV) and different biochemical assays, including irreversible labeling with a GTP analog followed by a proteomics analysis, to demonstrate the manganese-dependent covalent binding of guanosine and uridine phosphates to a lysine/histidine residue. Most likely this was the invariant lysine of the newly identified domain, named nidovirus RdRp-associated nucleotidyltransferase (NiRAN), whose substitution with alanine severely diminished the described binding. Furthermore, this mutation crippled EAV and prevented the replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in cell culture, indicating that NiRAN is essential for nidoviruses. Potential functions supported by NiRAN may include nucleic acid ligation, mRNA capping and protein-primed RNA synthesis, possibilities that remain to be explored in future studies. PMID:26304538

  19. Continuous In Vitro Evolution of a Ribozyme that Catalyzes Three Successive Nucleotidyl Addition Reactions

    NASA Technical Reports Server (NTRS)

    McGinness, Kathleen E.; Wright, Martin C.; Joyce, Gerald F.

    2002-01-01

    Variants of the class I ligase ribozyme, which catalyzes joining of the 3' end of a template bound oligonucleotide to its own 5' end, have been made to evolve in a continuous manner by a simple serial transfer procedure that can be carried out indefinitely. This process was expanded to allow the evolution of ribozymes that catalyze three successive nucleotidyl addition reactions, two template-directed mononucleotide additions followed by RNA ligation. During the development of this behavior, a population of ribozymes was maintained against an overall dilution of more than 10(exp 406). The resulting ribozymes were capable of catalyzing the three-step reaction pathway, with nucleotide addition occurring in either a 5' yieldig 3' or a 3' yielding 5' direction. This purely chemical system provides a functional model of a multi-step reaction pathway that is undergoing Darwinian evolution.

  20. Mechanism of the nucleotidyl-transfer reaction in DNA polymerase revealed by time-resolved protein crystallography

    PubMed Central

    Nakamura, Teruya; Zhao, Ye; Yamagata, Yuriko; Hua, Yue-jin; Yang, Wei

    2013-01-01

    Nucleotidyl-transfer reaction catalyzed by DNA polymerase is a fundamental enzymatic reaction for DNA synthesis. Until now, a number of structural and kinetic studies on DNA polymerases have proposed a two-metalion mechanism of the nucleotidyl-transfer reaction. However, the actual reaction process has never been visualized. Recently, we have followed the nucleotidyl-transfer reaction process by human DNA polymerase η using time-resolved protein crystallography. In sequence, two Mg2+ ions bind to the active site, the nucleophile 3′-OH is deprotonated, the deoxyribose at the primer end converts from C2′-endo to C3′-endo, and the nucleophile and the α-phosphate of the substrate dATP approach each other to form the new bond. In this process, we observed transient elements, which are a water molecule to deprotonate the 3′-OH and an additional Mg2+ ion to stabilize the intermediate state. Particularly, the third Mg2+ ion observed in this study may be a general feature of the two-metalion mechanism.

  1. Interrelationship between anionic and cationic forms of glutathione S-transferases of human liver.

    PubMed Central

    Awasthi, Y C; Dao, D D; Saneto, R P

    1980-01-01

    Human liver glutathione S-transferases (GSH S-transferases) were fractionated into cationic and anionic proteins. During fractionation with (NH4)2SO4 the anionic GSH S-transferases are concentrated in the 65%-saturated-(NH4)2SO4 fraction, whereas the cationic GSH S-transferases separate in the 80%-saturated-(NH4)2SO4 fraction. From the 65%-saturated-(NH4)2SO4 fraction two new anionic GSH S-transferases, omega and psi, were purified to homogeneity by using ion-exchange chromatography on DEAE-cellulose, Sephadex G-200 gel filtration, affinity chromatography on GSH bound to epoxy-activated Sepharose and isoelectric focusing. By a similar procedure, cationic GSH S-transferases were purified from the 80%-saturated-(NH4)2SO4 fraction. Isoelectric points of GSH S-transferases omega and psi are 4.6 and 5.4 respectively. GSH S-transferase omega is the major anionic GSH S-transferase of human liver, whereas GSH S-transferase psi is present only in traces. The subunit mol.wt. of GSH S-transferase omega is about 22500, whereas that of cationic GSH S-transferases is about 24500. Kinetic and structural properties as well as the amino acid composition of GSH S-transferase omega are described. The antibodies raised against cationic GSH S-transferases cross-react with GSH S-transferase omega. There are significant differences between the catalytic properties of GSH S-transferase omega and the cationic GSH S-transferases. GSH peroxidase II activity is displayed by all five cationic GSH S-transferases, whereas both anionic GSH S-transferases do not display this activity. Images Fig. 3. PMID:7470087

  2. Structure of the RNA 30-Phosphate Cyclase-Adenylate Intermediate Illuminates Nucleotide Specificity and Covalent Nucleotidyl Transfer

    SciTech Connect

    Tanaka, N.; Smith, P; Shuman, S

    2010-01-01

    RNA 3-phosphate cyclase (RtcA) synthesizes RNA 2,3 cyclic phosphate ends via three steps: reaction with ATP to form a covalent RtcA-AMP intermediate; transfer of adenylate to an RNA 3-phosphate to form RNA(3)pp(5)A; and attack of the vicinal O2 on the 3-phosphorus to form a 2,3 cyclic phosphate. Here we report the 1.7 {angstrom} crystal structure of the RtcA-AMP intermediate, which reveals the mechanism of nucleotidyl transfer. Adenylate is linked via a phosphoamide bond to the His309 N{var_epsilon} atom. A network of hydrogen bonds to the ribose O2 and O3 accounts for the stringent ribonucleotide preference. Adenine is sandwiched in a hydrophobic pocket between Tyr284 and Pro131 and the preference for adenine is enforced by Phe135, which packs against the purine C2 edge. Two sulfates bound near the adenylate plausibly mimic the 3-terminal and penultimate phosphates of RNA. The structure illuminates how the four {alpha}2/{beta}4 domains contribute to substrate binding and catalysis.

  3. Nomenclature for mammalian soluble glutathione transferases.

    PubMed

    Mannervik, Bengt; Board, Philip G; Hayes, John D; Listowsky, Irving; Pearson, William R

    2005-01-01

    The nomenclature for human soluble glutathione transferases (GSTs) is extended to include new members of the GST superfamily that have been discovered, sequenced, and shown to be expressed. The GST nomenclature is based on primary structure similarities and the division of GSTs into classes of more closely related sequences. The classes are designated by the names of the Greek letters: Alpha, Mu, Pi, etc., abbreviated in Roman capitals: A, M, P, and so on. (The Greek characters should not be used.) Class members are distinguished by Arabic numerals and the native dimeric protein structures are named according to their subunit composition (e.g., GST A1-2 is the enzyme composed of subunits 1 and 2 in the Alpha class). Soluble GSTs from other mammalian species can be classified in the same manner as the human enzymes, and this chapter presents the application of the nomenclature to the rat and mouse GSTs. PMID:16399376

  4. Active site of the mRNA-capping enzyme guanylyltransferase from Saccharomyces cerevisiae: similarity to the nucleotidyl attachment motif of DNA and RNA ligases.

    PubMed Central

    Fresco, L D; Buratowski, S

    1994-01-01

    Nascent mRNA chains are capped at the 5' end by the addition of a guanylyl residue to form a G(5')ppp(5')N ... structure. During the capping reaction, the guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) is reversibly and covalently guanylylated. In this enzyme-GMP (E-GMP) intermediate, GMP is linked to the epsilon-amino group of a lysine residue via a phosphoamide bond. Lys-70 was identified as the GMP attachment site of the Saccharomyces cerevisiae guanylyltransferase (encoded by the CEG1 gene) by guanylylpeptide sequencing. CEG1 genes with substitutions at Lys-70 were unable to support viability in yeast and produced proteins that were not guanylylated in vitro. The CEG1 active site exhibits sequence similarity to the active sites of viral guanylyltransferases and polynucleotide ligases, suggesting similarity in the mechanisms of nucleotidyl transfer catalyzed by these enzymes. Images PMID:8022828

  5. Purification and characterization of a DNA strand transferase from broccoli.

    PubMed

    Tissier, A F; Lopez, M F; Signer, E R

    1995-05-01

    A protein with DNA binding, renaturation, and strand-transfer activities has been purified to homogeneity from broccoli (Brassica oleracea var italica). The enzyme, broccoli DNA strand transferase, has a native molecular mass of at least 200 kD and an apparent subunit molecular mass of 95 kD and is isolated as a set of isoforms differing only in charge. All three activities are saturated at very low stoichiometry, one monomer per approximately 1000 nucleotides of single-stranded DNA. Strand transfer is not effected by nuclease activity and reannealing, is only slightly dependent on ATP, and is independent of added Mg2+. Transfer requires homologous single- and double-stranded DNA and at higher enzyme concentrations results in very high molecular mass complexes. As with Escherichia coli RecA, transfer by broccoli DNA strand transferase depends strongly on the presence of 3' homologous ends. PMID:7784508

  6. tRNA[superscript His] guanylyltransferase (THG1), a unique 3;#8242;-5;#8242; nucleotidyl transferase, shares unexpected structural homology with canonical 5;#8242;-3;#8242; DNA polymerases

    SciTech Connect

    Hyde, Samantha J.; Eckenroth, Brian E.; Smith, Brian A.; Eberley, William A.; Heintz, Nicholas H.; Jackman, Jane E.; Doublié, Sylvie

    2011-11-07

    All known DNA and RNA polymerases catalyze the formation of phosphodiester bonds in a 5' to 3' direction, suggesting this property is a fundamental feature of maintaining and dispersing genetic information. The tRNA{sup His} guanylyltransferase (Thg1) is a member of a unique enzyme family whose members catalyze an unprecedented reaction in biology: 3'-5' addition of nucleotides to nucleic acid substrates. The 2.3-{angstrom} crystal structure of human THG1 (hTHG1) reported here shows that, despite the lack of sequence similarity, hTHG1 shares unexpected structural homology with canonical 5'-3' DNA polymerases and adenylyl/guanylyl cyclases, two enzyme families known to use a two-metal-ion mechanism for catalysis. The ability of the same structural architecture to catalyze both 5'-3' and 3'-5' reactions raises important questions concerning selection of the 5'-3' mechanism during the evolution of nucleotide polymerases.

  7. Cloning and expression of clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli

    SciTech Connect

    Cary, J.W.; Petersen, D.J.; Bennett, G.N. ); Papoutsakis, E.T. )

    1990-06-01

    Coenzyme A (CoA)-transferase (acetoacetyl-CoA:acetate/butyrate:CoA-transferase (butyrate-acetoacetate CoA-transferase) (EC 2.8.3.9)) of Clostridium acetobutylicum ATCC 824 is an important enzyme in the metabolic shift between the acid-producing and solvent-forming states of this organism. The genes encoding the two subunits of this enzyme have been cloned and subsequent subcloning experiments established the position of the structural genes for CoA-transferase. Complementation of Escherichia coli ato mutants with the recombinant plasmid pCoAT4 (pUC19 carrying a 1.8-kilobase insert of C. acetobutylicum DNA encoding CoA-transferase activity) enabled the transformants to grow on butyrate as a sole carbon source. Despite the ability of CoA-transferase to complement the ato defect in E. coli mutants, Southern blot and Western blot (immunoblot) analyses showed showed that neither the C. acetobutylicum genes encoding CoA-transferase nor the enzyme itself shared any apparent homology with its E. coli counterpart. Polypeptides of M{sub r} of the purified CoA-transferase subunits were observed by Western blot and maxicell analysis of whole-cell extracts of E.coli harboring pCoAT4. The proximity and orientation of the genes suggest that the genes encoding the two subunits of CoA-transferase may form an operon similar to that found in E. coli. In the plasmid, however, transcription appears to be primarily from the lac promoter of the vector.

  8. Complementary DNA cloning, messenger RNA expression, and induction of alpha-class glutathione S-transferases in mouse tissues.

    PubMed

    Buetler, T M; Eaton, D L

    1992-01-15

    Glutathione S-transferases (EC 2.5.1.18) are a multigene family of related proteins divided into four classes. Each class has multiple isoforms that exhibit tissue-specific expression, which may be an important determinant of susceptibility of that tissue to toxic injury or cancer. Recent studies have suggested that alpha-class glutathione S-transferase isoforms may play an important role in the development of cancers. Several alpha-class glutathione S-transferase isozymes have been characterized, purified, and cloned from a number of species, including rats, mice, and humans. Here we report on the cloning, sequencing, and mRNA expression of two alpha-class glutathione S-transferases from mouse liver, termed mYa and mYc. While mYa was shown to be identical to the known alpha-class glutathione S-transferase complementary DNA clone pGT41 (W. R. Pearson et al., J. Biol. Chem., 263: 13324-13332, 1988), the other clone, mYc, was demonstrated to be a novel complementary DNA clone encoding a glutathione S-transferase homologous to rat Yc (subunit 2). The mRNA for this novel complementary DNA is expressed constitutively in mouse liver. It also is the major alpha-class glutathione S-transferase isoform expressed in lung. The levels of expression of the butylated hydroxyanisole-inducible form (mYa) are highest in kidney and intestine. Treatment of mice with butylated hydroxyanisole had little effect on the expression levels of mYc but strongly induced mYa expression in liver. Butylated hydroxyanisole treatment increased expression levels for both mYa and mYc to varying degrees in kidney, lung, and intestine. The importance of the novel mouse liver alpha-class glutathione S-transferase isoform (mYc) in the metabolism of aflatoxin B1 and other carcinogens is discussed. PMID:1728405

  9. Roles for glutathione transferases in antioxidant recycling

    PubMed Central

    Dixon, David P; Steel, Patrick G

    2011-01-01

    Uniquely among the plant glutathione transferases, two classes possess a catalytic cysteine capable of performing glutathione-dependent reductions. These are the dehydroascorbate reductases (DHARs) and the lambda-class glutathione transferases (GSTLs). Using immobilized GSTLs probed with crude plant extracts we have identified flavonols as high affinity ligands and subsequently demonstrated a novel glutathione-dependent role for these enzymes in recycling oxidized quercetin. By comparing the activities of DHARs and GSTLs we now propose a unified catalytic mechanism that suggests oxidized anthocyanidins and tocopherols may be alternative polyphenolic substrates of GSTLs. PMID:21778824

  10. Chemoenzymatic synthesis of glycopeptides with PglB, a bacterial oligosaccharyl transferase from Campylobacter jejuni.

    PubMed

    Glover, Kerney Jebrell; Weerapana, Eranthie; Numao, Shin; Imperiali, Barbara

    2005-12-01

    The gram-negative bacterium Campylobacter jejuni has a general N-linked glycosylation pathway encoded by the pgl gene cluster. One of the proteins in this cluster, PgIB, is thought to be the oligosaccharyl transferase due to its significant homology to Stt3p, a subunit of the yeast oligosaccharyl transferase complex. PgIB has been shown to be involved in catalyzing the transfer of an undecaprenyl-linked heptasaccharide to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Using a synthetic disaccharide glycan donor (GaINAc-alpha1,3-bacillosamine-pyrophosphate-undecaprenyl) and a peptide acceptor substrate (KDFNVSKA), we can observe the oligosaccharyl transferase activity of PgIB in vitro. Furthermore, the preparation of additional undecaprenyl-linked glycan variants reveals the ability of PgIB to transfer a wide variety of saccharides. With the demonstration of PgIB activity in vitro, fundamental questions surrounding the mechanism of N-linked glycosylation can now be addressed. PMID:16356848

  11. Purification and characterization of the Oligosaccharyl transferase

    SciTech Connect

    Kapoor, T.M.

    1990-11-01

    Oligosaccharyl transferase was characterized to be a glycoprotein with at least one saccharide unit that had a D-manno or D- glucopyranose configuration with unmodified hydroxy groups at C-3, C-4 and C-6, using a Concanavalin A affinity column. This afforded a 100 fold increase in the transferase purity in the solubilized microsomal sample and also removed over 90% of the microsomal proteins (the cytosolic ones being removed before solubilization). The detergent, N,N-Dimethyldodecylamine N-oxide (LDAO) was used for solubilization and it yielded a system compatible with the assay and the purification steps. An efficient method for detergent extraction without dilution of sample or protein precipitation was also developed.

  12. Effects of microsomal enzyme inducers on glutathione S-transferase isoenzymes in livers of rats and hamsters.

    PubMed

    Foliot, A; Beaune, P

    1994-07-19

    The effects of microsomal enzyme inducers on glutathione S-transferase (GST) isoenzymes were studied in livers of rats and hamsters using three hypolipidemic drugs of the peroxisome proliferator type and the two model substances phenobarbital (PB) and 3-methylcholanthrene (MC). The effects were investigated by immunoblot analysis of the various GST subunits using polyclonal antibodies directed to rat subunits 1-4. In untreated animals the subunit composition was different, with hamsters having a much higher content of class mu isoenzymes. Administration of all three hypolipidemic drugs reduced the protein concentration of both alpha and mu class GSTs in rats but reduced only class mu subunits in hamsters. This reduction was in good agreement with the decreased activity observed with the broad-spectrum substrate 1-chloro-2,4-dinitrobenzene (CDNB) in both species. As expected, PB and MC increased GST activity together with the concentration of subunits 1 and 3 in rats. In hamsters, PB significantly increased subunit 1 and slightly reduced subunits 3 and 4, although this decrease was not significant. Total GST, measured with CDNB, was reduced by 17%. In contrast, MC slightly decreased subunit 1 and markedly raised subunits 3 and 4, resulting in a net increase in total GST activity. All drugs increased relative liver weight, microsomal protein concentration and total P450 in both species; in contrast, total cytosolic proteins were raised by all drugs in rats but not in hamsters, except for MC. The results obtained in these two species show that GST activity is not always increased by microsomal enzyme inducers. The response may depend in part on isoenzyme profile, and varies with the subunit considered. PMID:8053925

  13. Methods for purification of glutathione transferases in the earthworm genus Eisenia, and their characterization.

    PubMed

    Borgeraas, J; Nilsen, K; Stenersen, J

    1996-06-01

    Isoenzymes of glutathione transferase (GST) were partially purified from the earthworm species Eisenia andrei and E. veneta using affinity chromatography followed by ion exchange chromatography and reversed-phase HPLC. In E. veneta, five activity peaks, named EvGST Ia, Ib, II, III and IV, were separated by anion exchange chromatography. The GSTs in E. andrei were resolved by cation exchange chromatography into six groups, named EaGST I-VI. Using reversed-phase HPLC, the affinity-purified GSTs from E. andrei and E. veneta were resolved into 14 subunits, named Ea1-Ea14 and Ev1-Ev14, respectively. EaGST I, II, IV and EvGST Ia were further characterized. These forms displayed different substrate specificity towards the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, ethacrynic acid (ETHA) and cumene hydroperoxide, as well as different subunit composition determined by SDS-PAGE and reversed-phase HPLC. EaGST IV and EvGST Ia showed exceptionally high ETHA activity compared with the other forms. EaGST IV consisted of a homodimeric protein involving subunit Ea6 with an apparent molecular weight of 26.5 kDa, whereas EvGST Ia is composed of two different subunits (Ev9 and Ev10). Amino acid composition and N-terminal analysis of the first 33 residues of Ea6 indicated that the enzyme is most related to the pi class. Subunit Ev10 had 67% identity with Ea6, over the region sequenced (12 residues), but up to 90% identity with GSTs from several nematodes. Exposure of both species to trans-stilbene oxide, 3-methylcholanthrene and phenobarbital for three weeks did not elevate the activity of GST measured with CDNB and ETHA. PMID:8760608

  14. Genetic and functional analyses of PptA, a phospho-form transferase targeting type IV pili in Neisseria gonorrhoeae.

    PubMed

    Naessan, Cecilia L; Egge-Jacobsen, Wolfgang; Heiniger, Ryan W; Wolfgang, Matthew C; Aas, Finn Erik; Røhr, Asmund; Winther-Larsen, Hanne C; Koomey, Michael

    2008-01-01

    The PilE pilin subunit protein of Neisseria gonorrhoeae undergoes unique covalent modifications with phosphoethanolamine (PE) and phosphocholine (PC). The pilin phospho-form transferase A (PptA) protein, required for these modifications, shows sequence relatedness with and architectural similarities to lipopolysaccharide PE transferases. Here, we used regulated expression and mutagenesis as means to better define the relationships between PptA structure and function, as well as to probe the mechanisms by which other factors impact the system. We show here that pptA expression is coupled at the level of transcription to its distal gene, murF, in a division/cell wall gene operon and that PptA can act in a dose-dependent fashion in PilE phospho-form modification. Molecular modeling and site-directed mutagenesis provided the first direct evidence that PptA is a member of the alkaline phosphatase superfamily of metalloenzymes with similar metal-binding sites and conserved structural folds. Through phylogenetic analyses and sequence alignments, these conclusions were extended to include the lipopolysaccharide PE transferases, including members of the disparate Lpt6 subfamily, and the MdoB family of phosphoglycerol transferases. Each of these enzymes thus likely acts as a phospholipid head group transferase whose catalytic mechanism involves a trans-esterification step generating a protein-phospho-form ester intermediate. Coexpression of PptA with PilE in Pseudomonas aeruginosa resulted in high levels of PE modification but was not sufficient for PC modification. This and other findings show that PptA-associated PC modification is governed by as-yet-undefined ancillary factors unique to N. gonorrhoeae. PMID:17951381

  15. Structural basis for the interaction of antibiotics with peptidyl transferase center in eubacteria

    SciTech Connect

    Schlunzen, Frank; Zarivach, Raz; Harms, Jörg; Bashan, Anat; Tocilj, Ante; Albrecht, Renate; Yonath, Ada; Franceschi, Francois

    2009-10-07

    Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics. Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action. Conversely, drugs are excellent tools for studying the ribosome function. To elucidate the structural basis of ribosome-antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin. We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavity and do not involve any interaction of the drugs with ribosomal proteins. Here we report the details of antibiotic interactions with the components of their binding sites. Our results also show the importance of putative Mg{sup +2} ions for the binding of some drugs. This structural analysis should facilitate rational drug design.

  16. Design of a monomeric human glutathione transferase GSTP1, a structurally stable but catalytically inactive protein.

    PubMed

    Abdalla, Abdel-Monem; Bruns, Christopher M; Tainer, John A; Mannervik, Bengt; Stenberg, Gun

    2002-10-01

    By the introduction of 10 site-specific mutations in the dimer interface of human glutathione transferase P1-1 (GSTP1-1), a stable monomeric protein variant, GSTP1, was obtained. The monomer had lost the catalytic activity but retained the affinity for a number of electrophilic compounds normally serving as substrates for GSTP1-1. Fluorescence and circular dichroism spectra of the monomer and wild-type proteins were similar, indicating that there are no large structural differences between the subunits of the respective proteins. The GSTs have potential as targets for in vitro evolution and redesign with the aim of developing proteins with novel properties. To this end, a monomeric GST variant may have distinct advantages. PMID:12468717

  17. Glucose-induced expression of MIP-1 genes requires O-GlcNAc transferase in monocytes

    SciTech Connect

    Chikanishi, Toshihiro; Fujiki, Ryoji; Hashiba, Waka; Sekine, Hiroki; Yokoyama, Atsushi; Kato, Shigeaki

    2010-04-16

    O-glycosylation has emerged as an important modification of nuclear proteins, and it appears to be involved in gene regulation. Recently, we have shown that one of the histone methyl transferases (MLL5) is activated through O-glycosylation by O-GlcNAc transferase (OGT). Addition of this monosaccharide is essential for forming a functional complex. However, in spite of the abundance of OGT in the nucleus, the impact of nuclear O-glycosylation by OGT remains largely unclear. To address this issue, the present study was undertaken to test the impact of nuclear O-glycosylation in a monocytic cell line, THP-1. Using a cytokine array, MIP-1{alpha} and -1{beta} genes were found to be regulated by nuclear O-glycosylation. Biochemical purification of the OGT interactants from THP-1 revealed that OGT is an associating partner for distinct co-regulatory complexes. OGT recruitment and protein O-glycosylation were observed at the MIP-1{alpha} gene promoter; however, the known OGT partner (HCF-1) was absent when the MIP-1{alpha} gene promoter was not activated. From these findings, we suggest that OGT could be a co-regulatory subunit shared by functionally distinct complexes supporting epigenetic regulation.

  18. Kinetics and catalytic properties of coenzyme A transferase from Peptostreptococcus elsdenii.

    PubMed Central

    Schulman, M; Valentino, D

    1976-01-01

    Coenzyme A (CoA) transferase from Peptostreptococcus elsdenii was purified to homogeneity, and some of its physical and catalytic properties were determined. The native enzyme has a molecular weight of 181,000 and is composed of two alpha subunits (molecular weight, 65,000) and one beta subunit (molecular weight 50,000). Heat treatment of the crude cell extract to 58 degrees C causes proteolysis of the native enzyme and yields a catalytically active enzyme with an approximate molecular weight of 120,000. The native CoA transferase is specific for CoA esters of short-chain alkyl monocarboxylic acids. With acetate as CoA acceptor the enzyme is active with propionyl-, butyryl-, isobutyryl-, valeryl-, isovaleryl,- and hexanoyl-CoA but not with heptanoyl or longer-chain CoA esters. There is no activity with acetoacetyl-CoA or the CoA esters of dicarboxylic acids. Steady-state kinetics indicated that the reaction proceeds via a classical bi-, bi-ping-pong mechanism. Maximal activity is obtained with propionyl- or butyryl-CoA, and both the Vmax and Km decrease as the alkyl chain length of the CoA ester increases. All CoA esters apompetitive inhibitor although it is not active as a substrate. Evidence for an enzyme CoA intermediate was provided by demonstration of an exchange between 14C-free acids (acetate and butyrate) and their corresponding CoA esters and by isolation of a 3H-labeled CoA enzyme after incubation of the enzyme with 3H-labeled acetyl-CoA. Approximately 2 mol of CoA was bound per mol of enzyme. Images PMID:977540

  19. Single prenyl-binding site on protein prenyl transferases

    PubMed Central

    Desnoyers, Luc; Seabra, Miguel C.

    1998-01-01

    Three distinct protein prenyl transferases, one protein farnesyl transferase (FTase) and two protein geranylgeranyl transferases (GGTase), catalyze prenylation of many cellular proteins. One group of protein substrates contains a C-terminal CAAX motif (C is Cys, A is aliphatic, and X is a variety of amino acids) in which the single cysteine residue is modified with either farnesyl or geranylgeranyl (GG) by FTase or GGTase type-I (GGTase-I), respectively. Rab proteins constitute a second group of substrates that contain a C-terminal double-cysteine motif (such as XXCC in Rab1a) in which both cysteines are geranylgeranylated by Rab GG transferase (RabGGTase). Previous characterization of CAAX prenyl transferases showed that the enzymes form stable complexes with their prenyl pyrophosphate substrates, acting as prenyl carriers. We developed a prenyl-binding assay and show that RabGGTase has a prenyl carrier function similar to the CAAX prenyl transferases. Stable RabGGTase:GG pyrophosphate (GGPP), FTase:GGPP, and GGTase-I:GGPP complexes show 1:1 (enzyme:GGPP) stoichiometry. Chromatographic analysis of prenylated products after single turnover reactions by using isolated RabGGTase:GGPP complex revealed that Rab is mono-geranylgeranylated. This study establishes that all three protein prenyl transferases contain a single prenyl-binding site and suggests that RabGGTase transfers two GG groups to Rabs in independent and consecutive reactions. PMID:9770475

  20. Properties of succinyl-coenzyme A:L-malate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus.

    PubMed

    Friedmann, Silke; Steindorf, Astrid; Alber, Birgit E; Fuchs, Georg

    2006-04-01

    The 3-hydroxypropionate cycle has been proposed to operate as the autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. In this pathway, acetyl coenzyme A (acetyl-CoA) and two bicarbonate molecules are converted to malate. Acetyl-CoA is regenerated from malyl-CoA by L-malyl-CoA lyase. The enzyme forming malyl-CoA, succinyl-CoA:L-malate coenzyme A transferase, was purified. Based on the N-terminal amino acid sequence of its two subunits, the corresponding genes were identified on a gene cluster which also contains the gene for L-malyl-CoA lyase, the subsequent enzyme in the pathway. Both enzymes were severalfold up-regulated under autotrophic conditions, which is in line with their proposed function in CO2 fixation. The two CoA transferase genes were cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Succinyl-CoA:L-malate CoA transferase forms a large (alphabeta)n complex consisting of 46- and 44-kDa subunits and catalyzes the reversible reaction succinyl-CoA + L-malate --> succinate + L-malyl-CoA. It is specific for succinyl-CoA as the CoA donor but accepts L-citramalate instead of L-malate as the CoA acceptor; the corresponding d-stereoisomers are not accepted. The enzyme is a member of the class III of the CoA transferase family. The demonstration of the missing CoA transferase closes the last gap in the proposed 3-hydroxypropionate cycle. PMID:16547052

  1. The Genetic Architecture of Murine Glutathione Transferases

    PubMed Central

    Lu, Lu; Pandey, Ashutosh K.; Houseal, M. Trevor; Mulligan, Megan K.

    2016-01-01

    Glutathione S-transferase (GST) genes play a protective role against oxidative stress and may influence disease risk and drug pharmacokinetics. In this study, massive multiscalar trait profiling across a large population of mice derived from a cross between C57BL/6J (B6) and DBA2/J (D2)—the BXD family—was combined with linkage and bioinformatic analyses to characterize mechanisms controlling GST expression and to identify downstream consequences of this variation. Similar to humans, mice show a wide range in expression of GST family members. Variation in the expression of Gsta4, Gstt2, Gstz1, Gsto1, and Mgst3 is modulated by local expression QTLs (eQTLs) in several tissues. Higher expression of Gsto1 in brain and liver of BXD strains is strongly associated (P < 0.01) with inheritance of the B6 parental allele whereas higher expression of Gsta4 and Mgst3 in brain and liver, and Gstt2 and Gstz1 in brain is strongly associated with inheritance of the D2 parental allele. Allele-specific assays confirmed that expression of Gsto1, Gsta4, and Mgst3 are modulated by sequence variants within or near each gene locus. We exploited this endogenous variation to identify coexpression networks and downstream targets in mouse and human. Through a combined systems genetics approach, we provide new insight into the biological role of naturally occurring variants in GST genes. PMID:26829228

  2. High resolution structure of the large ribosomal subunit from a Mesophilic Eubacterium

    SciTech Connect

    Harms, Joerg; Schluenzen, Frank; Zarivach, Raz; Bashan, Anat; Gat, Sharon; Agmon, Ilana; Bartels, Heike; Franceschi, Francois; Yonath, Ada

    2009-10-07

    We describe the high resolution structure of the large ribosomal subunit from Deinococcus radiodurans (D50S), a gram-positive mesophile suitable for binding of antibiotics and functionally relevant ligands. The over-all structure of D50S is similar to that from the archae bacterium Haloarcula marismortui (H50S); however, a detailed comparison revealed significant differences, for example, in the orientation of nucleotides in peptidyl transferase center and in the structures of many ribosomal proteins. Analysis of ribosomal features involved in dynamic aspects of protein biosynthesis that are partially or fully disordered in H50S revealed the conformations of intersubunit bridges in unbound subunits, suggesting how they may change upon subunit association and how movements of the L1-stalk may facilitate the exit of tRNA.

  3. Proteomic Profiling of Cytosolic Glutathione Transferases from Three Bivalve Species: Corbicula fluminea, Mytilus galloprovincialis and Anodonta cygnea

    PubMed Central

    Martins, José Carlos; Campos, Alexandre; Osório, Hugo; da Fonseca, Rute; Vasconcelos, Vítor

    2014-01-01

    Suspension-feeding bivalves are considered efficient toxin vectors with a relative insensitivity to toxicants compared to other aquatic organisms. This fact highlights the potential role of detoxification enzymes, such as glutathione transferases (GSTs), in this bivalve resistance. Nevertheless, the GST system has not been extensively described in these organisms. In the present study, cytosolic GSTs isoforms (cGST) were surveyed in three bivalves with different habitats and life strategies: Corbicula fluminea, Anodonta cygnea and Mytilus galloprovincialis. GSTs were purified by glutathione-agarose affinity chromatography, and the collection of expressed cGST classes of each bivalve were identified using a proteomic approach. All the purified extracts were also characterized kinetically. Results reveal variations in cGST subunits collection (diversity and properties) between the three tested bivalves. Using proteomics, four pi-class and two sigma-class GST subunits were identified in M. galloprovincialis. C. fluminea also yielded four pi-class and one sigma-class GST subunits. For A. cygnea, two mu-class and one pi-class GST subunits were identified, these being the first record of GSTs from these freshwater mussels. The affinity purified extracts also show differences regarding enzymatic behavior among species. The variations found in cGST collection and kinetics might justify diverse selective advantages for each bivalve organism. PMID:24473139

  4. Proteomic profiling of cytosolic glutathione transferases from three bivalve species: Corbicula fluminea, Mytilus galloprovincialis and Anodonta cygnea.

    PubMed

    Martins, José Carlos; Campos, Alexandre; Osório, Hugo; da Fonseca, Rute; Vasconcelos, Vítor

    2014-01-01

    Suspension-feeding bivalves are considered efficient toxin vectors with a relative insensitivity to toxicants compared to other aquatic organisms. This fact highlights the potential role of detoxification enzymes, such as glutathione transferases (GSTs), in this bivalve resistance. Nevertheless, the GST system has not been extensively described in these organisms. In the present study, cytosolic GSTs isoforms (cGST) were surveyed in three bivalves with different habitats and life strategies: Corbicula fluminea, Anodonta cygnea and Mytilus galloprovincialis. GSTs were purified by glutathione-agarose affinity chromatography, and the collection of expressed cGST classes of each bivalve were identified using a proteomic approach. All the purified extracts were also characterized kinetically. Results reveal variations in cGST subunits collection (diversity and properties) between the three tested bivalves. Using proteomics, four pi-class and two sigma-class GST subunits were identified in M. galloprovincialis. C. fluminea also yielded four pi-class and one sigma-class GST subunits. For A. cygnea, two mu-class and one pi-class GST subunits were identified, these being the first record of GSTs from these freshwater mussels. The affinity purified extracts also show differences regarding enzymatic behavior among species. The variations found in cGST collection and kinetics might justify diverse selective advantages for each bivalve organism. PMID:24473139

  5. Detection of glutathione transferase activity on polyacrylamide gels.

    PubMed

    Ricci, G; Lo Bello, M; Caccuri, A M; Galiazzo, F; Federici, G

    1984-12-01

    A simple and sensitive assay for glutathione transferase activity on polyacrylamide gel is described. The method is based on the fast reduction of nitroblue tetrazolium salt by glutathione. Blue insoluble formazan colors the gel except in the glutathione transferase area. The stable and defined colorless zone is still detectable with 0.005 unit enzyme. This technique has been successfully applied with enzyme preparations of human heart and other tissues. PMID:6532239

  6. Properties of succinyl-coenzyme A:D-citramalate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus.

    PubMed

    Friedmann, Silke; Alber, Birgit E; Fuchs, Georg

    2006-09-01

    The phototrophic bacterium Chloroflexus aurantiacus uses the 3-hydroxypropionate cycle for autotrophic CO(2) fixation. This cycle starts with acetyl-coenzyme A (CoA) and produces glyoxylate. Glyoxylate is an unconventional cell carbon precursor that needs special enzymes for assimilation. Glyoxylate is combined with propionyl-CoA to beta-methylmalyl-CoA, which is converted to citramalate. Cell extracts catalyzed the succinyl-CoA-dependent conversion of citramalate to acetyl-CoA and pyruvate, the central cell carbon precursor. This reaction is due to the combined action of enzymes that were upregulated during autotrophic growth, a coenzyme A transferase with the use of succinyl-CoA as the CoA donor and a lyase cleaving citramalyl-CoA to acetyl-CoA and pyruvate. Genomic analysis identified a gene coding for a putative coenzyme A transferase. The gene was heterologously expressed in Escherichia coli and shown to code for succinyl-CoA:d-citramalate coenzyme A transferase. This enzyme, which catalyzes the reaction d-citramalate + succinyl-CoA --> d-citramalyl-CoA + succinate, was purified and studied. It belongs to class III of the coenzyme A transferase enzyme family, with an aspartate residue in the active site. The homodimeric enzyme composed of 44-kDa subunits was specific for succinyl-CoA as a CoA donor but also accepted d-malate and itaconate instead of d-citramalate. The CoA transferase gene is part of a cluster of genes which are cotranscribed, including the gene for d-citramalyl-CoA lyase. It is proposed that the CoA transferase and the lyase catalyze the last two steps in the glyoxylate assimilation route. PMID:16952935

  7. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

    PubMed Central

    Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

    2014-01-01

    All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

  8. Terminal Deoxynucleotidyl Transferase: The Story of a Misguided DNA Polymerase

    PubMed Central

    Motea, Edward A.; Berdis, Anthony J.

    2009-01-01

    Nearly every DNA polymerase characterized to date exclusively catalyzes the incorporation of mononucleotides into a growing primer using a DNA or RNA template as a guide to direct each incorporation event. There is, however, one unique DNA polymerase designated terminal deoxynucleotidyl transferase that performs DNA synthesis using only single-stranded DNA as the nucleic acid substrate. In this chapter, we review the biological role of this enigmatic DNA polymerase and the biochemical mechanism for its ability to perform DNA synthesis in the absence of a templating strand. We compare and contrast the molecular events for template-independent DNA synthesis catalyzed by terminal deoxynucleotidyl transferase with other well-characterized DNA polymerases that perform template-dependent synthesis. This includes a quantitative inspection of how terminal deoxynucleotidyl transferase binds DNA and dNTP substrates, the possible involvement of a conformational change that precedes phosphoryl transfer, and kinetic steps that are associated with the release of products. These enzymatic steps are discussed within the context of the available structures of terminal deoxynucleotidyl transferase in the presence of DNA or nucleotide substrate. In addition, we discuss the ability of proteins involved in replication and recombination to regulate the activity of the terminal deoxynucleotidyl transferase. Finally, the biomedical role of this specialized DNA polymerase is discussed focusing on its involvement in cancer development and its use in biomedical applications such as labeling DNA for detecting apoptosis. PMID:19596089

  9. Inhibition of hepatic glutathione transferases by propylthiouracil and its metabolites.

    PubMed

    Kariya, K; Sawahata, T; Okuno, S; Lee, E

    1986-05-01

    The effects of propylthiouracil (PTU) and its metabolites on the activity of GSH transferases were examined using rat liver cytosol. PTU inhibited the enzyme activity toward both CDNB and DCNB in a concentration-dependent manner. At the concentration of 10 mM, PTU caused 25% inhibition, which was the maximum effect. PTU derivatives such as propyluracil and thiouracil showed the same effect as the parent compound. On the other hand, S-oxides of PTU such as PTU-SO2 and PTU-SO3, which were chemically synthesized by the oxidation of PTU, were more potent inhibitors of GSH transferases than the parent PTU. A significant inhibition was observed at a concentration of 0.1 mM of PTU S-oxides. At a concentration of 10 mM the S-oxides caused an 80% inhibition of the enzyme activity. PTU inhibited the transferase activity by competing with GSH but the S-oxides of PTU acted by another mechanism. In contrast to the effect on GSH transferases, PTU-SO3 had a weak inhibitory effect on GSH peroxidase activity. Thus, oxidation of PTU leads to products which are potent inhibitors of GSH transferases. PMID:3707612

  10. Partial characterization of glutathione S-transferases from wheat (Triticum spp.) and purification of a safener-induced glutathione S-transferase from Triticum tauschii.

    PubMed Central

    Riechers, D E; Irzyk, G P; Jones, S S; Fuerst, E P

    1997-01-01

    Hexaploid wheat (Triticum aestivum L.) has very low constitutive glutathione S-transferase (GST) activity when assayed with the chloroacetamide herbicide dimethenamid as a substrate, which may account for its low tolerance to dimethenamid in the field. Treatment of seeds with the herbicide safener fluxofenim increased the total GST activity extracted from T. aestivum shoots 9-fold when assayed with dimethenamid as a substrate, but had no effect on glutathione levels. Total GST activity in crude protein extracts from T. aestivum, Triticum durum, and Triticum tauschii was separated into several component GST activities by anion-exchange fast-protein liquid chromatography. These activities (isozymes) differed with respect to their activities toward dimethenamid or 1-chloro-2,4-dinitrobenzene as substrates and in their levels of induction by safener treatment. A safener-induced GST isozyme was subsequently purified by anion-exchange and affinity chromatography from etiolated shoots of the diploid wheat species T. tauschii (a progenitor of hexaploid wheat) treated with the herbicide safener cloquintocet-mexyl. The isozyme bound to a dimethenamid-affinity column and had a subunit molecular mass of 26 kD based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme (designated GST TSI-1) was recognized by an antiserum raised against a mixture of maize (Zea mays) GSTs. Amino acid sequences obtained from protease-digested GST TSI-1 had significant homology with the safener-inducible maize GST V and two auxin-regulated tobacco (Nicotiana tabacum) GST isozymes. PMID:9276955

  11. Regulation of aflatoxin B1-metabolizing aldehyde reductase and glutathione S-transferase by chemoprotectors.

    PubMed Central

    McLellan, L I; Judah, D J; Neal, G E; Hayes, J D

    1994-01-01

    Ingestion of aflatoxin B1 (AFB1) represents a major risk factor in the aetiology of human hepatocellular carcinoma. In the rat, the harmful effects of AFB1 can be prevented by the administration of certain drugs which induce hepatic detoxification enzymes. We have previously shown that treatment of rats with the chemoprotector ethoxyquin (EQ) results in a marked increase in expression of the Alpha-class glutathione S-transferase (GST) Yc2 subunit which has high activity towards AFB1-8,9-epoxide [Hayes, Judah, McLellan, Kerr, Peacock and Neal (1991) Biochem. J. 279, 385-398]. To allow an assessment of whether the increased expression of GST Yc2 represents a general adaptive resistance mechanism to chemical stress, that is invoked by both chemoprotectors and carcinogens, we have examined the effects of EQ, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), phenobarbital (PB), AFB1, 3-methylcholanthrene (3-MC) and clofibrate on the AFB1-glutathione-conjugating activity and the GST subunit levels in rat liver. In addition, the effect of these drugs on the hepatic levels of an aldehyde reductase (AFB1-AR) that metabolizes the cytotoxic dialdehydic form of AFB1 has been studied as this enzyme also appears to be important in chemoprotection. Administration of the antioxidants EQ, BHA or BHT, as well as PB, led to a marked increase in levels of the GST Yc2 subunit in rat liver, and this increase coincided with a substantial rise in the GST activity towards AFB1-8,9-epoxide; neither AFB1, 3-MC nor clofibrate caused induction of Yc2 or any of the GST subunits examined. Among the xenobiotics studied, EQ was found to be the most effective inducing agent for the Yc2 subunit as well as Yc1, Yb1 and Yf. However, PB was equally as effective as EQ in increasing levels of the Ya-type subunits, although it was not found to be as potent an inducer of the other GST subunits, including Yc2. In addition to induction of GST, EQ caused a substantial increase in the hepatic

  12. Fluorescent techniques for discovery and characterization of phosphopantetheinyl transferase inhibitors

    PubMed Central

    Kosa, Nicolas M.; Foley, Timothy L.; Burkart, Michael D.

    2016-01-01

    Phosphopantetheinyl transferase (E.C. 2.7.8.-) activates biosynthetic pathways that synthesize both primary and secondary metabolites in bacteria. Inhibitors of these enzymes have the potential to serve as antibiotic compounds that function through a unique mode of action and possess clinical utility. Here we report a direct and continuous assay for this enzyme class based upon monitoring polarization of a fluorescent phosphopantetheine analog as it is transferred from a low molecular weight coenzyme A substrate to higher molecular weight protein acceptor. We demonstrate the utility of this method for the biochemical characterization of phosphopantetheinyl transferase Sfp, a canonical representative from this class. We also establish the portability of this technique to other homologs by adapting the assay to function with the human phosphopantetheinyl transferase, a target for which a microplate detection method does not currently exist. Comparison of these targets provides a basis to predict therapeutic index of inhibitor candidates and offers a valuable characterization of enzyme activity. PMID:24192555

  13. Thioltransferase activity of bovine lens glutathione S-transferase.

    PubMed Central

    Dal Monte, M; Cecconi, I; Buono, F; Vilardo, P G; Del Corso, A; Mura, U

    1998-01-01

    A Mu-class glutathione S-transferase purified to electrophoretic homogeneity from bovine lens displayed thioltransferase activity, catalysing the transthiolation reaction between GSH and hydroxyethyldisulphide. The thiol-transfer reaction is composed of two steps, the formation of GSSG occurring through the generation of an intermediate mixed disulphide between GSH and the target disulphide. Unlike glutaredoxin, which is only able to catalyse the second step of the transthiolation process, glutathioneS-transferase catalyses both steps of the reaction. Data are presented showing that bovine lens glutathione S-transferase and rat liver glutaredoxin, which was used as a thioltransferase enzyme model, can operate in synergy to catalyse the GSH-dependent reduction of hydroxyethyldisulphide. PMID:9693102

  14. Interaction of factor XIII subunits.

    PubMed

    Katona, Eva; Pénzes, Krisztina; Csapó, Andrea; Fazakas, Ferenc; Udvardy, Miklós L; Bagoly, Zsuzsa; Orosz, Zsuzsanna Z; Muszbek, László

    2014-03-13

    Coagulation factor XIII (FXIII) is a heterotetramer consisting of 2 catalytic A subunits (FXIII-A2) and 2 protective/inhibitory B subunits (FXIII-B2). FXIII-B, a mosaic protein consisting of 10 sushi domains, significantly prolongs the lifespan of catalytic subunits in the circulation and prevents their slow progressive activation in plasmatic conditions. In this study, the biochemistry of the interaction between the 2 FXIII subunits was investigated. Using a surface plasmon resonance technique and an enzyme-linked immunosorbent assay-type binding assay, the equilibrium dissociation constant (Kd) for the interaction was established in the range of 10(-10) M. Based on the measured Kd, it was calculated that in plasma approximately 1% of FXIII-A2 should be in free form. This value was confirmed experimentally by measuring FXIII-A2 in plasma samples immunodepleted of FXIII-A2B2. Free plasma FXIII-A2 is functionally active, and when activated by thrombin and Ca(2+), it can cross-link fibrin. In cerebrospinal fluid and tears with much lower FXIII subunit concentrations, >80% of FXIII-A2 existed in free form. A monoclonal anti-FXIII-B antibody that prevented the interaction between the 2 subunits reacted with the recombinant combined first and second sushi domains of FXIII-B, and its epitope was localized to the peptide spanning positions 96 to 103 in the second sushi domain. PMID:24408323

  15. Effect of three xenobiotic compounds on Glutathione S-Transferase in the clam Ruditapes decussatus.

    PubMed

    Hoarau, Pascal; Garello, Ginette; Gnassia-Barelli, Mauricette; Roméo, Michèle; Girard, Jean-Pierre

    2004-05-28

    The effects of 4,4'DDE, methoxychlor and imidazole were studied on glutathione S-transferase activities in the gills and hepatopancreas of the clam Ruditapes decussatus. The contamination doses were 0.14 microM for 4,4'DDE, 0.14 microM for methoxychlor and 25 microM for imidazole. GST activities were spectrophotometrically measured. SDS-PAGE and isoelectric focusing (IEF) were used to separate the different GST isoforms in control and treated animals, followed by Western blotting performed with anti-alpha, anti-mu and anti-pi GST anti-sera. In the hepatopancreas, GST-CDNB activities were always two to five-fold lower than in the gills where the activities were significantly increased after exposure to 4,4'DDE (ca. 1.6-fold) and to methoxychlor (ca. 1.3-fold) compared to the controls (ca. 3 micromolmin(-1)mg(-1)protein) whereas they remained unchanged after treatment with imidazole. When using glutathione S-transferase anti-alpha, anti-mu and anti-pi anti-sera, a single 26 kDa polypeptide was observed in the hepatopancreas and in the gills in all the tested conditions. Five GST subunits were observed after IEF showing greater immuno-reactivity with the anti-pi mammalian class antiserum than with the anti-alpha or anti-mu mammalian anti-sera. One isoform of pI 5.77 was particularly induced by 4,4'DDE and methoxychlor; it was recognized by the three anti-sera tested and seemed to be more efficient in the gills than in the hepatopancreas. This isoform may play a role in organochlorine detoxication. PMID:15110472

  16. The complete structure of the large subunit of the mammalian mitochondrial ribosome.

    PubMed

    Greber, Basil J; Boehringer, Daniel; Leibundgut, Marc; Bieri, Philipp; Leitner, Alexander; Schmitz, Nikolaus; Aebersold, Ruedi; Ban, Nenad

    2014-11-13

    Mitochondrial ribosomes (mitoribosomes) are extensively modified ribosomes of bacterial descent specialized for the synthesis and insertion of membrane proteins that are critical for energy conversion and ATP production inside mitochondria. Mammalian mitoribosomes, which comprise 39S and 28S subunits, have diverged markedly from the bacterial ribosomes from which they are derived, rendering them unique compared to bacterial, eukaryotic cytosolic and fungal mitochondrial ribosomes. We have previously determined at 4.9 Å resolution the architecture of the porcine (Sus scrofa) 39S subunit, which is highly homologous to the human mitoribosomal large subunit. Here we present the complete atomic structure of the porcine 39S large mitoribosomal subunit determined in the context of a stalled translating mitoribosome at 3.4 Å resolution by cryo-electron microscopy and chemical crosslinking/mass spectrometry. The structure reveals the locations and the detailed folds of 50 mitoribosomal proteins, shows the highly conserved mitoribosomal peptidyl transferase active site in complex with its substrate transfer RNAs, and defines the path of the nascent chain in mammalian mitoribosomes along their idiosyncratic exit tunnel. Furthermore, we present evidence that a mitochondrial tRNA has become an integral component of the central protuberance of the 39S subunit where it architecturally substitutes for the absence of the 5S ribosomal RNA, a ubiquitous component of all cytoplasmic ribosomes. PMID:25271403

  17. The N-end rule in Escherichia coli: cloning and analysis of the leucyl, phenylalanyl-tRNA-protein transferase gene aat.

    PubMed Central

    Shrader, T E; Tobias, J W; Varshavsky, A

    1993-01-01

    The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Distinct versions of the N-end rule operate in bacteria, fungi, and mammals. We report the cloning and analysis of aat, the Escherichia coli gene that encodes leucyl, phenylalanyl-tRNA-protein transferase (L/F-transferase), a component of the bacterial N-end rule pathway. L/F-transferase is required for the degradation of N-end rule substrates bearing an N-terminal arginine or lysine. The aat gene maps to the 19-min region of the E. coli chromosome and encodes a 234-residue protein whose sequence lacks significant similarities to sequences in data bases. In vitro, L/F-transferase catalyzes the posttranslational conjugation of leucine or phenylalanine to the N termini of proteins that bear an N-terminal arginine or lysine. However, the isolation and sequence analysis of a beta-galactosidase variant engineered to expose an N-terminal arginine in vivo revealed the conjugation of leucine but not of phenylalanine to the N terminus of the beta-galactosidase variant. Thus, the specificity of L/F-transferase in vivo may be greater than that in vitro. The aat gene is located approximately 1 kb from clpA, which encodes a subunit of ATP-dependent protease Clp. Although both aat and clpA are required for the degradation of certain N-end rule substrates, their nearly adjacent genes are convergently transcribed. The aat gene lies downstream of an open reading frame that encodes a homolog of the mammalian multidrug resistance P glycoproteins. PMID:8331068

  18. GLUTATHIONE S-TRANSFERASE-MEDIATED METABOLISM OF BROMODICHLOROMETHANE

    EPA Science Inventory

    GLUTATHIONE s-TRANSFERASE-MEDIATED METABOLISM OF BROMODICHLOROMETHANE. M K Ross1 and R A Pegram2. 1Curriculum in Toxicology, University of North Carolina at Chapel Hill; 2Experimental Toxicology Division, NHEERL/ORD, United States Environmental Protection Agency, Research Triangl...

  19. 21 CFR 862.1535 - Ornithine carbamyl transferase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Ornithine carbamyl transferase test system. 862.1535 Section 862.1535 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1535 Ornithine...

  20. Rational design of an organometallic glutathione transferase inhibitor

    SciTech Connect

    Ang, W.H.; Parker, L.J.; De Luca, A.; Juillerat-Jeanneret, L.; Morton, C.J.; LoBello, M.; Parker, M.W.; Dyson, P.J.

    2010-08-17

    A hybrid organic-inorganic (organometallic) inhibitor was designed to target glutathione transferases. The metal center is used to direct protein binding, while the organic moiety acts as the active-site inhibitor. The mechanism of inhibition was studied using a range of biophysical and biochemical methods.

  1. METAL-INDUCED INHIBITION OF GLUTATHIONE S-TRANSFERASES

    EPA Science Inventory

    The glutathione S-transferases comprise a group of multi-functional enzymes involved in the biotransformation/detoxication of a broad spectrum of hydrophobic compounds bearing an electrophilic center. The enzymes facilitate the nucleophilic attack of the -SH group of reduced glut...

  2. Deficiency of glutathione transferase zeta causes oxidative stress and activation of antioxidant response pathways.

    PubMed

    Blackburn, Anneke C; Matthaei, Klaus I; Lim, Cindy; Taylor, Matthew C; Cappello, Jean Y; Hayes, John D; Anders, M W; Board, Philip G

    2006-02-01

    Glutathione S-transferase (GST) zeta (GSTZ1-1) plays a significant role in the catabolism of phenylalanine and tyrosine, and a deficiency of GSTZ1-1 results in the accumulation of maleylacetoacetate and its derivatives maleylacetone (MA) and succinylacetone. Induction of GST subunits was detected in the liver of Gstz1(-/-) mice by Western blotting with specific antisera and high-performance liquid chromatography analysis of glutathione affinity column-purified proteins. The greatest induction was observed in members of the mu class. Induction of NAD(P)H:quinone oxidoreductase 1 and the catalytic and modifier subunits of glutamate-cysteine ligase was also observed. Many of the enzymes that are induced in Gstz1(-/-) mice are regulated by antioxidant response elements that respond to oxidative stress via the Keap1/Nrf2 pathway. It is significant that diminished glutathione concentrations were also observed in the liver of Gstz1(-/-) mice, which supports the conclusion that under normal dietary conditions, the accumulation of electrophilic intermediates such as maleylacetoacetate and MA results in a high level of oxidative stress. Elevated GST activities in the livers of Gstz1(-/-) mice suggest that GSTZ1-1 deficiency may alter the metabolism of some drugs and xenobiotics. Gstz1(-/-) mice given acetaminophen demonstrated increased hepatotoxicity compared with wild-type mice. This toxicity may be attributed to the increased GST activity or the decreased hepatic concentrations of glutathione, or both. Patients with acquired deficiency of GSTZ1-1 caused by therapeutic exposure to dichloroacetic acid for the clinical treatment of lactic acidosis may be at increased risk of drug- and chemical-induced toxicity. PMID:16278372

  3. Differential regulation of AMPA receptor subunit trafficking by palmitoylation of two distinct sites.

    PubMed

    Hayashi, Takashi; Rumbaugh, Gavin; Huganir, Richard L

    2005-09-01

    Modification of AMPA receptor function is a major mechanism for the regulation of synaptic transmission and underlies several forms of synaptic plasticity. Post-translational palmitoylation is a reversible modification that regulates localization of many proteins. Here, we report that palmitoylation of the AMPA receptor regulates receptor trafficking. All AMPA receptor subunits are palmitoylated on two cysteine residues in their transmembrane domain (TMD) 2 and in their C-terminal region. Palmitoylation on TMD 2 is upregulated by the palmitoyl acyl transferase GODZ and leads to an accumulation of the receptor in the Golgi and a reduction of receptor surface expression. C-terminal palmitoylation decreases interaction of the AMPA receptor with the 4.1N protein and regulates AMPA- and NMDA-induced AMPA receptor internalization. Moreover, depalmitoylation of the receptor is regulated by activation of glutamate receptors. These data suggest that regulated palmitoylation of AMPA receptor subunits modulates receptor trafficking and may be important for synaptic plasticity. PMID:16129400

  4. Genetics Home Reference: succinyl-CoA:3-ketoacid CoA transferase deficiency

    MedlinePlus

    ... CoA:3-ketoacid CoA transferase deficiency succinyl-CoA:3-ketoacid CoA transferase deficiency Enable Javascript to view ... PDF Open All Close All Description Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is an inherited ...

  5. Purification and characterization of a glutathione S-transferase from Mucor mucedo.

    PubMed

    Hamed, Ragaa R; Abu-Shady, Mohamed R; El-Beih, Fawkia M; Abdalla, Abdel-Monem A; Afifi, Ola M

    2005-01-01

    An intracellular glutathione transferase was purified to homogenity from the fungus, Mucor mucedo, using DEAE-cellulose ion-exchange and glutathione affinity chromatography. Gel filtration chromatography and SDS-PAGE revealed that the purified GST is a homodimer with approximate native and subunit molecular mass of 53 kDa and 23.4 kDa, respectively. The enzyme has a pI value of 4.8, a pH optimum at pH 8.0 and apparent activation energy (Ea) of 1.42 kcal mol(-1). The purified GST acts readily on CDNB with almost negligible peroxidase activity and the activity was inhibited by Cibacron Blue (IC50 0.252 microM) and hematin (IC50 3.55 microM). M. mucedo GST displayed a non-Michaelian behavior. At low (0.1-0.3 mM) and high (0.3-2 mM) substrate concentration, Km (GSH) was calculated to be 0.179 and 0.65 mM, whereas Km(CDNB) was 0.531 and 11 mM and k(cat) was 39.8 and 552 s(-1), respectively. The enzyme showed apparent pKa values of 6-6.5 and 8.0. PMID:16209109

  6. Purification and kinetic mechanism of the major glutathione S-transferase from bovine brain.

    PubMed Central

    Young, P R; Briedis, A V

    1989-01-01

    The major glutathione S-transferase isoenzyme from bovine brain was isolated and purified approx. 500-fold. The enzyme has a pI of 7.39 +/- 0.02 and consists of two non-identical subunits having apparent Mr values of 22,000 and 24,000. The enzyme is uniformly distributed in brain, and kinetic data at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate suggest a random rapid-equilibrium mechanism. The kinetics of inhibition by product, by GSH analogues and by NADH are consistent with the suggested mechanism and require inhibitor binding to several different enzyme forms. Long-chain fatty acids are excellent inhibitors of the enzyme, and values of 1nKi for hexanoic acid, octanoic acid, decanoic acid and lauric acid form a linear series when plotted as a function of alkyl chain length. A free-energy change of -1900 J/mol (-455 cal/mol) per CH2 unit is calculated for the contribution of hydrophobic binding energy to the inhibition constants. The turnover number of the purified enzyme dimer is approx. 3400/min. When compared with the second-order rate constant for the reaction between CDNB and GSH, the enzyme is providing a rate acceleration of about 1000-fold. The role of entropic contributions to this small rate acceleration is discussed. PMID:2930465

  7. 3-Methyleneoxindole: an affinity label of glutathione S-transferase pi which targets tryptophan 38.

    PubMed

    Pettigrew, N E; Brush, E J; Colman, R F

    2001-06-26

    The compound 3-methyleneoxindole (MOI), a photooxidation product of the plant auxin indole-3-acetic acid, functions as an affinity label of the dimeric pi class glutathione S-transferase (GST) isolated from pig lung. MOI inactivates the enzyme to a limit of 14% activity. The k for inactivation by MOI is decreased 20-fold by S-hexylglutathione but only 2-fold by S-methylglutathione, suggesting that MOI does not react entirely within the glutathione site. The striking protection against inactivation provided by S-(hydroxyethyl)ethacrynic acid indicates that MOI reacts in the active site region involving both the glutathione and the xenobiotic substrate sites. Incorporation of [(3)H]MOI up to approximately 1 mol/mol of enzyme dimer concomitant with maximum inactivation suggests that there are interactions between subunits. Fractionation of the proteolytic digest of [(3)H]MOI-modified GST pi yielded Trp38 as the only labeled amino acid. The crystal structure of the human GST pi-ethacrynic acid complex (2GSS) shows that the indole of Trp38 is less than 4 A from ethacrynic acid. Similarly, MOI may bind in this substrate site. In contrast to its effect on the pi class GST, MOI inactivates much less rapidly and extensively alpha and mu class GSTs isolated from the rat. These results show that MOI reacts preferentially with GST pi. Such a compound may be useful in novel combination chemotherapy to enhance the efficacy of alkylating cancer drugs while minimizing toxic side effects. PMID:11412109

  8. The HTLV-1-encoded protein HBZ directly inhibits the acetyl transferase activity of p300/CBP

    PubMed Central

    Wurm, Torsten; Wright, Diana G.; Polakowski, Nicholas; Mesnard, Jean-Michel; Lemasson, Isabelle

    2012-01-01

    The homologous cellular coactivators p300 and CBP contain intrinsic lysine acetyl transferase (termed HAT) activity. This activity is responsible for acetylation of several sites on the histones as well as modification of transcription factors. In a previous study, we found that HBZ, encoded by the Human T-cell Leukemia Virus type 1 (HTLV-1), binds to multiple domains of p300/CBP, including the HAT domain. In this study, we found that HBZ inhibits the HAT activity of p300/CBP through the bZIP domain of the viral protein. This effect correlated with a reduction of H3K18 acetylation, a specific target of p300/CBP, in cells expressing HBZ. Interestingly, lower levels of H3K18 acetylation were detected in HTLV-1 infected cells compared to non-infected cells. The inhibitory effect of HBZ was not limited to histones, as HBZ also inhibited acetylation of the NF-κB subunit, p65, and the tumor suppressor, p53. Recent studies reported that mutations in the HAT domain of p300/CBP that cause a defect in acetylation are found in certain types of leukemia. These observations suggest that inhibition of the HAT activity by HBZ is important for the development of adult T-cell leukemia associated with HTLV-1 infection. PMID:22434882

  9. The ribosomal subunit assembly line

    PubMed Central

    Dlakić, Mensur

    2005-01-01

    Recent proteomic studies in Saccharomyces cerevisiae have identified nearly 200 proteins, other than the structural ribosomal proteins, that participate in the assembly of ribosomal subunits and their transport from the nucleus. In a separate line of research, proteomic studies of mature plant ribosomes have revealed considerable variability in the protein composition of individual ribosomes. PMID:16207363

  10. S-(4-bromo-2,3-dioxobutyl)glutathione: A new affinity label for the 4-4 isoenzyme of rat liver glutathione S-transferase

    SciTech Connect

    Katusz, R.M.; Colman, R.F. )

    1991-11-26

    S-(4-Bromo-2,3-dioxobutyl)glutathione (S-BDB-G), a reactive analogue of glutathione, has been synthesized and characterized by UV spectroscopy and thin-layer chromatography, as well as by bromide and primary amine analysis. Incubation of S-BDB-G (200 {mu}M) with the 4-4 isoenzyme of rat liver glutathione S-transferase at pH 6.5 and 25C results in a time-dependent inactivation of the enzyme. The k{sub obs} exhibits a nonlinear dependence on S-BDB-G concentration from 50 to 1000 {mu}M. Modified enzyme, prepared by incubating glutathione S-transferase with ({sup 3}H)S-BDB-G in the absence or in the presence of S-hexylglutathione, was reduced with NaBH{sub 4}, carboxymethylated, and digested with trypsin. The tryptic digest was fractionated by reverse-phase high-performance liquid chromatography. Two radioactive peptides were identified. These results suggest that S-BDB-G functions as an affinity label at or near the active site of glutathione S-transferase and that modification of one site per enzyme subunit causes inactivation. It is proposed that the new compound, S-(4-bromo-2,3-dioxobutyl)glutathione, may have general applicability as an affinity label of other enzymes with glutathione binding sites.

  11. Developmental aspects of glutathione S-transferase B (ligandin) in rat liver.

    PubMed Central

    Hales, B F; Neims, A H

    1976-01-01

    The postnatal development in male Sprague-Dawley rats of hepatic glutathione S-transferase B (ligandin) in relation to the other glutathione S-transferases is described. The concentration of glutathione S-transferase B in 1-day-old male rats is about one-fifth of that in adult animals. The enzyme reaches adult concentrations 4-5 weeks later. When assessed by substrate specificity or immunologically, the proportion of transferase B relative to the other glutathione S-transferases is high during the first week after birth. At this age, 67.5% of the transferase activity towards 1-chloro-2,4-dinitrobenzene is immunoprecipitable by anti-(transferase B), compared with about 50% in adults and older pups. Between the second and the fifth postnatal week, the fraction of transferase B increases in parallel fashion with the other transferases in hepatic cytosol. Neither L-thyroxine nor cortisol induce a precocious increase in glutathione S-transferase activity. Phenobarbital did induce transferase activity towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene in both pups and adults. The extent of induction by phenobarbital was a function of basal activity during development such that the percentage stimulation remained constant from 5 days postnatally to adulthood. PMID:1008852

  12. Photolabelling of mutant forms of the S1 subunit of pertussis toxin with NAD+.

    PubMed Central

    Cieplak, W; Locht, C; Mar, V L; Burnette, W N; Keith, J M

    1990-01-01

    The S1 subunit of pertussis toxin catalyses the hydrolysis of NAD+ (NAD+ glycohydrolysis) and the NAD(+)-dependent ADP-ribosylation of guanine-nucleotide-binding proteins. Recently, the S1 subunit of pertussis toxin was shown to be photolabelled by using radiolabelled NAD+ and u.v.; the primary labelled residue was Glu-129, thereby implicating this residue in the binding of NAD+. Studies from various laboratories have shown that the N-terminal portion of the S1 subunit, which shows sequence similarity to cholera toxin and Escherichia coli heat-labile toxin, is important to the maintenance of both glycohydrolase and transferase activity. In the present study the photolabelling technique was applied to the analysis of a series of recombinant-derived S1 molecules that possessed deletions or substitutions near the N-terminus of the S1 molecule. The results revealed a positive correlation between the extent of photolabelling with NAD+ and the magnitude of specific NAD+ glycohydrolase activity exhibited by the mutants. Enzyme kinetic analyses of the N-terminal mutants also identified a mutant with substantially reduced activity, a depressed photolabelling efficiency and a markedly increased Km for NAD+. The results support a direct role for the N-terminal region of the S1 subunit in the binding of NAD+, thereby providing a rationale for the effect of mutations in this region on enzymic activity. Images Fig. 1. PMID:2363691

  13. Determination of Activity of the Enzymes Hypoxanthine Phosphoribosyl Transferase (HPRT) and Adenine Phosphoribosyl Transferase (APRT) in Blood Spots on Filter Paper.

    PubMed

    Auler, Kasie; Broock, Robyn; Nyhan, William L

    2015-01-01

    Hypoxanthine-guanine phosphoribosyl-transferase (HPRT) deficiency is the cause of Lesch-Nyhan disease. Adenine phosphoribosyl-transferase (APRT) deficiency causes renal calculi. The activity of each enzyme is readily determined on spots of whole blood on filter paper. This unit describes a method for detecting deficiencies of HPRT and APRT. PMID:26132002

  14. Effect of ethanol on the expression of hepatic glutathione S-transferase: an in vivo/in vitro study.

    PubMed

    Vanhaecke, T; Lindros, K O; Oinonen, T; Coecke, S; DeBast, G; Phillips, I R; Shephard, E A; Vercruysse, A; Rogiers, V

    2000-11-15

    Ethanol, a human toxicant and a solvent in pharmacological research, is known to interfere with biotransformation of xenobiotics. We compared the in vivo and in vitro long-term effects of ethanol exposure on the expression of glutathione S-transferases (GST, EC 2. 5.1.18) in rat liver. Long-term in vivo ethanol treatment to achieve blood ethanol levels ranging between 10-50 mM was by liquid diet feeding. For in vitro experiments, rat hepatocytes co-cultured with rat liver epithelial cells were exposed to 17 and 68 mM ethanol for up to 10 days. Two weeks of liquid diet ethanol treatment increased total GST activity. Both Mu and Alpha classes and in particular the A1 and A2 subunits and the amount of their corresponding mRNAs were increased. Total GST activity was also increased in co-cultures after exposure to 68 mM ethanol for 10 days. However, the Mu class subunits M1 and M2 and the corresponding mRNAs were increased, rather than the Alpha class subunits. Thus, long-term exposure to ethanol induces hepatic GST both in vivo and in vitro, but different isoenzymes are affected. Consequently, extrapolation of in vitro data on GST expression and regulation to the in vivo situation must be judicious. During xenobiotic metabolism in cell culture, a shift in relative expression and induction of different GST forms may occur, resulting in either an under- or overestimation of effects. PMID:11020451

  15. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1315 Galactose-1-phosphate uridyl transferase test system. (a)...

  16. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1315 Galactose-1-phosphate uridyl transferase test system. (a)...

  17. Cloning, expression and analysis of the olfactory glutathione S-transferases in coho salmon

    PubMed Central

    Espinoza, Herbert M.; Shireman, Laura M.; McClain, Valerie; Atkins, William; Gallagher, Evan P.

    2013-01-01

    The glutathione S-transferases (GSTs) provide cellular protection by detoxifying xenobiotics, maintaining redox status, and modulating secondary messengers, all of which are critical to maintaining olfaction in salmonids. Here, we characterized the major coho salmon olfactory GSTs (OlfGSTs), namely omega, pi, and rho subclasses. OlfGST omega contained an open reading frame of 720 bp and encoded a protein of 239 amino acids. OlfGST pi and OlfGST rho contained open reading frames of 727 and 681 bp, respectively, and encoded proteins of 208 and 226 amino acids. Whole-protein mass spectrometry yielded molecular weights of 29,950, 23,354, and 26,655 Da, respectively, for the GST omega, pi, and rho subunits. Homology modeling using four protein-structure prediction algorithms suggest that the active sites in all three OlfGST isoforms resembled counterparts in other species. The olfactory GSTs conjugated prototypical GST substrates, but only OlfGST rho catalyzed the demethylation of the pesticide methyl parathion. OlfGST pi and rho exhibited thiol oxidoreductase activity towards 2-hydroxyethyl disulfide (2-HEDS) and conjugated 4-hydroxynonenal (HNE), a toxic aldehyde with neurodegenerative properties. The kinetic parameters for OlfGST pi conjugation of HNE were KM = 0.16 ± 0.06 mM and Vmax = 0.5 ± 0.1 μmol min−1 mg−1 for OlfGST pi, whereas OlfGST rho was more efficient at catalyzing HNE conjugation (KM = 0.022 ± 0.008 mM and Vmax = 0.47 ± 0.05 μmol min−1 mg−1). Our findings indicate that the peripheral olfactory system of coho expresses GST isoforms that detoxify certain electrophiles and pesticides and that help maintain redox statusand signal transduction. PMID:23261526

  18. Cloning, expression and analysis of the olfactory glutathione S-transferases in coho salmon.

    PubMed

    Espinoza, Herbert M; Shireman, Laura M; McClain, Valerie; Atkins, William; Gallagher, Evan P

    2013-03-15

    The glutathione S-transferases (GSTs) provide cellular protection by detoxifying xenobiotics, maintaining redox status, and modulating secondary messengers, all of which are critical to maintaining olfaction in salmonids. Here, we characterized the major coho salmon olfactory GSTs (OlfGSTs), namely omega, pi, and rho subclasses. OlfGST omega contained an open reading frame of 720bp and encoded a protein of 239 amino acids. OlfGST pi and OlfGST rho contained open reading frames of 627 and 681nt, respectively, and encoded proteins of 208 and 226 amino acids. Whole-protein mass spectrometry yielded molecular weights of 29,950, 23,354, and 26,655Da, respectively, for the GST omega, pi, and rho subunits. Homology modeling using four protein-structure prediction algorithms suggest that the active sites in all three OlfGST isoforms resembled counterparts in other species. The olfactory GSTs conjugated prototypical GST substrates, but only OlfGST rho catalyzed the demethylation of the pesticide methyl parathion. OlfGST pi and rho exhibited thiol oxidoreductase activity toward 2-hydroxyethyl disulfide (2-HEDS) and conjugated 4-hydroxynonenal (HNE), a toxic aldehyde with neurodegenerative properties. The kinetic parameters for OlfGST pi conjugation of HNE were K(M)=0.16 ± 0.06mM and V(max)=0.5 ± 0.1μmolmin⁻¹mg⁻¹, whereas OlfGST rho was more efficient at catalyzing HNE conjugation (K(M)=0.022 ± 0.008 mM and V(max)=0.47 ± 0.05μmolmin⁻¹mg⁻¹). Our findings indicate that the peripheral olfactory system of coho expresses GST isoforms that detoxify certain electrophiles and pesticides and that help maintain redox status and signal transduction. PMID:23261526

  19. Biochemical studies on glutathione S-transferase from the bovine filarial worm Setaria digitata.

    PubMed

    Srinivasan, Lakshmy; Mathew, Nisha; Karunan, Twinkle; Muthuswamy, Kalyanasundaram

    2011-07-01

    Setaria digitata is a filarial worm of the cattle used as a model system for antifilarial drug screening, due to its similarity to the human filarial parasites Wuchereria bancrofti and Brugia malayi. Since filarial glutathione S-transferase (GST) is a good biochemical target for antifilarial drug development, a study has been undertaken for the biochemical characterization of GST from S. digitata. Cytosolic fraction was separated from the crude S.digitata worm homogenate by ultracentrifugation at 100,000 g and subjected to ammonium sulfate precipitation followed by affinity chromatography using GSH-agarose column. The kinetic parameters K (m) and V (max) values with respect to GSH were 0.45 mM and 0.105 μmol min(-1) mL(-1) respectively. With respect to 1-chloro-2,4-dinitrobenzene, the K (m) and V (max) values were 1.21 and 0.117 μmol min(-1) mL(-1) respectively. The effect of temperature and pH on GST enzyme activity was studied. The protein retained its enzyme activity between 0°C and 40°C, beyond which it showed a decreasing tendency, and at 80°C, the activity was lost completely. The enzyme activity was varying with change in pH, and the maximum GST activity was observed at pH 7.5. Gel filtration chromatographic studies indicated that the protein has a native molecular mass of about 54 kDa. The single band of GST subunit appeared in sodium dodecyl sulfate polyacrylamide gel electrophoresis was found to have molecular mass of ∼27 kDa. This shows that cytosolic S. digitata GST protein is homodimeric in nature. PMID:21207063

  20. AMP-activated protein kinase regulates nicotinamide phosphoribosyl transferase expression in skeletal muscle

    PubMed Central

    Brandauer, Josef; Vienberg, Sara G; Andersen, Marianne A; Ringholm, Stine; Risis, Steve; Larsen, Per S; Kristensen, Jonas M; Frøsig, Christian; Leick, Lotte; Fentz, Joachim; Jørgensen, Sebastian; Kiens, Bente; Wojtaszewski, Jørgen F P; Richter, Erik A; Zierath, Juleen R; Goodyear, Laurie J; Pilegaard, Henriette; Treebak, Jonas T

    2013-01-01

    Deacetylases such as sirtuins (SIRTs) convert NAD to nicotinamide (NAM). Nicotinamide phosphoribosyl transferase (Nampt) is the rate-limiting enzyme in the NAD salvage pathway responsible for converting NAM to NAD to maintain cellular redox state. Activation of AMP-activated protein kinase (AMPK) increases SIRT activity by elevating NAD levels. As NAM directly inhibits SIRTs, increased Nampt activation or expression could be a metabolic stress response. Evidence suggests that AMPK regulates Nampt mRNA content, but whether repeated AMPK activation is necessary for increasing Nampt protein levels is unknown. To this end, we assessed whether exercise training- or 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR)-mediated increases in skeletal muscle Nampt abundance are AMPK dependent. One-legged knee-extensor exercise training in humans increased Nampt protein by 16% (P < 0.05) in the trained, but not the untrained leg. Moreover, increases in Nampt mRNA following acute exercise or AICAR treatment (P < 0.05 for both) were maintained in mouse skeletal muscle lacking a functional AMPK α2 subunit. Nampt protein was reduced in skeletal muscle of sedentary AMPK α2 kinase dead (KD), but 6.5 weeks of endurance exercise training increased skeletal muscle Nampt protein to a similar extent in both wild-type (WT) (24%) and AMPK α2 KD (18%) mice. In contrast, 4 weeks of daily AICAR treatment increased Nampt protein in skeletal muscle in WT mice (27%), but this effect did not occur in AMPK α2 KD mice. In conclusion, functional α2-containing AMPK heterotrimers are required for elevation of skeletal muscle Nampt protein, but not mRNA induction. These findings suggest AMPK plays a post-translational role in the regulation of skeletal muscle Nampt protein abundance, and further indicate that the regulation of cellular energy charge and nutrient sensing is mechanistically related. PMID:23918774

  1. Biochemical Warfare on the Reef: The Role of Glutathione Transferases in Consumer Tolerance of Dietary Prostaglandins

    PubMed Central

    Whalen, Kristen E.; Lane, Amy L.; Kubanek, Julia; Hahn, Mark E.

    2010-01-01

    Background Despite the profound variation among marine consumers in tolerance for allelochemically-rich foods, few studies have examined the biochemical adaptations underlying diet choice. Here we examine the role of glutathione S-transferases (GSTs) in the detoxification of dietary allelochemicals in the digestive gland of the predatory gastropod Cyphoma gibbosum, a generalist consumer of gorgonian corals. Controlled laboratory feeding experiments were used to investigate the influence of gorgonian diet on Cyphoma GST activity and isoform expression. Gorgonian extracts and semi-purified fractions were also screened to identify inhibitors and possible substrates of Cyphoma GSTs. In addition, we investigated the inhibitory properties of prostaglandins (PGs) structurally similar to antipredatory PGs found in high concentrations in the Caribbean gorgonian Plexaura homomalla. Principal Findings Cyphoma GST subunit composition was invariant and activity was constitutively high regardless of gorgonian diet. Bioassay-guided fractionation of gorgonian extracts revealed that moderately hydrophobic fractions from all eight gorgonian species examined contained putative GST substrates/inhibitors. LC-MS and NMR spectral analysis of the most inhibitory fraction from P. homomalla subsequently identified prostaglandin A2 (PGA2) as the dominant component. A similar screening of commercially available prostaglandins in series A, E, and F revealed that those prostaglandins most abundant in gorgonian tissues (e.g., PGA2) were also the most potent inhibitors. In vivo estimates of PGA2 concentration in digestive gland tissues calculated from snail grazing rates revealed that Cyphoma GSTs would be saturated with respect to PGA2 and operating at or near physiological capacity. Significance The high, constitutive activity of Cyphoma GSTs is likely necessitated by the ubiquitous presence of GST substrates and/or inhibitors in this consumer's gorgonian diet. This generalist's GSTs may

  2. Glutathione S-transferases in rat olfactory epithelium: purification, molecular properties and odorant biotransformation.

    PubMed Central

    Ben-Arie, N; Khen, M; Lancet, D

    1993-01-01

    The olfactory epithelium is exposed to a variety of xenobiotic chemicals, including odorants and airborne toxic compounds. Recently, two novel, highly abundant, olfactory-specific biotransformation enzymes have been identified: cytochrome P-450olf1 and olfactory UDP-glucuronosyltransferase (UGT(olf)). The latter is a phase II biotransformation enzyme which catalyses the glucuronidation of alcohols, thiols, amines and carboxylic acids. Such covalent modification, which markedly affects lipid solubility and agonist potency, may be particularly important in the rapid termination of odorant signals. We report here the identification and characterization of a second olfactory phase II biotransformation enzyme, a glutathione S-transferase (GST). The olfactory epithelial cytosol shows the highest GST activity among the extrahepatic tissues examined. Significantly, olfactory epithelium had an activity 4-7 times higher than in other airway tissues, suggesting a role for this enzyme in chemoreception. The olfactory GST has been affinity-purified to homogeneity, and shown by h.p.l.c. and N-terminal amino acid sequencing to constitute mainly the Yb1 and Yb2 subunits, different from most other tissues that have mixtures of more enzyme classes. The identity of the olfactory enzymes was confirmed by PCR cloning and restriction enzyme analysis. Most importantly, the olfactory GSTs were found to catalyse glutathione conjugation of several odorant classes, including many unsaturated aldehydes and ketones, as well as epoxides. Together with UGT(olf), olfactory GST provides the necessary broad coverage of covalent modification capacity, which may be crucial for the acuity of the olfactory process. Images Figure 1 Figure 4 Figure 5 PMID:8503873

  3. Activities of the peptidyl transferase center of ribosomes lacking protein L27

    PubMed Central

    Maracci, Cristina; Wohlgemuth, Ingo; Rodnina, Marina V.

    2015-01-01

    The ribosome is the molecular machine responsible for protein synthesis in all living organisms. Its catalytic core, the peptidyl transferase center (PTC), is built of rRNA, although several proteins reach close to the inner rRNA shell. In the Escherichia coli ribosome, the flexible N-terminal tail of the ribosomal protein L27 contacts the A- and P-site tRNA. Based on computer simulations of the PTC and on previous biochemical evidence, the N-terminal α-amino group of L27 was suggested to take part in the peptidyl-transfer reaction. However, the contribution of this group to catalysis has not been tested experimentally. Here we investigate the role of L27 in peptide-bond formation using fast kinetics approaches. We show that the rate of peptide-bond formation at physiological pH, both with aminoacyl-tRNA or with the substrate analog puromycin, is independent of the presence of L27; furthermore, translation of natural mRNAs is only marginally affected in the absence of L27. The pH dependence of the puromycin reaction is unaltered in the absence of L27, indicating that the N-terminal α-amine is not the ionizing group taking part in catalysis. Likewise, L27 is not required for the peptidyl-tRNA hydrolysis during termination. Thus, apart from the known effect on subunit association, which most likely explains the phenotype of the deletion strains, L27 does not appear to be a key player in the core mechanism of peptide-bond formation on the ribosome. PMID:26475831

  4. Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin

    PubMed Central

    Ralat, Luis A.; Misquitta, Stephanie A.; Manevich, Yefim; Fisher, Aron B.; Colman, Roberta F.

    2016-01-01

    Glutathione S-transferase pi has been shown to reactivate 1-cysteine peroxiredoxin (1-Cys Prx) by formation of a complex. A model of the complex was proposed based on the crystal structures of the two enzymes. We have now characterized the complex of GST pi/1-Cys Prx by determining the Mw of the complex, by measuring the catalytic activity of the GST pi monomer, and by identifying the interaction sites between GST pi and 1-Cys Prx. The Mw of the purified GST pi/1-Cys Prx complex is 50,200 at pH 8.0 in the presence of 2.5 mM glutathione, as measured by light scattering, providing direct evidence that the active complex is a heterodimer composed of equimolar amounts of the two proteins. In the presence of 4 M KBr, GST pi is dissociated to monomer and retains catalytic activity, but the Km value for GSH is increased substantially. To identify the peptides of GST pi that interact with 1-Cys Prx, GST pi was digested with V8 protease and the peptides were purified. The binding by 1-Cys Prx of each of four pure GST pi peptides (residues 41–85, 115–124, 131–163, and 164–197) was investigated by protein fluorescence titration. An apparent stoichiometry of 1 mol/subunit 1-Cys Prx was measured for each peptide and the formation of the heterodimer is decreased when these peptides are included in the incubation mixture. These results support our proposed model of the heterodimer. PMID:18358825

  5. Glutathione transferase mimics: micellar catalysis of an enzymic reaction.

    PubMed Central

    Lindkvist, B; Weinander, R; Engman, L; Koetse, M; Engberts, J B; Morgenstern, R

    1997-01-01

    Substances that mimic the enzyme action of glutathione transferases (which serve in detoxification) are described. These micellar catalysts enhance the reaction rate between thiols and activated halogenated nitroarenes as well as alpha,beta-unsaturated carbonyls. The nucleophilic aromatic substitution reaction is enhanced by the following surfactants in descending order: poly(dimethyldiallylammonium - co - dodecylmethyldiallylammonium) bromide (86/14) >>cetyltrimethylammonium bromide>zwittergent 3-16 (n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate)>zwittergent+ ++ 3-14 (n-tetradecyl-N,N-dimethyl - 3 - ammonio -1 - propanesulphonate) approximately N,N - dimethyl - laurylamine N-oxide>N,N-dimethyloctylamine N-oxide. The most efficient catalyst studied is a polymeric material that incorporates surfactant properties (n-dodecylmethyldiallylammonium bromide) and opens up possibilities for engineering sequences of reactions on a polymeric support. Michael addition to alpha,beta-unsaturated carbonyls is exemplified by a model substance, trans-4-phenylbut-3-en-2-one, and a toxic compound that is formed during oxidative stress, 4-hydroxy-2-undecenal. The latter compound is conjugated with the highest efficiency of those tested. Micellar catalysts can thus be viewed as simple models for the glutathione transferases highlighting the influence of a positive electrostatic field and a non-specific hydrophobic binding site, pertaining to two catalytic aspects, namely thiolate anion stabilization and solvent shielding. PMID:9173899

  6. Caffeine Junkie: an Unprecedented Glutathione S-Transferase-Dependent Oxygenase Required for Caffeine Degradation by Pseudomonas putida CBB5

    PubMed Central

    Summers, Ryan M.; Seffernick, Jennifer L.; Quandt, Erik M.; Yu, Chi Li; Barrick, Jeffrey E.

    2013-01-01

    Caffeine and other N-methylated xanthines are natural products found in many foods, beverages, and pharmaceuticals. Therefore, it is not surprising that bacteria have evolved to live on caffeine as a sole carbon and nitrogen source. The caffeine degradation pathway of Pseudomonas putida CBB5 utilizes an unprecedented glutathione-S-transferase-dependent Rieske oxygenase for demethylation of 7-methylxanthine to xanthine, the final step in caffeine N-demethylation. The gene coding this function is unusual, in that the iron-sulfur and non-heme iron domains that compose the normally functional Rieske oxygenase (RO) are encoded by separate proteins. The non-heme iron domain is located in the monooxygenase, ndmC, while the Rieske [2Fe-2S] domain is fused to the RO reductase gene, ndmD. This fusion, however, does not interfere with the interaction of the reductase with N1- and N3-demethylase RO oxygenases, which are involved in the initial reactions of caffeine degradation. We demonstrate that the N7-demethylation reaction absolutely requires a unique, tightly bound protein complex composed of NdmC, NdmD, and NdmE, a novel glutathione-S-transferase (GST). NdmE is proposed to function as a noncatalytic subunit that serves a structural role in the complexation of the oxygenase (NdmC) and Rieske domains (NdmD). Genome analyses found this gene organization of a split RO and GST gene cluster to occur more broadly, implying a larger function for RO-GST protein partners. PMID:23813729

  7. Modelling and bioinformatics studies of the human Kappa-class glutathione transferase predict a novel third glutathione transferase family with similarity to prokaryotic 2-hydroxychromene-2-carboxylate isomerases.

    PubMed Central

    Robinson, Anna; Huttley, Gavin A; Booth, Hilary S; Board, Philip G

    2004-01-01

    The Kappa class of GSTs (glutathione transferases) comprises soluble enzymes originally isolated from the mitochondrial matrix of rats. We have characterized a Kappa class cDNA from human breast. The cDNA is derived from a single gene comprising eight exons and seven introns located on chromosome 7q34-35. Recombinant hGSTK1-1 was expressed in Escherichia coli as a homodimer (subunit molecular mass approximately 25.5 kDa). Significant glutathione-conjugating activity was found only with the model substrate CDNB (1-chloro-2,4-ditnitrobenzene). Hyperbolic kinetics were obtained for GSH (parameters: K(m)app, 3.3+/-0.95 mM; V(max)app, 21.4+/-1.8 micromol/min per mg of enzyme), while sigmoidal kinetics were obtained for CDNB (parameters: S0.5app, 1.5+/-1.0 mM; V(max)app, 40.3+/-0.3 micromol/min per mg of enzyme; Hill coefficient, 1.3), reflecting low affinities for both substrates. Sequence analyses, homology modelling and secondary structure predictions show that hGSTK1 has (a) most similarity to bacterial HCCA (2-hydroxychromene-2-carboxylate) isomerases and (b) a predicted C-terminal domain structure that is almost identical to that of bacterial disulphide-bond-forming DsbA oxidoreductase (root mean square deviation 0.5-0.6 A). The structures of hGSTK1 and HCCA isomerase are predicted to possess a thioredoxin fold with a polyhelical domain (alpha(x)) embedded between the beta-strands (betaalphabetaalpha(x)betabetaalpha, where the underlined elements represent the N and C motifs of the thioredoxin fold), as occurs in the bacterial disulphide-bond-forming oxidoreductases. This is in contrast with the cytosolic GSTs, where the helical domain occurs exclusively at the C-terminus (betaalphabetaalphabetabetaalphaalpha(x)). Although hGSTK1-1 catalyses some typical GST reactions, we propose that it is structurally distinct from other classes of cytosolic GSTs. The present study suggests that the Kappa class may have arisen in prokaryotes well before the divergence of the

  8. Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family

    PubMed Central

    Schürmann, Marc; Hirsch, Beatrice; Wübbeler, Jan Hendrik; Stöveken, Nadine

    2013-01-01

    The act gene of Variovorax paradoxus TBEA6 encodes a succinyl-CoA:3-sulfinopropionate coenzyme A (CoA)-transferase, ActTBEA6 (2.8.3.x), which catalyzes the activation of 3-sulfinopropionate (3SP), an intermediate during 3,3′-thiodipropionate (TDP) degradation. In a previous study, accumulation of 3SP was observed in a Tn5::mob-induced mutant defective in growth on TDP. In contrast to the wild type and all other obtained mutants, this mutant showed no growth when 3SP was applied as the sole source of carbon and energy. The transposon Tn5::mob was inserted in a gene showing high homology to class III CoA-transferases. In the present study, analyses of the translation product clearly allocated ActTBEA6 to this protein family. The predicted secondary structure indicates the lack of a C-terminal α-helix. ActTBEA6 was heterologously expressed in Escherichia coli Lemo21(DE3) and was then purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography. Analytical size exclusion chromatography revealed a homodimeric structure with a molecular mass of 96 ± 3 kDa. Enzyme assays identified succinyl-CoA, itaconyl-CoA, and glutaryl-CoA as potential CoA donors and unequivocally verified the conversion of 3SP to 3SP-CoA. Kinetic studies revealed an apparent Vmax of 44.6 μmol min−1 mg−1 for succinyl-CoA, which corresponds to a turnover number of 36.0 s−1 per subunit of ActTBEA6. For 3SP, the apparent Vmax was determined as 46.8 μmol min−1 mg−1, which corresponds to a turnover number of 37.7 s−1 per subunit of ActTBEA6. The apparent Km values were 0.08 mM for succinyl-CoA and 5.9 mM for 3SP. Nonetheless, the V. paradoxus Δact mutant did not reproduce the phenotype of the Tn5::mob-induced mutant. This defined deletion mutant was able to utilize TDP or 3SP as the sole carbon source, like the wild type. Complementation of the Tn5::mob-induced mutant with pBBR1MCS5::acdDPN7 partially restored growth on 3SP, which indicated a polar effect of the Tn5::mob transposon

  9. 1-3-A Resolution Structure of Human Glutathione S-Transferase With S-Hexyl Glutathione Bound Reveals Possible Extended Ligandin Binding Site

    SciTech Connect

    Trong, I.Le; Stenkamp, R.E.; Ibarra, C.; Atkins, W.M.; Adman, E.T.

    2005-08-22

    Cytosolic glutathione S-transferases (GSTs) play a critical role in xenobiotic binding and metabolism, as well as in modulation of oxidative stress. Here, the high-resolution X-ray crystal structures of homodimeric human GSTA1-1 in the apo form and in complex with S-hexyl glutathione (two data sets) are reported at 1.8, 1.5, and 1.3A respectively. At this level of resolution, distinct conformations of the alkyl chain of S-hexyl glutathione are observed, reflecting the nonspecific nature of the hydrophobic substrate binding site (H-site). Also, an extensive network of ordered water, including 75 discrete solvent molecules, traverses the open subunit-subunit interface and connects the glutathione binding sites in each subunit. In the highest-resolution structure, three glycerol moieties lie within this network and directly connect the amino termini of the glutathione molecules. A search for ligand binding sites with the docking program Molecular Operating Environment identified the ordered water network binding site, lined mainly with hydrophobic residues, suggesting an extended ligand binding surface for nonsubstrate ligands, the so-called ligandin site. Finally, detailed comparison of the structures reported here with previously published X-ray structures reveal a possible reaction coordinate for ligand-dependent conformational changes in the active site and the C-terminus.

  10. [Selective N-heterylazimine inhibition of reactions catalyzed by rat liver glutathione transferase].

    PubMed

    Stulovskiĭ, A V; Voznyĭ, I V; Rozengart, E V; Suvorov, A A; Khovanskikh, A E

    1992-01-01

    Three reactions (nucleophile substitution, thiolysis and N-deoxygenation) catalyzed by rat liver glutathione transferase have been studied using several N-heterylazimine inhibitors. The inhibitors are sharply different in their effectiveness in the transferase reactions. Their efficiency depends on their structure. The mechanism which underlies the found regularities is suggested. PMID:1413125

  11. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 28 Judicial Administration 2 2012-07-01 2012-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  12. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  13. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 28 Judicial Administration 2 2011-07-01 2011-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  14. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 28 Judicial Administration 2 2013-07-01 2013-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  15. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 28 Judicial Administration 2 2014-07-01 2014-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  16. Glutathione S-transferase class {pi} polymorphism in baboons

    SciTech Connect

    Aivaliotis, M.J.; Cantu, T.; Gilligan, R.

    1995-02-01

    Glutathione S-transferase (GST) comprises a family of isozymes with broad substrate specificities. One or more GST isozymes are present in most animal tissues and function in several detoxification pathways through the conjugation of reduced glutathione with various electrophiles, thereby reducing their potential toxicity. Four soluble GST isozymes encoded by genes on different chromosomes have been identified in humans. The acidic class pi GST, GSTP (previously designated GST-3), is widely distributed in adult tissues and appears to be the only GST isozyme present in leukocytes and placenta. Previously reported electrophoretic analyses of erythrocyte and leukocyte extracts revealed single bands of activity, which differed slightly in mobility between the two cell types, or under other conditions, a two-banded pattern. To our knowledge, no genetically determined polymorphisms have previously been reported in GSTP from any species. We now report a polymorphism of GSTP in baboon leukocytes, and present family data that verifies autosomal codominant inheritance. 14 refs., 2 figs., 1 tab.

  17. Recombinant baculovirus vectors expressing glutathione-S-transferase fusion proteins.

    PubMed

    Davies, A H; Jowett, J B; Jones, I M

    1993-08-01

    Recombinant baculoviruses are a popular means of producing heterologous protein in eukaryotic cells. Purification of recombinant proteins away from the insect cell background can, however, remain an obstacle for many developments. Recently, prokaryotic fusion protein expression systems have been developed allowing single-step purification of the heterologous protein and specific proteolytic cleavage of the affinity tag moiety from the desired antigen. Here we report the introduction of these attributes to the baculovirus system. "Baculo-GEX" vectors enable baculovirus production of fusion proteins with the above advantages, but in a eukaryotic post-translational processing environment. Glutathione-S-transferase (GST) fusions are stable cytoplasmic proteins in insect cells and may therefore be released by sonication alone, avoiding the solubility problems and detergent requirements of bacterial systems. Thus large amounts of authentic antigen may be purified in a single, non-denaturing step. PMID:7763917

  18. Functional analysis and localisation of a delta-class glutathione S-transferase from Sarcoptes scabiei.

    PubMed

    Pettersson, Eva U; Ljunggren, Erland L; Morrison, David A; Mattsson, Jens G

    2005-01-01

    The mite Sarcoptes scabiei causes sarcoptic mange, or scabies, a disease that affects both animals and humans worldwide. Our interest in S. scabiei led us to further characterise a glutathione S-transferase. This multifunctional enzyme is a target for vaccine and drug development in several parasitic diseases. The S. scabiei glutathione S-transferase open reading frame reported here is 684 nucleotides long and yields a protein with a predicted molecular mass of 26 kDa. Through phylogenetic analysis the enzyme was classified as a delta-class glutathione S-transferase, and our paper is the first to report that delta-class glutathione S-transferases occur in organisms other than insects. The recombinant S. scabiei glutathione S-transferase was expressed in Escherichia coli via three different constructs and purified for biochemical analysis. The S. scabiei glutathione S-transferase was active towards the substrate 1-chloro-2,4-dinitrobenzene, though the positioning of fusion partners influenced the kinetic activity of the enzyme. Polyclonal antibodies raised against S. scabiei glutathione S-transferase specifically localised the enzyme to the integument of the epidermis and cavities surrounding internal organs in adult parasites. However, some minor staining of parasite intestines was observed. No staining was seen in host tissues, nor could we detect any antibody response against S. scabiei glutathione S-transferase in sera from naturally S. scabiei infected dogs or pigs. Additionally, the polyclonal sera raised against recombinant S. scabiei glutathione S-transferase readily detected a protein from mites, corresponding to the predicted size of native glutathione S-transferase. PMID:15619514

  19. Protein degradation corrects for imbalanced subunit stoichiometry in OST complex assembly

    PubMed Central

    Mueller, Susanne; Wahlander, Asa; Selevsek, Nathalie; Otto, Claudia; Ngwa, Elsy Mankah; Poljak, Kristina; Frey, Alexander D.; Aebi, Markus; Gauss, Robert

    2015-01-01

    Protein degradation is essential for cellular homeostasis. We developed a sensitive approach to examining protein degradation rates in Saccharomyces cerevisiae by coupling a SILAC approach to selected reaction monitoring (SRM) mass spectrometry. Combined with genetic tools, this analysis made it possible to study the assembly of the oligosaccharyl transferase complex. The ER-associated degradation machinery compensated for disturbed homeostasis of complex components by degradation of subunits in excess. On a larger scale, protein degradation in the ER was found to be a minor factor in the regulation of protein homeostasis in exponentially growing cells, but ERAD became relevant when the gene dosage was affected, as demonstrated in heterozygous diploid cells. Hence the alleviation of fitness defects due to abnormal gene copy numbers might be an important function of protein degradation. PMID:25995378

  20. Impact of Ancillary Subunits on Ventricular Repolarization

    PubMed Central

    Abbott, Geoffrey W.; Xu, Xianghua; Roepke, Torsten K.

    2007-01-01

    Voltage-gated potassium (Kv) channels generate the outward K+ ion currents that constitute the primary force in ventricular repolarization. Kv channels comprise tetramers of pore-forming α subunits and, in probably the majority of cases in vivo, ancillary or β subunits that help define the properties of the Kv current generated. Ancillary subunits can be broadly categorized as cytoplasmic or transmembrane, and can modify Kv channel trafficking, conductance, gating, ion selectivity, regulation and pharmacology. Because of their often profound effects on Kv channel function, studies of the molecular correlates of ventricular repolarization must take into account ancillary subunits as well as α subunits. Cytoplasmic ancillary subunits include the Kvβ subunits, which regulate a range of Kv channels and may link channel gating to redox potential; and the KChIPs, which appear most often associated with Kv4 subfamily channels that generate the ventricular Ito current. Transmembrane ancillary subunits include the MinK-related proteins (MiRPs) encoded by KCNE genes, which modulate members of most Kv α subunit subfamilies; and the putative 12-transmembrane domain KCR1 protein which modulates hERG. In some cases, such as the ventricular IKs channel complex, it is well-established that the KCNQ1 α subunit must co-assemble with the MinK (KCNE1) single transmembrane domain ancillary subunit for recapitulation of the characteristic, unusually slowly-activating IKs current. In other cases it is not so clear-cut, and in particular the roles of the other MinK-related proteins (MiRPs 1–4) in regulating cardiac Kv channels such as KCNQ1 and hERG in vivo are under debate. MiRP1 alters hERG function and pharmacology, and inherited MiRP1 mutations are associated with inherited and acquired arrhythmias, but controversy exists over the native role of MiRP1 in regulating hERG (and therefore ventricular IKr) in vivo. Some ancillary subunits may exhibit varied expression to shape

  1. Exploiting topological constraints to reveal buried sequence motifs in the membrane-bound N-linked oligosaccharyl transferases.

    PubMed

    Jaffee, Marcie B; Imperiali, Barbara

    2011-09-01

    The central enzyme in N-linked glycosylation is the oligosaccharyl transferase (OTase), which catalyzes glycan transfer from a polyprenyldiphosphate-linked carrier to select asparagines within acceptor proteins. PglB from Campylobacter jejuni is a single-subunit OTase with homology to the Stt3 subunit of the complex multimeric yeast OTase. Sequence identity between PglB and Stt3 is low (17.9%); however, both have a similar predicted architecture and contain the conserved WWDxG motif. To investigate the relationship between PglB and other Stt3 proteins, sequence analysis was performed using 28 homologues from evolutionarily distant organisms. Since detection of small conserved motifs within large membrane-associated proteins is complicated by divergent sequences surrounding the motifs, we developed a program to parse sequences according to predicted topology and then analyze topologically related regions. This approach identified three conserved motifs that served as the basis for subsequent mutagenesis and functional studies. This work reveals that several inter-transmembrane loop regions of PglB/Stt3 contain strictly conserved motifs that are essential for PglB function. The recent publication of a 3.4 Å resolution structure of full-length C. lari OTase provides clear structural evidence that these loops play a fundamental role in catalysis [ Lizak , C. ; ( 2011 ) Nature 474 , 350 - 355 ]. The current study provides biochemical support for the role of the inter-transmembrane domain loops in OTase catalysis and demonstrates the utility of combining topology prediction and sequence analysis for exposing buried pockets of homology in large membrane proteins. The described approach allowed detection of the catalytic motifs prior to availability of structural data and reveals additional catalytically relevant residues that are not predicted by structural data alone. PMID:21812456

  2. Plasma glutathione S-transferase and F protein are more sensitive than alanine aminotransferase as markers of paracetamol (acetaminophen)-induced liver damage.

    PubMed

    Beckett, G J; Foster, G R; Hussey, A J; Oliveira, D B; Donovan, J W; Prescott, L F; Proudfoot, A T

    1989-11-01

    Concentrations of glutathione S-transferase (GST; glutathione transferase; EC 2.5.1.18) B1 subunits, F protein, and the activity of alanine aminotransferase (ALT; EC 2.6.1.2) were measured in sequential plasma samples taken from nine patients with self-administered paracetamol (acetaminophen) poisoning. GST exceeded the reference interval in all patients at the time of admission, and F protein was increased in seven. In contrast, abnormal activities of ALT in plasma were found in only one of the nine on admission, a patient admitted 12 h after poisoning. Subsequent to admission nine, eight, and five patients, respectively, had abnormal concentrations of GST, F protein, and ALT. When expressed as multiples of the upper reference limit, the highest values for GST measured in each patient always far exceeded the greatest abnormalities in ALT; this was true for F protein in only five patients. Patients in whom the concentration of GST exceeded 10 micrograms/L on admission subsequently went on to develop moderate or severe liver damage, despite treatment with N-acetylcysteine. F protein and ALT measurements on admission were not as efficient as GST at predicting the clinical outcome of the patients. We conclude that GST and F protein offer clear advantages over ALT for detecting minor degrees of acute liver dysfunction, particularly when only centrilobular damage may be involved. PMID:2582614

  3. Identification and Characterization of an Alternatively Spliced Isoform of the Human Protein Phosphatase 2Aα Catalytic Subunit*

    PubMed Central

    Migueleti, Deivid L. S.; Smetana, Juliana H. C.; Nunes, Hugo F.; Kobarg, Jörg; Zanchin, Nilson I. T.

    2012-01-01

    PP2A is the main serine/threonine-specific phosphatase in animal cells. The active phosphatase has been described as a holoenzyme consisting of a catalytic, a scaffolding, and a variable regulatory subunit, all encoded by multiple genes, allowing for the assembly of more than 70 different holoenzymes. The catalytic subunit can also interact with α4, TIPRL (TIP41, TOR signaling pathway regulator-like), the methyl-transferase LCMT-1, and the methyl-esterase PME-1. Here, we report that the gene encoding the catalytic subunit PP2Acα can generate two mRNA types, the standard mRNA and a shorter isoform, lacking exon 5, which we termed PP2Acα2. Higher levels of the PP2Acα2 mRNA, equivalent to the level of the longer PP2Acα mRNA, were detected in peripheral blood mononuclear cells that were left to rest for 24 h. After this time, the peripheral blood mononuclear cells are still viable and the PP2Acα2 mRNA decreases soon after they are transferred to culture medium, showing that generation of the shorter isoform depends on the incubation conditions. FLAG-tagged PP2Acα2 expressed in HEK293 is catalytically inactive. It displays a specific interaction profile with enhanced binding to the α4 regulatory subunit, but no binding to the scaffolding subunit and PME-1. Consistently, α4 out-competes PME-1 and LCMT-1 for binding to both PP2Acα isoforms in pulldown assays. Together with molecular modeling studies, this suggests that all three regulators share a common binding surface on the catalytic subunit. Our findings add important new insights into the complex mechanisms of PP2A regulation. PMID:22167190

  4. Cleft Lip Repair: The Hybrid Subunit Method.

    PubMed

    Tollefson, Travis T

    2016-04-01

    The unilateral cleft lip repair is one of the most rewarding and challenging of plastic surgery procedures. Surgeons have introduced a variety of straight line, geometric, and rotation-advancement designs, while in practice the majority of North American surgeons have been using hybrids of the rotation-advancement techniques. The anatomic subunit approach was introduced in 2005 by Fisher and has gained popularity, with early adopters of the design touting its simplicity and effectiveness. The objectives of this article are to summarize the basic tenets of respecting the philtral subunit, accurate measurement and planning, and tips for transitioning to this subunit approach. PMID:27097136

  5. Specific Prenylation of Tomato Rab Proteins by Geranylgeranyl Type-II Transferase Requires a Conserved Cysteine-Cysteine Motif.

    PubMed Central

    Yalovsky, S.; Loraine, A. E.; Gruissem, W.

    1996-01-01

    Posttranslational isoprenylation of some small GTP-binding proteins is required for their biological activity. Rab geranylgeranyl transferase (Rab GGTase) uses geranylgeranyl pyrophosphate to modify Rab proteins, its only known substrates. Geranylgeranylation of Rabs is believed to promote their association with target membranes and interaction with other proteins. Plants, like other eukaryotes, contain Rab-like proteins that are associated with intracellular membranes. However, to our knowledge, the geranylgeranylation of Rab proteins has not yet been characterized from any plant source. This report presents an activity assay that allows the characterization of prenylation of Rab-like proteins in vitro, by protein extracts prepared from plants. Tomato Rab1 proteins and mammalian Rab1a were modified by geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate. This modification required a conserved cysteine-cysteine motif. A mutant form lacking the cysteine-cysteine motif could not be modified, but inhibited the geranylgeranylation of its wild-type homolog. The tomato Rab proteins were modified in vitro by protein extract prepared from yeast, but failed to become modified when the protein extract was prepared from a yeast strain containing a mutant allele for the [alpha] subunit of yeast Rab GGTase (bet4 ts). These results demonstrate that plant cells, like other eukaryotes, contain Rab GGTase-like activity. PMID:12226265

  6. Inactivation of Anopheles gambiae Glutathione Transferase ε2 by Epiphyllocoumarin

    PubMed Central

    Marimo, Patience; Hayeshi, Rose; Mukanganyama, Stanley

    2016-01-01

    Glutathione transferases (GSTs) are part of a major family of detoxifying enzymes that can catalyze the reductive dehydrochlorination of dichlorodiphenyltrichloroethane (DDT). The delta and epsilon classes of insect GSTs have been implicated in conferring resistance to this insecticide. In this study, the inactivation of Anopheles gambiae GSTε2 by epiphyllocoumarin (Tral 1) was investigated. Recombinant AgGSTε2 was expressed in Escherichia coli cells containing a pET3a-AGSTε2 plasmid and purified by affinity chromatography. Tral 1 was shown to inactivate GSTε2 both in a time-dependent manner and in a concentration-dependent manner. The half-life of GSTε2 in the presence of 25 μM ethacrynic acid (ETA) was 22 minutes and with Tral 1 was 30 minutes, indicating that Tral 1 was not as efficient as ETA as an inactivator. The inactivation parameters kinact and KI were found to be 0.020 ± 0.001 min−1 and 7.5 ± 2.1 μM, respectively, after 90 minutes of incubation. Inactivation of GSTε2 by Tral 1 implies that Tral 1 covalently binds to this enzyme in vitro and would be expected to exhibit time-dependent effects on the enzyme in vivo. Tral 1, therefore, would produce irreversible effects when used together with dichlorodiphenyltrichloroethane (DDT) in malaria control programmes where resistance is mediated by GSTs. PMID:26925266

  7. Crystal structure of E. coli lipoprotein diacylglyceryl transferase

    PubMed Central

    Mao, Guotao; Zhao, Yan; Kang, Xusheng; Li, Zhijie; Zhang, Yan; Wang, Xianping; Sun, Fei; Sankaran, Krishnan; Zhang, Xuejun C.

    2016-01-01

    Lipoprotein biogenesis is essential for bacterial survival. Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is an integral membrane enzyme that catalyses the first reaction of the three-step post-translational lipid modification. Deletion of the lgt gene is lethal to most Gram-negative bacteria. Here we present the crystal structures of Escherichia coli Lgt in complex with phosphatidylglycerol and the inhibitor palmitic acid at 1.9 and 1.6 Å resolution, respectively. The structures reveal the presence of two binding sites and support the previously reported structure–function relationships of Lgt. Complementation results of lgt-knockout cells with different mutant Lgt variants revealed critical residues, including Arg143 and Arg239, that are essential for diacylglyceryl transfer. Using a GFP-based in vitro assay, we correlated the activities of Lgt with structural observations. Together, the structural and biochemical data support a mechanism whereby substrate and product, lipid-modified lipobox-containing peptide, enter and leave the enzyme laterally relative to the lipid bilayer. PMID:26729647

  8. Crystal structure of E. coli lipoprotein diacylglyceryl transferase.

    PubMed

    Mao, Guotao; Zhao, Yan; Kang, Xusheng; Li, Zhijie; Zhang, Yan; Wang, Xianping; Sun, Fei; Sankaran, Krishnan; Zhang, Xuejun C

    2016-01-01

    Lipoprotein biogenesis is essential for bacterial survival. Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is an integral membrane enzyme that catalyses the first reaction of the three-step post-translational lipid modification. Deletion of the lgt gene is lethal to most Gram-negative bacteria. Here we present the crystal structures of Escherichia coli Lgt in complex with phosphatidylglycerol and the inhibitor palmitic acid at 1.9 and 1.6 Å resolution, respectively. The structures reveal the presence of two binding sites and support the previously reported structure-function relationships of Lgt. Complementation results of lgt-knockout cells with different mutant Lgt variants revealed critical residues, including Arg143 and Arg239, that are essential for diacylglyceryl transfer. Using a GFP-based in vitro assay, we correlated the activities of Lgt with structural observations. Together, the structural and biochemical data support a mechanism whereby substrate and product, lipid-modified lipobox-containing peptide, enter and leave the enzyme laterally relative to the lipid bilayer. PMID:26729647

  9. Benzene oxide is a substrate for glutathione S-transferases.

    PubMed

    Zarth, Adam T; Murphy, Sharon E; Hecht, Stephen S

    2015-12-01

    Benzene is a known human carcinogen which must be activated to benzene oxide (BO) to exert its carcinogenic potential. BO can be detoxified in vivo by reaction with glutathione and excretion in the urine as S-phenylmercapturic acid. This process may be catalyzed by glutathione S-transferases (GSTs), but kinetic data for this reaction have not been published. Therefore, we incubated GSTA1, GSTT1, GSTM1, and GSTP1 with glutathione and BO and quantified the formation of S-phenylglutathione. Kinetic parameters were determined for GSTT1 and GSTP1. At 37 °C, the putative Km and Vmax values for GSTT1 were 420 μM and 450 fmol/s, respectively, while those for GSTP1 were 3600 μM and 3100 fmol/s. GSTA1 and GSTM1 did not exhibit sufficient activity for determination of kinetic parameters. We conclude that GSTT1 is a critical enzyme in the detoxification of BO and that GSTP1 may also play an important role, while GSTA1 and GSTM1 seem to be less important. PMID:26554337

  10. Modulation of Rab GTPase function by a protein phosphocholine transferase.

    PubMed

    Mukherjee, Shaeri; Liu, Xiaoyun; Arasaki, Kohei; McDonough, Justin; Galán, Jorge E; Roy, Craig R

    2011-09-01

    The intracellular pathogen Legionella pneumophila modulates the activity of host GTPases to direct the transport and assembly of the membrane-bound compartment in which it resides. In vitro studies have indicated that the Legionella protein DrrA post-translationally modifies the GTPase Rab1 by a process called AMPylation. Here we used mass spectrometry to investigate post-translational modifications to Rab1 that occur during infection of host cells by Legionella. Consistent with in vitro studies, DrrA-mediated AMPylation of a conserved tyrosine residue in the switch II region of Rab1 was detected during infection. In addition, a modification to an adjacent serine residue in Rab1 was discovered, which was independent of DrrA. The Legionella effector protein AnkX was required for this modification. Biochemical studies determined that AnkX directly mediates the covalent attachment of a phosphocholine moiety to Rab1. This phosphocholine transferase activity used CDP-choline as a substrate and required a conserved histidine residue located in the FIC domain of the AnkX protein. During infection, AnkX modified both Rab1 and Rab35, which explains how this protein modulates membrane transport through both the endocytic and exocytic pathways of the host cell. Thus, phosphocholination of Rab GTPases represents a mechanism by which bacterial FIC-domain-containing proteins can alter host-cell functions. PMID:21822290

  11. Glutathione S-transferase activity and glutathione S-transferase mu expression in subjects with risk for colorectal cancer.

    PubMed

    Szarka, C E; Pfeiffer, G R; Hum, S T; Everley, L C; Balshem, A M; Moore, D F; Litwin, S; Goosenberg, E B; Frucht, H; Engstrom, P F

    1995-07-01

    The glutathione S-transferases (alpha, mu, and pi), a family of Phase II detoxication enzymes, play a critical role in protecting the colon mucosa by catalyzing the conjugation of dietary carcinogens with glutathione. We investigated the efficacy of using the glutathione S-transferase (GST) activity of blood lymphocytes and GST-mu expression as biomarkers of risk for colorectal cancer. GST activity was measured in the blood lymphocytes of control individuals (n = 67) and in the blood lymphocytes (n = 60) and colon tissue (n = 34) of individuals at increased risk for colon cancer. Total GST activity was determined spectrophotometrically with the use of 1-chloro-2,4-dinitrobenzene as a substrate. The ability to express the um subclass of GST was determined with the use of an ELISA. Although interindividual variability in the GST activity of blood lymphocytes was greater than 8-fold (range, 16.7-146.8 nmol/min/mg), the GST activity of blood lymphocytes and colon tissue within an individual was constant over time and was unrelated to sex, age, or race. The GST activity of blood lymphocytes from high-risk individuals was significantly lower than that of blood lymphocytes from control individuals (P < or = 0.004). No association was observed between the frequency of GST-mu phenotype and risk for colorectal cancer. Blood lymphocytes from high-risk individuals unable to express GST-mu had lower levels of GST activity than did those from control subjects with the GST-mu null phenotype; however, this difference was significant in male subjects only (P < or = 0.006). Analysis of paired samples of blood lymphocytes and colon tissue indicated a strong correlation between the GST activity of the two tissue types (Spearman's rank correlation, r = 0.87; P < or = 0.0001). The GST activity of blood lymphocytes may be used to identify high-risk individuals with decreased protection from this Phase II detoxication enzyme who may benefit from clinical trials evaluating GST modulators

  12. The K+-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation

    PubMed Central

    Manikas, Rizos-Georgios; Thomson, Emma; Thoms, Matthias; Hurt, Ed

    2016-01-01

    Ribosome synthesis employs a number of energy-consuming enzymes in both eukaryotes and prokaryotes. One such enzyme is the conserved circularly permuted GTPase Nug1 (nucleostemin in human). Nug1 is essential for 60S subunit assembly and nuclear export, but its role and time of action during maturation remained unclear. Based on in vitro enzymatic assays using the Chaetomium thermophilum (Ct) orthologue, we show that Nug1 exhibits a low intrinsic GTPase activity that is stimulated by potassium ions, rendering Nug1 a cation-dependent GTPase. In vivo we observe 60S biogenesis defects upon depletion of yeast Nug1 or expression of a Nug1 nucleotide-binding mutant. Most prominently, the RNA helicase Dbp10 was lost from early pre-60S particles, which suggested a physical interaction that could be reconstituted in vitro using CtNug1 and CtDbp10. In vivo rRNA–protein crosslinking revealed that Nug1 and Dbp10 bind at proximal and partially overlapping sites on the 60S pre-ribosome, most prominently to H89 that will constitute part of the peptidyl transferase center (PTC). The binding sites of Dbp10 are the same as those identified for the prokaryotic helicase DbpA bound to the 50S subunit. We suggest that Dbp10 and DbpA are performing a conserved role during PTC formation in all organisms. PMID:26823502

  13. The K⁺-dependent GTPase Nug1 is implicated in the association of the helicase Dbp10 to the immature peptidyl transferase centre during ribosome maturation.

    PubMed

    Manikas, Rizos-Georgios; Thomson, Emma; Thoms, Matthias; Hurt, Ed

    2016-02-29

    Ribosome synthesis employs a number of energy-consuming enzymes in both eukaryotes and prokaryotes. One such enzyme is the conserved circularly permuted GTPase Nug1 (nucleostemin in human). Nug1 is essential for 60S subunit assembly and nuclear export, but its role and time of action during maturation remained unclear. Based on in vitro enzymatic assays using the Chaetomium thermophilum (Ct) orthologue, we show that Nug1 exhibits a low intrinsic GTPase activity that is stimulated by potassium ions, rendering Nug1 a cation-dependent GTPase. In vivo we observe 60S biogenesis defects upon depletion of yeast Nug1 or expression of a Nug1 nucleotide-binding mutant. Most prominently, the RNA helicase Dbp10 was lost from early pre-60S particles, which suggested a physical interaction that could be reconstituted in vitro using CtNug1 and CtDbp10. In vivo rRNA-protein crosslinking revealed that Nug1 and Dbp10 bind at proximal and partially overlapping sites on the 60S pre-ribosome, most prominently to H89 that will constitute part of the peptidyl transferase center (PTC). The binding sites of Dbp10 are the same as those identified for the prokaryotic helicase DbpA bound to the 50S subunit. We suggest that Dbp10 and DbpA are performing a conserved role during PTC formation in all organisms. PMID:26823502

  14. Selection of antisense oligodeoxynucleotides against glutathione S-transferase Mu.

    PubMed Central

    't Hoen, Peter A C; Out, Ruud; Commandeur, Jan N M; Vermeulen, Nico P E; van Batenburg, F H D; Manoharan, Muthiah; van Berkel, Theo J C; Biessen, Erik A L; Bijsterbosch, Martin K

    2002-01-01

    The aim of the present study was to identify functional antisense oligodeoxynucleotides (ODNs) against the rat glutathione S-transferase Mu (GSTM) isoforms, GSTM1 and GSTM2. These antisense ODNs would enable the study of the physiological consequences of GSTM deficiency. Because it has been suggested that the effectiveness of antisense ODNs is dependent on the secondary mRNA structures of their target sites, we made mRNA secondary structure predictions with two software packages, Mfold and STAR. The two programs produced only marginally similar structures, which can probably be attributed to differences in the algorithms used. The effectiveness of a set of 18 antisense ODNs was evaluated with a cell-free transcription/translation assay, and their activity was correlated with the predicted secondary RNA structures. Four phosphodiester ODNs specific for GSTM1, two ODNs specific for GSTM2, and four ODNs targeted at both GSTM isoforms were found to be potent, sequence-specific, and RNase H-dependent inhibitors of protein expression. The IC50 value of the most potent ODN was approximately 100 nM. Antisense ODNs targeted against regions that were predicted by STAR to be predominantly single stranded were more potent than antisense ODNs against double-stranded regions. Such a correlation was not found for the Mfold prediction. Our data suggest that simulation of the local folding of RNA facilitates the discovery of potent antisense sequences. In conclusion, we selected several promising antisense sequences, which, when synthesized as biologically stable oligonucleotides, can be applied for study of the physiological impact of reduced GSTM expression. PMID:12515389

  15. Analysis of Arabidopsis glutathione-transferases in yeast.

    PubMed

    Krajewski, Matthias P; Kanawati, Basem; Fekete, Agnes; Kowalski, Natalie; Schmitt-Kopplin, Philippe; Grill, Erwin

    2013-07-01

    The genome of Arabidopsis thaliana encodes 54 functional glutathione transferases (GSTs), classified in seven clades. Although plant GSTs have been implicated in the detoxification of xenobiotics, such as herbicides, extensive redundancy within this large gene family impedes a functional analysis in planta. In this study, a GST-deficient yeast strain was established as a system for analyzing plant GSTs that allows screening for GST substrates and identifying substrate preferences within the plant GST family. To this end, five yeast genes encoding GSTs and GST-related proteins were simultaneously disrupted. The resulting yeast quintuple mutant showed a strongly reduced conjugation of the GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl). Consistently, the quintuple mutant was hypersensitive to CDNB, and this phenotype was complemented by the inducible expression of Arabidopsis GSTs. The conjugating activity of the plant GSTs was assessed by in vitro enzymatic assays and via analysis of exposed yeast cells. The formation of glutathione adducts with dinitrobenzene was unequivocally verified by stable isotope labeling and subsequent accurate ultrahigh-resolution mass spectrometry (ICR-FTMS). Analysis of Arabidopsis GSTs encompassing six clades and 42 members demonstrated functional expression in yeast by using CDNB and NBD-Cl as model substrates. Subsequently, the established yeast system was explored for its potential to screen the Arabidopsis GST family for conjugation of the fungicide anilazine. Thirty Arabidopsis GSTs were identified that conferred increased levels of glutathionylated anilazine. Efficient anilazine conjugation was observed in the presence of the phi, tau, and theta clade GSTs including AtGSTF2, AtGSTF4, AtGSTF6, AtGSTF8, AtGSTF10, and AtGSTT2, none of which had previously been known to contribute to fungicide detoxification. ICR-FTMS analysis of yeast extracts allowed the simultaneous detection and

  16. Phosphonocarboxylates Inhibit the Second Geranylgeranyl Addition by Rab Geranylgeranyl Transferase*

    PubMed Central

    Baron, Rudi A.; Tavaré, Richard; Figueiredo, Ana C.; Błażewska, Katarzyna M.; Kashemirov, Boris A.; McKenna, Charles E.; Ebetino, Frank H.; Taylor, Adam; Rogers, Michael J.; Coxon, Fraser P.; Seabra, Miguel C.

    2009-01-01

    Rab geranylgeranyl transferase (RGGT) catalyzes the post-translational geranylgeranyl (GG) modification of (usually) two C-terminal cysteines in Rab GTPases. Here we studied the mechanism of the Rab geranylgeranylation reaction by bisphosphonate analogs in which one phosphonate group is replaced by a carboxylate (phosphonocarboxylate, PC). The phosphonocarboxylates used were 3-PEHPC, which was previously reported, and 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid ((+)-3-IPEHPC), a >25-fold more potent related compound as measured by both IC50 and Ki.(+)-3-IPEHPC behaves as a mixed-type inhibitor with respect to GG pyrophosphate (GGPP) and an uncompetitive inhibitor with respect to Rab substrates. We propose that phosphonocarboxylates prevent only the second GG transfer onto Rabs based on the following evidence. First, geranylgeranylation of Rab proteins ending with a single cysteine motif such as CAAX, is not affected by the inhibitors, either in vitro or in vivo. Second, the addition of an -AAX sequence onto Rab-CC proteins protects the substrate from inhibition by the inhibitors. Third, we demonstrate directly that in the presence of (+)-3-IPEHPC, Rab-CC and Rab-CXC proteins are modified by only a single GG addition. The presence of (+)-3-IPEHPC resulted in a preference for the Rab N-terminal cysteine to be modified first, suggesting an order of cysteine geranylgeranylation in RGGT catalysis. Our results further suggest that the inhibitor binds to a site distinct from the GGPP-binding site on RGGT. We suggest that phosphonocarboxylate inhibitors bind to a GG-cysteine binding site adjacent to the active site, which is necessary to align the mono-GG-Rab for the second GG addition. These inhibitors may represent a novel therapeutic approach in Rab-mediated diseases. PMID:19074143

  17. Amaranth (Amaranthus hypochondriacus) vicilin subunit structure.

    PubMed

    Quiroga, Alejandra; Martínez, E Nora; Rogniaux, Hélène; Geairon, Audrey; Añón, M Cristina

    2010-12-22

    The 7S-globulin fraction is a minor component of the amaranth storage proteins. The present work provides new information about this protein. The amaranth 7S-globulin or vicilin presented a sedimentation coefficient of 8.6 ± 0.6 S and was composed of main subunits of 66, 52, 38, and 16 kDa. On the basis of mass spectrometry (MS) analysis of tryptic fragments, the 52, 38, and 16 kDa subunits presented sequence homology with sesame vicilin, whereas the 66 kDa subunit showed sequence similarity with a putative vicilin. Several characteristics of the 66 kDa subunit were similar to members of the convicilin family. Results support the hypothesis that the 7S-globulin molecules are composed of subunits coming from at least two gene families with primary products of 66 and 52 kDa, respectively. According to the present information, amaranth vicilin may be classified into the vicilin group that includes pea, broad bean, and sesame vicilins, among others. PMID:21117690

  18. Modulation of the skeletal muscle sodium channel alpha-subunit by the beta 1-subunit.

    PubMed

    Wallner, M; Weigl, L; Meera, P; Lotan, I

    1993-12-28

    Co-expression of cloned sodium channel beta 1-subunit with the rat skeletal muscle-subunit (alpha microI) accelerated the macroscopic current decay, enhanced the current amplitude, shifted the steady state inactivation curve to more negative potentials and decreased the time required for complete recovery from inactivation. Sodium channels expressed from skeletal muscle mRNA showed a similar behaviour to that observed from alpha microI/beta 1, indicating that beta 1 restores 'physiological' behaviour. Northern blot analysis revealed that the Na+ channel beta 1-subunit is present in high abundance (about 0.1%) in rat heart, brain and skeletal muscle, and the hybridization with untranslated region of the 'brain' beta 1 cDNA to skeletal muscle and heart mRNA indicated that the different Na+ channel alpha-subunits in brain, skeletal muscle and heart may share a common beta 1-subunit. PMID:8282123

  19. Apo- and Cellopentaose-bound Structures of the Bacterial Cellulose Synthase Subunit BcsZ

    SciTech Connect

    Mazur, Olga; Zimmer, Jochen

    2012-10-25

    Cellulose, a very abundant extracellular polysaccharide, is synthesized in a finely tuned process that involves the activity of glycosyl-transferases and hydrolases. The cellulose microfibril consists of bundles of linear {beta}-1,4-glucan chains that are synthesized inside the cell; however, the mechanism by which these polymers traverse the cell membrane is currently unknown. In Gram-negative bacteria, the cellulose synthase complex forms a trans-envelope complex consisting of at least four subunits. Although three of these subunits account for the synthesis and translocation of the polysaccharide, the fourth subunit, BcsZ, is a periplasmic protein with endo-{beta}-1,4-glucanase activity. BcsZ belongs to family eight of glycosyl-hydrolases, and its activity is required for optimal synthesis and membrane translocation of cellulose. In this study we report two crystal structures of BcsZ from Escherichia coli. One structure shows the wild-type enzyme in its apo form, and the second structure is for a catalytically inactive mutant of BcsZ in complex with the substrate cellopentaose. The structures demonstrate that BcsZ adopts an ({alpha}/{alpha}){sub 6}-barrel fold and that it binds four glucan moieties of cellopentaose via highly conserved residues exclusively on the nonreducing side of its catalytic center. Thus, the BcsZ-cellopentaose structure most likely represents a posthydrolysis state in which the newly formed nonreducing end has already left the substrate binding pocket while the enzyme remains attached to the truncated polysaccharide chain. We further show that BcsZ efficiently degrades {beta}-1,4-glucans in in vitro cellulase assays with carboxymethyl-cellulose as substrate.

  20. Subunit architecture of general transcription factor TFIIH.

    PubMed

    Gibbons, Brian J; Brignole, Edward J; Azubel, Maia; Murakami, Kenji; Voss, Neil R; Bushnell, David A; Asturias, Francisco J; Kornberg, Roger D

    2012-02-01

    Structures of complete 10-subunit yeast TFIIH and of a nested set of subcomplexes, containing 5, 6, and 7 subunits, have been determined by electron microscopy (EM) and 3D reconstruction. Consistency among all the structures establishes the location of the "minimal core" subunits (Ssl1, Tfb1, Tfb2, Tfb4, and Tfb5), and additional densities can be specifically attributed to Rad3, Ssl2, and the TFIIK trimer. These results can be further interpreted by placement of previous X-ray structures into the additional densities to give a preliminary picture of the RNA polymerase II preinitiation complex. In this picture, the key catalytic components of TFIIH, the Ssl2 ATPase/helicase and the Kin28 protein kinase are in proximity to their targets, downstream promoter DNA and the RNA polymerase C-terminal domain. PMID:22308316

  1. Heteromeric assembly of P2X subunits

    PubMed Central

    Saul, Anika; Hausmann, Ralf; Kless, Achim; Nicke, Annette

    2013-01-01

    Transcripts and/or proteins of P2X receptor (P2XR) subunits have been found in virtually all mammalian tissues. Generally more than one of the seven known P2X subunits have been identified in a given cell type. Six of the seven cloned P2X subunits can efficiently form functional homotrimeric ion channels in recombinant expression systems. This is in contrast to other ligand-gated ion channel families, such as the Cys-loop or glutamate receptors, where homomeric assemblies seem to represent the exception rather than the rule. P2XR mediated responses recorded from native tissues rarely match exactly the biophysical and pharmacological properties of heterologously expressed homomeric P2XRs. Heterotrimerization of P2X subunits is likely to account for this observed diversity. While the existence of heterotrimeric P2X2/3Rs and their role in physiological processes is well established, the composition of most other P2XR heteromers and/or the interplay between distinct trimeric receptor complexes in native tissues is not clear. After a description of P2XR assembly and the structure of the intersubunit ATP-binding site, this review summarizes the distribution of P2XR subunits in selected mammalian cell types and the biochemically and/or functionally characterized heteromeric P2XRs that have been observed upon heterologous co-expression of P2XR subunits. We further provide examples where the postulated heteromeric P2XRs have been suggested to occur in native tissues and an overview of the currently available pharmacological tools that have been used to discriminate between homo- and heteromeric P2XRs. PMID:24391538

  2. Post-transcriptional regulation of chloramphenicol acetyl transferase.

    PubMed Central

    Byeon, W H; Weisblum, B

    1984-01-01

    The +1 site for initiation of inducible chloramphenicol acetyl transferase (CAT) mRNA encoded by plasmid pC194 was determined experimentally by using [alpha-32P]ATP-labeled runoff transcripts partially digested with T1 RNase. By partial digestion of the in vitro transcripts with S1, T1, and cobra venom nucleases as probes of mRNA conformation, single- and double-stranded regions, respectively, were also identified. Thus, a prominent inverted complementary repeat sequence was demonstrated spanning the +14 to +50 positions, which contain the complementary sequences CCUCC and GGAGG (the Shine and Dalgarno sequence for synthesis of CAT) symmetrically apposed and paired as part of a perfect 12-base-pair inverted complementary repeat sequence (-19.5 kcal [ca. -81.7 kJ] per mol). The CAT mRNA was stable to digestion by T1 RNase at the four guanosine residues in the Shine and Dalgarno sequence GGAGG , even at 60 degrees C, suggesting that nascent CAT mRNA allows ribosomes to initiate protein synthesis inefficiently and that induction involves post-transcriptional unmasking of the Shine and Dalgarno sequence. Consistent with this model of regulation, we found that cells carrying pC194 , induced with chloramphenicol, contain about the same concentration of pulse-labeled CAT-specific RNA as do uninduced cells. Induction of CAT synthesis by the non- acetylatable chloramphenicol analog fluorothiamphenicol was tested by using minicells of Bacillus subtilis carrying pC194 as well as minicells containing the cloned pC194 derivatives in which parts of the CAT structural gene were deleted in vitro with BAL 31 exonuclease. Optimal induction of both full-length (active) and deleted (inactive) CAT required similar concentrations of fluorothiamphenicol, whereas induction by chloramphenicol required a higher concentration for the wild-type full-length (active) CAT than for the (inactive) deleted CAT. Because synthesis of deleted CAT was inducible, we infer that CAT plays no direct role

  3. Glucomannan synthesis in pea epicotyls: the mannose and glucose transferases.

    PubMed

    Piro, G; Zuppa, A; Dalessandro, G; Northcote, D H

    1993-01-01

    Membrane fractions and digitonin-solubilized enzymes prepared from stem segments isolated from the third internode of etiolated pea seedlings (Pisum sativum L. cv. Alaska) catalyzed the synthesis of a beta-1,4-[14C]mannan from GDP-D-[U-14C]-mannose, a mixed beta-1,3- and beta-1,4-[14C]glucan from GDP-D-[U-14C]-glucose and a beta-1,4-[14C]-glucomannan from both GDP-D-[U-14C]mannose and GDP-D-[U-14C]glucose. The kinetics of the membrane-bound and soluble mannan and glucan synthases were determined. The effects of ions, chelators, inhibitors of lipid-linked saccharides, polyamines, polyols, nucleotides, nucleoside-diphosphate sugars, acetyl-CoA, group-specific chemical probes, phospholipases and detergents on the membrane-bound mannan and glucan synthases were investigated. The beta-glucan synthase had different properties from other preparations which bring about the synthesis of beta-1,3-glucans (callose) and mixed beta-1,3- and beta-1,4- glucans and which use UDP-D-glucose as substrate. It also differed from xyloglucan synthase because in the presence of several concentrations of UDP-D-xylose in addition to GDP-D-glucose no xyloglucan was formed. Using either the membrane-bound or the soluble mannan synthase, GDP-D-glucose acted competitively in the presence of GDP-D-mannose to inhibit the incorporation of mannose into the polymer. This was not due to an inhibition of the transferase activity but was a result of the incorporation of glucose residues from GDP-D-glucose into a glucomannan. The kinetics and the composition of the synthesized glucomannan depended on the ratio of the concentrations of GDP-D-glucose and GDP-D-mannose that were available. Our data indicated that a single enzyme has an active centre that can use both GDP-D-mannose and GDP-D-glucose to bring about the synthesis of the heteropolysaccharide. PMID:7685647

  4. Oxidant regulated inter-subunit disulfide bond formation between ASIC1a subunits

    PubMed Central

    Zha, Xiang-ming; Wang, Runping; Collier, Dan M.; Snyder, Peter M.; Wemmie, John A.; Welsh, Michael J.

    2009-01-01

    The acid-sensing ion channel-1a (ASIC1a) is composed of 3 subunits and is activated by a decrease in extracellular pH. It plays an important role in diseases associated with a reduced pH and production of oxidants. Previous work showed that oxidants reduce ASIC1a currents. However, the effects on channel structure and composition are unknown. We found that ASIC1a formed inter-subunit disulfide bonds and the oxidant H2O2 increased this link between subunits. Cys-495 in the ASIC1a C terminus was particularly important for inter-subunit disulfide bond formation, although other C-terminal cysteines contributed. Inter-subunit disulfide bonds also produced some ASIC1a complexes larger than trimers. Inter-subunit disulfide bond formation reduced the proportion of ASIC1a located on the cell surface and contributed to the H2O2-induced decrease in H+-gated current. These results indicate that channel function is controlled by disulfide bond formation between intracellular residues on distinct ASIC1a subunits. They also suggest a mechanism by which the redox state can dynamically regulate membrane protein activity by forming intracellular bridges. PMID:19218436

  5. Characterization of prenyl protein transferase enzymes in a human keratinocyte cell line.

    PubMed

    MacNulty, E E; Ryder, N S

    1996-02-01

    Prenylation is a post-translational modification of proteins that involves the attachment of an isoprenoid group derived from mevalonic acid, either 15-carbon farnesyl or 20-carbon geranylgeranyl, to a specific carboxy-terminal domain of acceptor proteins. Three prenyl transferase enzymes have been identified so far. In this paper we report the presence of two prenyl transferases in the HaCaT human keratinocyte cell line. Chromatography of a cytosolic extract from these cells resolved a farnesyl protein transferase (FPT) and geranylgeranyl protein transferase-I (GGPT-I) whose activities were measured using a novel peptide-based assay. Both enzymes were inhibited dose dependently by zaragozic acids A and C. Zaragozic acid C was more active towards the FPT than GGPT-I while zaragozic acid A inhibited both enzymes with similar potency. Incubation of HaCaT cell homogenates with [3H] prenyl precursors resulted in the labelling of a number of proteins which was increased when the cells were pretreated with an inhibitor of hydroxymethylglutaryl CoA reductase. Given the role of prenylated proteins in proliferative and inflammatory processes, our finding that prenyl transferases capable of prenylating endogenous substrates are also present in keratinocytes suggests that these enzymes might provide novel therapeutic targets of dermatological importance. PMID:8605230

  6. Tight Binding of the Phosphorylated α Subunit of Initiation Factor 2 (eIF2α) to the Regulatory Subunits of Guanine Nucleotide Exchange Factor eIF2B Is Required for Inhibition of Translation Initiation

    PubMed Central

    Krishnamoorthy, Thanuja; Pavitt, Graham D.; Zhang, Fan; Dever, Thomas E.; Hinnebusch, Alan G.

    2001-01-01

    Translation initiation factor 2 (eIF2) is a heterotrimeric protein that transfers methionyl-initiator tRNAMet to the small ribosomal subunit in a ternary complex with GTP. The eIF2 phosphorylated on serine 51 of its α subunit [eIF2(αP)] acts as competitive inhibitor of its guanine nucleotide exchange factor, eIF2B, impairing formation of the ternary complex and thereby inhibiting translation initiation. eIF2B is comprised of catalytic and regulatory subcomplexes harboring independent eIF2 binding sites; however, it was unknown whether the α subunit of eIF2 directly contacts any eIF2B subunits or whether this interaction is modulated by phosphorylation. We found that recombinant eIF2α (glutathione S-transferase [GST]–SUI2) bound to the eIF2B regulatory subcomplex in vitro, in a manner stimulated by Ser-51 phosphorylation. Genetic data suggest that this direct interaction also occurred in vivo, allowing overexpressed SUI2 to compete with eIF2(αP) holoprotein for binding to the eIF2B regulatory subcomplex. Mutations in SUI2 and in the eIF2B regulatory subunit GCD7 that eliminated inhibition of eIF2B by eIF2(αP) also impaired binding of phosphorylated GST-SUI2 to the eIF2B regulatory subunits. These findings provide strong evidence that tight binding of phosphorylated SUI2 to the eIF2B regulatory subcomplex is crucial for the inhibition of eIF2B and attendant downregulation of protein synthesis exerted by eIF2(αP). We propose that this regulatory interaction prevents association of the eIF2B catalytic subcomplex with the β and γ subunits of eIF2 in the manner required for GDP-GTP exchange. PMID:11438658

  7. A polymorphic motif in the small subunit of ADP-glucose pyrophosphorylase modulates interactions between the small and large subunits.

    PubMed

    Cross, Joanna M; Clancy, Maureen; Shaw, Janine R; Boehlein, Susan K; Greene, Thomas W; Schmidt, Robert R; Okita, Thomas W; Hannah, L Curtis

    2005-02-01

    The heterotetrameric, allosterically regulated enzyme, adenosine-5'-diphosphoglucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step in starch synthesis. Despite vast differences in allosteric properties and a long evolutionary separation, heterotetramers of potato small subunit and maize large subunit have activity comparable to either parent in an Escherichia coli expression system. In contrast, co-expression of maize small subunit with the potato large subunit produces little activity as judged by in vivo activity stain. To pinpoint the region responsible for differential activity, we expressed chimeric maize/potato small subunits in E. coli. This identified a 55-amino acid motif of the potato small subunit that is critical for glycogen production when expressed with the potato large subunit. Potato and maize small subunit sequences differ at five amino acids in this motif. Replacement experiments revealed that at least four amino acids of maize origin were required to reduce staining. An AGPase composed of a chimeric potato small subunit containing the 55-amino acid maize motif with the potato large subunit exhibited substantially less affinity for the substrates, glucose-1-phosphate and ATP and an increased Ka for the activator, 3-phosphoglyceric acid. Placement of the potato motif into the maize small subunit restored glycogen synthesis with the potato large subunit. Hence, a small polymorphic motif within the small subunit influences both catalytic and allosteric properties by modulating subunit interactions. PMID:15686515

  8. Glutathione-S-transferase A3 knockout mice are sensitive to acute cytotoxic and genotoxic effects of aflatoxin B1

    SciTech Connect

    Ilic, Zoran; Crawford, Dana; Egner, Patricia A.; Sell, Stewart

    2010-02-01

    Aflatoxin B1 (AFB1) is a major risk factor for hepatocellular carcinoma (HCC) in humans. However, mice, a major animal model for the study of AFB1 carcinogenesis, are resistant, due to high constitutive expression, in the mouse liver, of glutathione S-transferase A3 subunit (mGSTA3) that is lacking in humans. Our objective was to establish that a mouse model for AFB1 toxicity could be used to study mechanisms of toxicity that are relevant for human disease, i.e., an mGSTA3 knockout (KO) mouse that responds to toxicants such as AFB1 in a manner similar to humans. Exons 3-6 of the mGSTA3 were replaced with a neomycin cassette by homologous recombination. Southern blotting, RT-PCR, Western blotting, and measurement of AFB1-N{sup 7}-DNA adduct formation were used to evaluate the mGSTA3 KO mice. The KO mice have deletion of exons 3-6 of the mGSTA3 gene, as expected, as well as a lack of mGSTA3 expression at the mRNA and protein levels. Three hours after injection of 5 mg/kg AFB1, mGSTA3 KO mice have more than 100-fold more AFB1-N{sup 7}-DNA adducts in their livers than do similarly treated wild-type (WT) mice. In addition, the mGSTA3 KO mice die of massive hepatic necrosis, at AFB1 doses that have minimal toxic effects in WT mice. We conclude that mGSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of AFB1, confirming the crucial role of GSTA3 subunit in protection of normal mice against AFB1 toxicity. We propose the mGSTA3 KO mouse as a useful model with which to study the interplay of risk factors leading to HCC development in humans, as well as for testing of additional possible functions of mGSTA3.

  9. Glutathione, glutathione S-transferases, and related redox enzymes in Adriamycin-resistant cell lines with a multidrug resistant phenotype.

    PubMed

    Schisselbauer, J C; Crescimanno, M; D'Alessandro, N; Clapper, M; Toulmond, S; Tapiero, H; Tew, K D

    1989-01-01

    Friend erythroleukemia cells (FLC) selected by exposure to Adriamycin (doxorubicin) express an approximate 2.5-fold (ARN1) or 13-fold (ARN2) resistance to the drug with various degrees of cross-resistance to other anthracyclines, vinca alkaloids, and epipodophyllotoxins. Because the redox cycling of the quinone moiety of Adriamycin is known to produce oxidative stress, however, an analysis of glutathione (GSH) and related enzyme systems was undertaken in the wild-type and selected resistant cells. In ARN1 and ARN2, superoxide dismutase (SOD) and catalase activities were slightly decreased, intracellular GSH and GSH reductase were essentially unchanged, and total GSH peroxidase, glutathione S-transferase (GST), and DT-diaphorase activities were slightly elevated. In each case there was no stoichiometric relationship between degree of resistance and level of activity. GST isozymes were purified from each cell line by HPLC GSH affinity column chromatography. Two-dimensional gel electrophoresis and western blot immunoreactivity against a battery of GST isozyme polyclonal antibodies determined that both the resistant and sensitive cells expressed isozymes of the alpha, pi, and mu classes (alternative murine nomenclature: M1, M2, M3). Of significance, both ARN1 and ARN2 cell lines expressed a unique alpha subunit which was absent from the parent FLC cell line. This isozyme presumably accounted for the increased GSH peroxidase activity (cumene hydroperoxide as substrate) found in ARN1 and ARN2 and may play a role in the small incremental resistance to melphalan found for both resistant lines. Expression of the isozyme was not stoichiometric with respect to degree of resistance. The presence of this isozyme may contribute to the resistant phenotype or may be the consequence of a more general cellular response to oxidative stress. PMID:2639724

  10. The Interaction of the Chemotherapeutic Drug Chlorambucil with Human Glutathione Transferase A1-1: Kinetic and Structural Analysis

    PubMed Central

    Karpusas, Michael; Axarli, Irine; Chiniadis, Lykourgos; Papakyriakou, Athanasios; Bethanis, Kostas; Scopelitou, Katholiki; Clonis, Yannis D.; Labrou, Nikolaos E.

    2013-01-01

    Glutathione transferases (GSTs) are enzymes that contribute to cellular detoxification by catalysing the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of xenobiotic compounds, including several chemotherapeutic drugs. In the present work we investigated the interaction of the chemotherapeutic drug chlorambucil (CBL) with human GSTA1-1 (hGSTA1-1) using kinetic analysis, protein crystallography and molecular dynamics. In the presence of GSH, CBL behaves as an efficient substrate for hGSTA1-1. The rate-limiting step of the catalytic reaction between CBL and GSH is viscosity-dependent and kinetic data suggest that product release is rate-limiting. The crystal structure of the hGSTA1-1/CBL-GSH complex was solved at 2.1 Å resolution by molecular replacement. CBL is bound at the H-site attached to the thiol group of GSH, is partially ordered and exposed to the solvent, making specific interactions with the enzyme. Molecular dynamics simulations based on the crystal structure indicated high mobility of the CBL moiety and stabilization of the C-terminal helix due to the presence of the adduct. In the absence of GSH, CBL is shown to be an alkylating irreversible inhibitor for hGSTA1-1. Inactivation of the enzyme by CBL followed a biphasic pseudo-first-order saturation kinetics with approximately 1 mol of CBL per mol of dimeric enzyme being incorporated. Structural analysis suggested that the modifying residue is Cys112 which is located at the entrance of the H-site. The results are indicative of a structural communication between the subunits on the basis of mutually exclusive modification of Cys112, indicating that the two enzyme active sites are presumably coordinated. PMID:23460799

  11. Modulation of the glutathione S-transferase in Ochrobactrum anthropi: function of xenobiotic substrates and other forms of stress.

    PubMed Central

    Favaloro, B; Tamburro, A; Trofino, M A; Bologna, L; Rotilio, D; Heipieper, H J

    2000-01-01

    The gluthathione S-transferase gene of the atrazine-degrading bacterium Ochrobactrum anthropi (OaGST) encodes a single-subunit polypeptide of 201 amino acid residues (Favaloro et al. 1998, Biochem. J. 335, 573-579). RNA blot analysis showed that the gene is transcribed into an mRNA of about 800 nucleotides, indicating a monocistronic transcription of the OaGST gene. The modulation of OaGST in this bacterium, in the presence of different stimulants, was investigated. The level of expression of OaGST was detected both by measuring the mRNA level and by immunoblotting experiments. OaGST is a constitutive enzyme which is also inducible by several stimulants. In fact, atrazine caused an increase in the expression of OaGST even at concentrations which had no effect on growth rates of the bacteria. Moreover, the presence of other aromatic substrates of this bacterium, such as phenol and chlorophenols, leads to a marked enhancement in OaGST expression. In this case, the expression of OaGST was related to growth inhibition and membrane damage caused by these hydrophobic compounds, and to the adaptive responses of the cell membranes. On the other hand, toluene and xylene, two aromatic compounds not degradable by this bacterium, did not induce the OaGST expression. The same was observed for other stress conditions such as low pH, heat shock, hydrogen peroxide, osmotic stress, starvation, the presence of aliphatic alcohols or heavy metals. These results suggest a co-regulation of the OaGST gene by the catabolic pathways of phenols and chlorophenols in this bacterium. Therefore, OaGST could function as a detoxifying agent within the catabolism of these xenobiotics. PMID:10677378

  12. O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis

    PubMed Central

    Schimpl, Marianne; Zheng, Xiaowei; Borodkin, Vladimir S.; Blair, David E.; Ferenbach, Andrew T.; Schüttelkopf, Alexander W.; Navratilova, Iva; Aristotelous, Tonia; Albarbarawi, Osama; Robinson, David A.; Macnaughtan, Megan A.; van Aalten, Daan M.F.

    2012-01-01

    Protein O-GlcNAcylation is an essential post-translational modification on hundreds of intracellular proteins in metazoa, catalyzed by O-GlcNAc transferase using unknown mechanisms of transfer and substrate recognition. Through crystallographic snapshots and mechanism-inspired chemical probes, we define how human O-GlcNAc transferase recognizes the sugar donor and acceptor peptide and employs a novel catalytic mechanism of glycosyl transfer, involving the sugar donor α-phosphate as the catalytic base, as well as an essential lysine. This mechanism appears to be a unique evolutionary solution to the spatial constraints imposed by a bulky protein acceptor substrate, and explains the unexpected specificity of a recently reported metabolic O-GlcNAc transferase inhibitor. PMID:23103942

  13. The yeast WBP1 is essential for oligosaccharyl transferase activity in vivo and in vitro.

    PubMed Central

    te Heesen, S; Janetzky, B; Lehle, L; Aebi, M

    1992-01-01

    Asparagine-linked N-glycosylation is a highly conserved and functionally important modification of proteins in eukaryotic cells. The central step in this process is a cotranslational transfer of lipid-linked core oligosaccharides to selected Asn-X-Ser/Thr-sequences of nascent polypeptide chains, catalysed by the enzyme N-oligosaccharyl transferase. In this report we show that the essential yeast protein WBP1 (te Heesen et al., 1991) is required for N-oligosaccharyl transferase in vivo and in vitro. Depletion of WBP1 correlates with a defect in transferring core oligosaccharides to carboxypeptidase Y and proteinase A in vivo. In addition, in vitro N-glycosylation of the acceptor peptide Tyr-Asn-Leu-Thr-Ser-Val using microsomal membranes from WBP1 depleted cells is reduced as compared with membranes from wild-type cells. We propose that WBP1 is an essential component of the oligosaccharyl transferase in yeast. Images PMID:1600939

  14. Terminal Deoxynucleotidyl Transferase in a Case of Childhood Acute Lymphoblastic Leukemia

    PubMed Central

    McCaffrey, Ronald; Smoler, Donna F.; Baltimore, David

    1973-01-01

    Cells from a patient with childhood acute lymphoblastic leukemia contain an apparent DNA polymerase activity that was not found in any other cells except thymus cells. The enzyme has the properties of terminal transferase, an enzyme known to be found in thymocytes. The cells also contain the three major DNA polymerases found in growing cells. The results suggest that these tumor cells arose from a block in the differentiation of thymocytes. Terminal transferase may be a marker for the origin of leukemic cells. PMID:4346893

  15. Type II Hydride Transferases from Different Microorganisms Yield Nitrite and Diarylamines from Polynitroaromatic Compounds▿ †

    PubMed Central

    van Dillewijn, Pieter; Wittich, Rolf-Michael; Caballero, Antonio; Ramos, Juan-Luis

    2008-01-01

    Homogenous preparations of XenB of Pseudomonas putida, pentaerythritol tetranitrate reductase of Enterobacter cloacae, and N-ethylmaleimide reductase of Escherichia coli, all type II hydride transferases of the Old Yellow Enzyme family of flavoproteins, are shown to reduce the polynitroaromatic compound 2,4,6-trinitrotoluene (TNT). The reduction of this compound yields hydroxylaminodinitrotoluenes and Meisenheimer dihydride complexes, which, upon condensation, yield stoichiometric amounts of nitrite and diarylamines, implying that type II hydride transferases are responsible for TNT denitration, a process with important environmental implications for TNT remediation. PMID:18791007

  16. Purification and Biochemical Characterization of Glutathione S-Transferase from Down Syndrome and Normal Children Erythrocytes: A Comparative Study

    ERIC Educational Resources Information Center

    Hamed, Ragaa R.; Maharem, Tahany M.; Abdel-Meguid, Nagwa; Sabry, Gilane M.; Abdalla, Abdel-Monem; Guneidy, Rasha A.

    2011-01-01

    Down syndrome (DS) is the phenotypic manifestation of trisomy 21. Our study was concerned with the characterization and purification of glutathione S-transferase enzyme (GST) from normal and Down syndrome (DS) erythrocytes to illustrate the difference in the role of this enzyme in the cell. Glutathione S-transferase and glutathione (GSH) was…

  17. Recent Advances in Subunit Vaccine Carriers.

    PubMed

    Vartak, Abhishek; Sucheck, Steven J

    2016-01-01

    The lower immunogenicity of synthetic subunit antigens, compared to live attenuated vaccines, is being addressed with improved vaccine carriers. Recent reports indicate that the physio-chemical properties of these carriers can be altered to achieve optimal antigen presentation, endosomal escape, particle bio-distribution, and cellular trafficking. The carriers can be modified with various antigens and ligands for dendritic cells targeting. They can also be modified with adjuvants, either covalently or entrapped in the matrix, to improve cellular and humoral immune responses against the antigen. As a result, these multi-functional carrier systems are being explored for use in active immunotherapy against cancer and infectious diseases. Advancing technology, improved analytical methods, and use of computational methodology have also contributed to the development of subunit vaccine carriers. This review details recent breakthroughs in the design of nano-particulate vaccine carriers, including liposomes, polymeric nanoparticles, and inorganic nanoparticles. PMID:27104575

  18. PKA regulatory subunit expression in tooth development.

    PubMed

    de Sousa, Sílvia Ferreira; Kawasaki, Katsushige; Kawasaki, Maiko; Volponi, Ana Angelova; Gomez, Ricardo Santiago; Gomes, Carolina Cavaliéri; Sharpe, Paul T; Ohazama, Atsushi

    2014-05-01

    Protein kinase A (PKA) plays critical roles in many biological processes including cell proliferation, cell differentiation, cellular metabolism and gene regulation. Mutation in PKA regulatory subunit, PRKAR1A has previously been identified in odontogenic myxomas, but it is unclear whether PKA is involved in tooth development. The aim of the present study was to assess the expression of alpha isoforms of PKA regulatory subunit (Prkar1a and Prkar2a) in mouse and human odontogenesis by in situ hybridization. PRKAR1A and PRKAR2A mRNA transcription was further confirmed in a human deciduous germ by qRT-PCR. Mouse Prkar1a and human PRKAR2A exhibited a dynamic spatio-temporal expression in tooth development, whereas neither human PRKAR1A nor mouse Prkar2a showed their expression in odontogenesis. These isoforms thus showed different expression pattern between human and mouse tooth germs. PMID:24755349

  19. Recent Advances in Subunit Vaccine Carriers

    PubMed Central

    Vartak, Abhishek; Sucheck, Steven J.

    2016-01-01

    The lower immunogenicity of synthetic subunit antigens, compared to live attenuated vaccines, is being addressed with improved vaccine carriers. Recent reports indicate that the physio-chemical properties of these carriers can be altered to achieve optimal antigen presentation, endosomal escape, particle bio-distribution, and cellular trafficking. The carriers can be modified with various antigens and ligands for dendritic cells targeting. They can also be modified with adjuvants, either covalently or entrapped in the matrix, to improve cellular and humoral immune responses against the antigen. As a result, these multi-functional carrier systems are being explored for use in active immunotherapy against cancer and infectious diseases. Advancing technology, improved analytical methods, and use of computational methodology have also contributed to the development of subunit vaccine carriers. This review details recent breakthroughs in the design of nano-particulate vaccine carriers, including liposomes, polymeric nanoparticles, and inorganic nanoparticles. PMID:27104575

  20. Staggering of subunits in NMDAR channels.

    PubMed Central

    Sobolevsky, Alexander I; Rooney, LeeAnn; Wollmuth, Lonnie P

    2002-01-01

    Functional N-methyl-D-aspartate receptors (NMDARs) are heteromultimers formed by NR1 and NR2 subunits. The M3 segment, as contributed by NR1, forms the core of the extracellular vestibule, including binding sites for channel blockers, and represents a critical molecular link between ligand binding and channel opening. Taking advantage of the substituted cysteine accessibility method along with channel block and multivalent coordination, we studied the contribution of the M3 segment in NR2C to the extracellular vestibule. We find that the M3 segment in NR2C, like that in NR1, contributes to the core of the extracellular vestibule. However, the M3 segments from the two subunits are staggered relative to each other in the vertical axis of the channel. Compared to NR1, homologous positions in NR2C, including those in the highly conserved SYTANLAAF motif, are located about four amino acids more externally. The staggering of subunits may represent a key structural feature underlying the distinct functional properties of NMDARs. PMID:12496098

  1. Na Channel β Subunits: Overachievers of the Ion Channel Family.

    PubMed

    Brackenbury, William J; Isom, Lori L

    2011-01-01

    Voltage-gated Na(+) channels (VGSCs) in mammals contain a pore-forming α subunit and one or more β subunits. There are five mammalian β subunits in total: β1, β1B, β2, β3, and β4, encoded by four genes: SCN1B-SCN4B. With the exception of the SCN1B splice variant, β1B, the β subunits are type I topology transmembrane proteins. In contrast, β1B lacks a transmembrane domain and is a secreted protein. A growing body of work shows that VGSC β subunits are multifunctional. While they do not form the ion channel pore, β subunits alter gating, voltage-dependence, and kinetics of VGSCα subunits and thus regulate cellular excitability in vivo. In addition to their roles in channel modulation, β subunits are members of the immunoglobulin superfamily of cell adhesion molecules and regulate cell adhesion and migration. β subunits are also substrates for sequential proteolytic cleavage by secretases. An example of the multifunctional nature of β subunits is β1, encoded by SCN1B, that plays a critical role in neuronal migration and pathfinding during brain development, and whose function is dependent on Na(+) current and γ-secretase activity. Functional deletion of SCN1B results in Dravet Syndrome, a severe and intractable pediatric epileptic encephalopathy. β subunits are emerging as key players in a wide variety of physiopathologies, including epilepsy, cardiac arrhythmia, multiple sclerosis, Huntington's disease, neuropsychiatric disorders, neuropathic and inflammatory pain, and cancer. β subunits mediate multiple signaling pathways on different timescales, regulating electrical excitability, adhesion, migration, pathfinding, and transcription. Importantly, some β subunit functions may operate independently of α subunits. Thus, β subunits perform critical roles during development and disease. As such, they may prove useful in disease diagnosis and therapy. PMID:22007171

  2. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  3. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  4. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  5. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  6. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  7. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  8. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  9. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  10. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  11. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  12. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed conditions... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Aminoglycoside 3â²-phospho- transferase II. 573.130 Section 573.130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  13. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed conditions... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Aminoglycoside 3â²-phospho- transferase II. 573.130 Section 573.130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  14. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed conditions... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Aminoglycoside 3â²-phospho- transferase II. 573.130 Section 573.130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  15. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed conditions... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Aminoglycoside 3â²-phospho- transferase II. 573.130 Section 573.130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  16. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... genetically modified cotton, oilseed rape, and tomatoes in accordance with the following prescribed conditions... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Aminoglycoside 3â²-phospho- transferase II. 573.130 Section 573.130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  17. Structural and biochemical analyses reveal how ornithine acetyl transferase binds acidic and basic amino acid substrates.

    PubMed

    Iqbal, Aman; Clifton, Ian J; Chowdhury, Rasheduzzaman; Ivison, David; Domene, Carmen; Schofield, Christopher J

    2011-09-21

    Structural and biochemical analyses reveal how ornithine acetyl-transferases catalyse the reversible transfer of an acetyl-group from a basic (ornithine) to an acidic (glutamate) amino acid by employing a common mechanism involving an acetyl-enzyme intermediate but using different side chain binding modes. PMID:21796301

  18. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Galactose-1-phosphate uridyl transferase test system. 862.1315 Section 862.1315 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES...

  19. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Galactose-1-phosphate uridyl transferase test system. 862.1315 Section 862.1315 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... hereditary disease galactosemia (disorder of galactose metabolism) in infants. (b) Classification. Class II....

  20. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Galactose-1-phosphate uridyl transferase test system. 862.1315 Section 862.1315 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... hereditary disease galactosemia (disorder of galactose metabolism) in infants. (b) Classification. Class II....

  1. [(1)H] magnetic resonance spectroscopy of urine: diagnosis of a guanidinoacetate methyl transferase deficiency case.

    PubMed

    Tassini, Maria; Zannolli, Raffaella; Buoni, Sabrina; Engelke, Udo; Vivi, Antonio; Valensin, Gianni; Salomons, Gajja S; De Nicola, Anna; Strambi, Mirella; Monti, Lucia; Morava, Eva; Wevers, Ron A; Hayek, Joseph

    2010-01-01

    For the first time, the use of urine [(1)H] magnetic resonance spectroscopy has allowed the detection of 1 case of guanidinoacetate methyl transferase in a database sample of 1500 pediatric patients with a diagnosis of central nervous system impairment of unknown origin. The urine [(1)H] magnetic resonance spectroscopy of a 9-year-old child, having severe epilepsy and nonprogressive mental and motor retardation with no apparent cause, revealed a possible guanidinoacetic acid increase. The definitive assignment of guanidinoacetic acid was checked by addition of pure substance to the urine sample and by measuring [(1)H]-[(1)H] correlation spectroscopy. Diagnosis of guanidinoacetate methyl transferase deficiency was further confirmed by liquid chromatography-mass spectrometry, brain [(1)H] magnetic resonance spectroscopy, and mutational analysis of the guanidinoacetate methyl transferase gene. The replacement therapy was promptly started and, after 1 year, the child was seizure free. We conclude that for this case, urine [(1)H] magnetic resonance spectroscopy screening was able to diagnose guanidinoacetate methyl transferase deficiency. PMID:19461121

  2. Maize white seedling 3 results from disruption of homogentisate solanesyl transferase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize white seedling 3 (w3) has served as a model albino-seedling mutant since its discovery in 1923. We show here that the w3 phenotype is caused by disruptions in homogentisate solanesyl transferase (HST), an enzyme that catalyzes the committed step in plastoquinone-9 (PQ9) biosynthesis. This re...

  3. Effect of salicylates on histamine and L-histidine metabolism. Inhibition of imidazoleacetate phosphoribosyl transferase.

    PubMed Central

    Moss, J; De Mello, M C; Vaughan, M; Beaven, M A

    1976-01-01

    In man and other animals, urinary excretion of the histidine and histamine metabolite, imidazoleacetate, is increased and that of its conjugated metabolite, ribosylimidazoleacetate, decreased by salicylates. Imidazoleacetate has been reported to produce analgesia and narcosis. Its accumulation as a result of transferase inhibition could play a part in the therapeutic effects of salicylates. To determine the locus of salicylate action, we have investigated the effect of anti-inflammatory drugs on imidazoleacetate phosphoribosyl transferase, the enzyme that catalyzes the ATP-dependent conjugation of imidazoleacetate with phosphoribosylpyrophosphate. As little as 0.2 mM aspirin produced 50% inhibition of the rat liver transferase. In vivo, a 30% decrease in the urinary excretion of ribosylimidazoleacetate has been observed with plasma salicylate concentrations of 0.4 mM. The enzyme was also inhibited by sodium salicylate but not by salicylamide, sodium gentisate, aminopyrine, phenacetin, phenylbutazone, or indomethacin. The last four drugs have been shown previously not to alter the excretion of ribosylimidazoleacetate when administered in vivo. Since both the drug specificity and inhibitory concentrations are similar in vivo and in vitro, it seems probable that the effect of salicylates on imidazoleacetate conjugation results from inhibition of imidazoleacetate phosphoribosyl transferase. PMID:180057

  4. GLUTATHIONE S-TRANSFERASE THETA 1-1-DEPENDENT METABOLISM OF THE DISINFECTION BYPRODUCT BROMODICHLOROMETHANE

    EPA Science Inventory

    ABSTRACT
    Bromodichloromethane (BDCM), a prevalent drinking water disinfection by-product, was previously shown to be mutagenic in Salmonella expressing glutathione S-transferase (GST) theta 1-1 (GST T1-1). In the present study, in vitro experiments were performed to study the...

  5. Preliminary X-ray crystallographic analysis of glutathione transferase zeta 1 (GSTZ1a-1a)

    SciTech Connect

    Boone, Christopher D.; Zhong, Guo; Smeltz, Marci; James, Margaret O. McKenna, Robert

    2014-01-21

    Crystals of glutathione transferase zeta 1 were grown and shown to diffract X-rays to 3.1 Å resolution. They belonged to space group P1, with unit-cell parameters a = 42.0, b = 49.6, c = 54.6 Å, α = 82.9, β = 69.9, γ = 73.4°.

  6. DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1

    EPA Science Inventory


    DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1. R A Pegram1 and M K Ross2. 2Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC; 1Pharmacokinetics Branch, NHEERL, ORD, United States Environmental Protection Ag...

  7. A tyrosine-reactive irreversible inhibitor for glutathione S-transferase Pi (GSTP1).

    PubMed

    Crawford, L A; Weerapana, E

    2016-05-24

    Glutathione S-transferase Pi (GSTP1) mediates cellular defense against reactive electrophiles. Here, we report LAS17, a dichlorotriazine-containing compound that irreversibly inhibits GSTP1 and is selective for GSTP1 within cellular proteomes. Mass spectrometry and mutational studies identified Y108 as the site of modification, providing a unique mode of GSTP1 inhibition. PMID:27113843

  8. How subunit coupling produces the γ-subunit rotary motion in F1-ATPase

    PubMed Central

    Pu, Jingzhi; Karplus, Martin

    2008-01-01

    FoF1-ATP synthase manufactures the energy “currency,” ATP, of living cells. The soluble F1 portion, called F1-ATPase, can act as a rotary motor, with ATP binding, hydrolysis, and product release, inducing a torque on the γ-subunit. A coarse-grained plastic network model is used to show at a residue level of detail how the conformational changes of the catalytic β-subunits act on the γ-subunit through repulsive van der Waals interactions to generate a torque that drives unidirectional rotation, as observed experimentally. The simulations suggest that the calculated 85° substep rotation is driven primarily by ATP binding and that the subsequent 35° substep rotation is produced by product release from one β-subunit and a concomitant binding pocket expansion of another β-subunit. The results of the simulation agree with single-molecule experiments [see, for example, Adachi K, et al. (2007) Cell 130:309–321] and support a tri-site rotary mechanism for F1-ATPase under physiological condition. PMID:18216260

  9. Studies on chromatin. II. Isolation and characterization of chromatin subunits.

    PubMed Central

    Bakayev, V V; Melnickov, A A; Osicka, V D; Varshausky, A J

    1975-01-01

    Earlier findings /1-10/ bearing on a subunit organization of chromatin were confirmed and in some points detailed. Besides this, a large-scale isolation of chromatin subunits; their protein composition, electron microscopic appearance and CsCl banding pattern are described. Although the purified chromatin subunit contains all five histones, the relative content of histone H1 i in it is two times lower than that in the original chromatin. tit is shown that a mild digestion of chromatin with staphylococcal nuclease produced not only separate chromatin subunits and their "oligomers' but also deoxyribonucleoprotein particles which sediment more slowly than subunits. It appears that these particles and subunits are produced from different initial structures in the chromatin. Finally, a crystallization of the purified chromatin subunit as a cetyltrimethyl ammonium salt is described. Images PMID:1178523

  10. Effect of glutathione S-transferase M1 polymorphisms on biomarkers of exposure and effects.

    PubMed Central

    Srám, R J

    1998-01-01

    Genotypes responsible for interindividual differences in ability to activate or detoxify genotoxic agents are recognized as biomarkers of susceptibility. Among the most studied genotypes are human glutathione transferases. The relationship of genetic susceptibility to biomarkers of exposure and effects was studied especially in relation to the genetic polymorphism of glutathione S-transferase M1 (GSTM1). For this review papers reporting the effect of GSTM1 genotype on DNA adducts, protein adducts, urine mutagenicity, Comet assay parameters, chromosomal aberrations, sister chromatid exchanges (SCE), micronuclei, and hypoxanthine-guanine phosphoribosyl transferase mutations were assessed. Subjects in groups occupationally exposed to polycyclic aromatic hydrocarbons, benzidine, pesticides, and 1,3-butadiene were included. As environmentally exposed populations, autopsy donors, coal tar-treated patients, smokers, nonsmokers, mothers, postal workers, and firefighters were followed. From all biomarkers the effect of GSTM1 and N-acetyl transferase 2 was seen in coke oven workers on mutagenicity of urine and of glutathione S-transferase T1 on the chromosomal aberrations in subjects from 1,3-butadiene monomer production units. Effects of genotypes on DNA adducts were found from lung tissue of autopsy donors and from placentas of mothers living in an air-polluted region. The GSTM1 genotype affected mutagenicity of urine in smokers and subjects from polluted regions, protein adducts in smokers, SCE in smokers and nonsmokers, and Comet assay parameters in postal workers. A review of all studies on GSTM1 polymorphisms suggests that research probably has not reached the stage where results can be interpreted to formulate preventive measures. The relationship between genotypes and biomarkers of exposure and effects may provide an important guide to the risk assessment of human exposure to mutagens and carcinogens. PMID:9539016

  11. Glycosyl transferases in chondroitin sulphate biosynthesis. Effect of acceptor structure on activity.

    PubMed Central

    Gundlach, M W; Conrad, H E

    1985-01-01

    The D-glucuronosyl (GlcA)- and N-acetyl-D-galactosaminyl (GalNAc)-transferases involved in chondroitin sulphate biosynthesis were studied in a microsomal preparation from chick-embryo chondrocytes. Transfer of GlcA and GalNAc from their UDP derivatives to 3H-labelled oligosaccharides prepared from chondroitin sulphate and hyaluronic acid was assayed by h.p.l.c. of the reaction mixture. Conditions required for maximal activities of the two enzymes were remarkably similar. Activities were stimulated 3.5-6-fold by neutral detergents. Both enzymes were completely inhibited by EDTA and maximally stimulated by MnCl2 or CoCl2. MgCl2 neither stimulated nor inhibited. The GlcA transferase showed a sharp pH optimum between pH5 and 6, whereas the GalNAc transferase gave a broad optimum from pH 5 to 8. At pH 7 under optimal conditions, the GalNAc transferase gave a velocity that was twice that of the GlcA transferase. Oligosaccharides prepared from chondroitin 4-sulphate and hyaluronic acid were almost inactive as acceptors for both enzymes, whereas oligosaccharides from chondroitin 6-sulphate and chondroitin gave similar rates that were 70-80-fold higher than those observed with the endogenous acceptors. Oligosaccharide acceptors with degrees of polymerization of 6 or higher gave similar Km and Vmax. values, but the smaller oligosaccharides were less effective acceptors. These results are discussed in terms of the implications for regulation of the overall rates of the chain-elongation fractions in chondroitin sulphate synthesis in vivo. PMID:3921015

  12. Inherent conformational flexibility of F1-ATPase α-subunit.

    PubMed

    Hahn-Herrera, Otto; Salcedo, Guillermo; Barril, Xavier; García-Hernández, Enrique

    2016-09-01

    The core of F1-ATPase consists of three catalytic (β) and three noncatalytic (α) subunits, forming a hexameric ring in alternating positions. A wealth of experimental and theoretical data has provided a detailed picture of the complex role played by catalytic subunits. Although major conformational changes have only been seen in β-subunits, it is clear that α-subunits have to respond to these changes in order to be able to transmit information during the rotary mechanism. However, the conformational behavior of α-subunits has not been explored in detail. Here, we have combined unbiased molecular dynamics (MD) simulations and calorimetrically measured thermodynamic signatures to investigate the conformational flexibility of isolated α-subunits, as a step toward deepening our understanding of its function inside the α3β3 ring. The simulations indicate that the open-to-closed conformational transition of the α-subunit is essentially barrierless, which is ideal to accompany and transmit the movement of the catalytic subunits. Calorimetric measurements of the recombinant α-subunit from Geobacillus kaustophilus indicate that the isolated subunit undergoes no significant conformational changes upon nucleotide binding. Simulations confirm that the nucleotide-free and nucleotide-bound subunits show average conformations similar to that observed in the F1 crystal structure, but they reveal an increased conformational flexibility of the isolated α-subunit upon MgATP binding, which might explain the evolutionary conserved capacity of α-subunits to recognize nucleotides with considerable strength. Furthermore, we elucidate the different dependencies that α- and β-subunits show on Mg(II) for recognizing ATP. PMID:27137408

  13. Cytokine induced changes in proteasome subunit composition are concentration dependent.

    PubMed

    Stohwasser, R; Kloetzel, P M

    1996-09-01

    In eukaryotes, 20S proteasome subunit composition is controlled by the cytokine interferon-gamma (IFN-gamma). IFN-gamma induces the synthesis of the beta-subunits LMP2, LMP7 and MECL-1, which in consequence replace their constitutive subunit homologs delta, MB1 and MC14/Z in the 20S complex. By pulse labeling mouse RMA cells and immunoprecipitation of proteasome complexes with the antibody MP3, we have analysed the effect of different IFN-gamma concentrations on proteasomal subunit composition. Our experiments show that IFN-gamma concentrations as low as 5 U/ml induce subunit substitutions and that overall proteasomal subunit composition is dependent on the cytokine concentration used. An IFN-gamma concentration of 50 U/ml is sufficient for complete replacement of subunit delta by LMP2. In contrast, IFN-gamma treatment never induces a complete replacement of subunit MC14 by MECL-1. These subunits are present at an approximate 1:1 molar ratio, suggesting that both subunits coexist in the same 20S proteasome complex. Furthermore, different regulatory mechanisms have to be postulated for the synthesis and incorporation of the three IFN-gamma inducible proteasome subunits. Both IFN-gamma as well as IL-2 also seem to influence the modification state of the alpha subunit C8. Since the subunit composition is dependent on the cytokine concentration used and strongly influences the proteolytic properties of the 20S proteasome complex, our experiments represent a caveat for experiments in which IFN-gamma dependent proteasomal enzyme characteristics have been analysed without monitoring the subunit composition. PMID:9067255

  14. PKA catalytic subunit mutations in adrenocortical Cushing's adenoma impair association with the regulatory subunit.

    PubMed

    Calebiro, Davide; Hannawacker, Annette; Lyga, Sandra; Bathon, Kerstin; Zabel, Ulrike; Ronchi, Cristina; Beuschlein, Felix; Reincke, Martin; Lorenz, Kristina; Allolio, Bruno; Kisker, Caroline; Fassnacht, Martin; Lohse, Martin J

    2014-01-01

    We recently identified a high prevalence of mutations affecting the catalytic (Cα) subunit of protein kinase A (PKA) in cortisol-secreting adrenocortical adenomas. The two identified mutations (Leu206Arg and Leu199_Cys200insTrp) are associated with increased PKA catalytic activity, but the underlying mechanisms are highly controversial. Here we utilize a combination of biochemical and optical assays, including fluorescence resonance energy transfer in living cells, to analyze the consequences of the two mutations with respect to the formation of the PKA holoenzyme and its regulation by cAMP. Our results indicate that neither mutant can form a stable PKA complex, due to the location of the mutations at the interface between the catalytic and the regulatory subunits. We conclude that the two mutations cause high basal catalytic activity and lack of regulation by cAMP through interference of complex formation between the regulatory and the catalytic subunits of PKA. PMID:25477193

  15. Cloning and characterization of GABAA α subunits and GABAB subunits in Xenopus laevis during development

    PubMed Central

    Kaeser, Gwendolyn E.; Rabe, Brian A.; Saha, Margaret S.

    2011-01-01

    Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult nervous system, acts via two classes of receptors, the ionotropic GABAA and metabotropic GABAB receptors. During the development of the nervous system GABA acts in a depolarizing, excitatory manner and plays an important role in various neural developmental processes including cell proliferation, migration, synapse formation and activity-dependent differentiation. Here we describe the spatial and temporal expression patterns of the GABAA and GABAB receptors during early development of Xenopus laevis. Using in situ hybridization and qRT-PCR, GABAA α2 was detected as a maternal mRNA. All other α-subunits were first detected by tailbud through hatching stages. Expression of the various subunits was seen in the brain, spinal cord, cranial ganglia, olfactory epithelium, pineal, and pituitary gland. Each receptor subunit showed a distinctive, unique expression pattern suggesting these receptors have specific functions and are regulated in a precise spatial and temporal manner. PMID:21384470

  16. MRM2 and MRM3 are involved in biogenesis of the large subunit of the mitochondrial ribosome

    PubMed Central

    Rorbach, Joanna; Boesch, Pierre; Gammage, Payam A.; Nicholls, Thomas J. J.; Pearce, Sarah F.; Patel, Dipali; Hauser, Andreas; Perocchi, Fabiana; Minczuk, Michal

    2014-01-01

    Defects of the translation apparatus in human mitochondria are known to cause disease, yet details of how protein synthesis is regulated in this organelle remain to be unveiled. Ribosome production in all organisms studied thus far entails a complex, multistep pathway involving a number of auxiliary factors. This includes several RNA processing and modification steps required for correct rRNA maturation. Little is known about the maturation of human mitochondrial 16S rRNA and its role in biogenesis of the mitoribosome. Here we investigate two methyltransferases, MRM2 (also known as RRMJ2, encoded by FTSJ2) and MRM3 (also known as RMTL1, encoded by RNMTL1), that are responsible for modification of nucleotides of the 16S rRNA A-loop, an essential component of the peptidyl transferase center. Our studies show that inactivation of MRM2 or MRM3 in human cells by RNA interference results in respiratory incompetence as a consequence of diminished mitochondrial translation. Ineffective translation in MRM2- and MRM3-depleted cells results from aberrant assembly of the large subunit of the mitochondrial ribosome (mt-LSU). Our findings show that MRM2 and MRM3 are human mitochondrial methyltransferases involved in the modification of 16S rRNA and are important factors for the biogenesis and function of the large subunit of the mitochondrial ribosome. PMID:25009282

  17. Pin1 Interacts with the Epstein-Barr Virus DNA Polymerase Catalytic Subunit and Regulates Viral DNA Replication

    PubMed Central

    Narita, Yohei; Ryo, Akihide; Kawashima, Daisuke; Sugimoto, Atsuko; Kanda, Teru; Kimura, Hiroshi

    2013-01-01

    Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) protein is known as a regulator which recognizes phosphorylated Ser/Thr-Pro motifs and increases the rate of cis and trans amide isomer interconversion, thereby altering the conformation of its substrates. We found that Pin1 knockdown using short hairpin RNA (shRNA) technology resulted in strong suppression of productive Epstein-Barr virus (EBV) DNA replication. We further identified the EBV DNA polymerase catalytic subunit, BALF5, as a Pin1 substrate in glutathione S-transferase (GST) pulldown and immunoprecipitation assays. Lambda protein phosphatase treatment abolished the binding of BALF5 to Pin1, and mutation analysis of BALF5 revealed that replacement of the Thr178 residue by Ala (BALF5 T178A) disrupted the interaction with Pin1. To further test the effects of Pin1 in the context of virus infection, we constructed a BALF5-deficient recombinant virus. Exogenous supply of wild-type BALF5 in HEK293 cells with knockout recombinant EBV allowed efficient synthesis of viral genome DNA, but BALF5 T178A could not provide support as efficiently as wild-type BALF5. In conclusion, we found that EBV DNA polymerase BALF5 subunit interacts with Pin1 through BALF5 Thr178 in a phosphorylation-dependent manner. Pin1 might modulate EBV DNA polymerase conformation for efficient, productive viral DNA replication. PMID:23221557

  18. Functional Dissection of the Bipartite Active Site of the Class I Coenzyme A (CoA)-Transferase Succinyl-CoA:Acetate CoA-Transferase.

    PubMed

    Murphy, Jesse R; Mullins, Elwood A; Kappock, T Joseph

    2016-01-01

    Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA. PMID:27242998

  19. Functional Dissection of the Bipartite Active Site of the Class I Coenzyme A (CoA)-Transferase Succinyl-CoA:Acetate CoA-Transferase

    PubMed Central

    Murphy, Jesse R.; Mullins, Elwood A.; Kappock, T. Joseph

    2016-01-01

    Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA. PMID:27242998

  20. Functional dissection of the bipartite active site of the class I coenzyme A (CoA)-transferase succinyl-CoA:acetate CoA-transferase

    NASA Astrophysics Data System (ADS)

    Murphy, Jesse; Mullins, Elwood; Kappock, T.

    2016-05-01

    Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates less than 3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analogue dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analogue of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA.

  1. [Nose surgical anatomy in six aesthetic subunits].

    PubMed

    Chaput, B; Lauwers, F; Lopez, R; Saboye, J; André, A; Grolleau, J-L; Chavoin, J-P

    2013-04-01

    The nose is a complex entity, combining aesthetic and functional roles. Descriptive anatomy is a fundamental science that it can be difficult to relate directly to our daily surgical activity. Reasoning in terms of aesthetic subunits to decide on his actions appeared to us so obvious. The aim of this paper is to resume the anatomical bases relevant to our daily practice in order to fully apprehend the restorative or cosmetic procedures. We discuss the limits of the systematization of these principles in nasal oncology. PMID:22699003

  2. MspA Nanopores from Subunit Dimers

    PubMed Central

    Pavlenok, Mikhail; Derrington, Ian M.; Gundlach, Jens H.; Niederweis, Michael

    2012-01-01

    Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore sequencing of DNA. PMID

  3. MspA nanopores from subunit dimers.

    PubMed

    Pavlenok, Mikhail; Derrington, Ian M; Gundlach, Jens H; Niederweis, Michael

    2012-01-01

    Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore sequencing of DNA. PMID

  4. Analysis of the phosphofructokinase subunits and isoenzymes in human tissues.

    PubMed Central

    Dunaway, G A; Kasten, T P; Sebo, T; Trapp, R

    1988-01-01

    The 6-phosphofructo-1-kinase (PFK) subunits and isoenzymes were studied in human muscle, heart, brain, liver, platelets, fibroblasts, erythrocytes, placenta and umbilical cord. In each tissue, the subunit types in the native isoenzymes were characterized by immunological titration with subunit-specific antibodies and by column chromatography on QAE (quaternary aminoethyl)-Sephadex. Further, the subunits of the partially purified native isoenzymes were resolved by SDS/polyacrylamide-gel electrophoresis, identified by immunoblotting, and quantified by scanning gel densitometry of silver-stained gels and immunoblots. Depending on the type of tissue, one to three subunits were detected. The Mr values of the L, M and C subunits regardless of tissue were 76,700 +/- 1400, 82,500 +/- 1640 and 86,500 +/- 1620. Of the tissues studied, only the muscle PFK isoenzymes exhibited one subunit, which was the M-type subunit. Of the other tissues studied, the PFK isoenzymes contained various amounts of all three subunits. Considering the properties of the native PFK isoenzymes, it is clear that, in human tissues, they are not simply various combinations of two or three homotetrameric isoenzymes, but complex mixtures of homotetramers and heterotetramers. The kinetic/regulatory properties of the various isoenzyme pools were found to be dependent on subunit composition. Images Fig. 1. PMID:2970843

  5. The role of the C-terminal region on the oligomeric state and enzymatic activity of Trypanosoma cruzi hypoxanthine phosphoribosyl transferase.

    PubMed

    Valsecchi, Wanda M; Cousido-Siah, Alexandra; Defelipe, Lucas A; Mitschler, André; Podjarny, Alberto; Santos, Javier; Delfino, José M

    2016-06-01

    Hypoxanthine phosphoribosyl transferase from Trypanosoma cruzi (TcHPRT) is a critical enzyme for the survival of the parasite. This work demonstrates that the full-length form in solution adopts a stable and enzymatically active tetrameric form, exhibiting large inter-subunit surfaces. Although this protein irreversibly aggregates during unfolding, oligomerization is reversible and can be modulated by low concentrations of urea. When the C-terminal region, which is predicted as a disordered stretch, is excised by proteolysis, TcHPRT adopts a dimeric state, suggesting that the C-terminal region acts as a main guide for the quaternary arrangement. These results are in agreement with X-ray crystallographic data presented in this work. On the other hand, the C-terminal region exhibits a modulatory role on the enzyme, as attested by the enhanced activity observed for the dimeric form. Bisphosphonates act as substrate-mimetics, uncovering long-range communications among the active sites. All in all, this work contributes to establish new ways applicable to the design of novel inhibitors that could eventually result in new drugs against parasitic diseases. PMID:26969784

  6. Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans

    PubMed Central

    Meyer, Britta; Wurm, Jan Philip; Sharma, Sunny; Immer, Carina; Pogoryelov, Denys; Kötter, Peter; Lafontaine, Denis L. J.; Wöhnert, Jens; Entian, Karl-Dieter

    2016-01-01

    The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m1acp3Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells. In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes. PMID:27084949

  7. Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans.

    PubMed

    Meyer, Britta; Wurm, Jan Philip; Sharma, Sunny; Immer, Carina; Pogoryelov, Denys; Kötter, Peter; Lafontaine, Denis L J; Wöhnert, Jens; Entian, Karl-Dieter

    2016-05-19

    The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m(1)acp(3)Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells. In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes. PMID:27084949

  8. Radiographic changes and lung function in relation to activity of the glutathione transferases theta and mu among asbestos cement workers.

    PubMed

    Jakobsson, K; Rannug, A; Alexandrie, A K; Warholm, M; Rylander, L; Hagmar, L

    1995-05-01

    Experimental data indicate that active oxygen species may be casually involved in the development of asbestos-related disease. Thus, it was hypothesized that individual differences in glutathione transferase activity, which may affect the ability to inactivate molecules formed in relation to oxidative stress, could influence the biological response to asbestos exposure. We could, however, not demonstrate an increased risk for radiographic changes or reduced lung function among asbestos cement workers deficient for glutathione transferase theta (GSTT1), glutathione transferase mu (GSTM1), or having a combined deficiency of enzyme activity. PMID:7618163

  9. Subunit Arrangement and Function in NMDA Receptors

    SciTech Connect

    Furukawa,H.; Singh, S.; Mancusso, R.; Gouaux, E.

    2005-01-01

    Excitatory neurotransmission mediated by NMDA (N-methyl-D-aspartate) receptors is fundamental to the physiology of the mammalian central nervous system. These receptors are heteromeric ion channels that for activation require binding of glycine and glutamate to the NR1 and NR2 subunits, respectively. NMDA receptor function is characterized by slow channel opening and deactivation, and the resulting influx of cations initiates signal transduction cascades that are crucial to higher functions including learning and memory. Here we report crystal structures of the ligand-binding core of NR2A with glutamate and that of the NR1-NR2A heterodimer with glutamate and glycine. The NR2A-glutamate complex defines the determinants of glutamate and NMDA recognition, and the NR1-NR2A heterodimer suggests a mechanism for ligand-induced ion channel opening. Analysis of the heterodimer interface, together with biochemical and electrophysiological experiments, confirms that the NR1-NR2A heterodimer is the functional unit in tetrameric NMDA receptors and that tyrosine 535 of NR1, located in the subunit interface, modulates the rate of ion channel deactivation.

  10. TSH Receptor Cleavage Into Subunits and Shedding of the A-Subunit; A Molecular and Clinical Perspective.

    PubMed

    Rapoport, Basil; McLachlan, Sandra M

    2016-04-01

    The TSH receptor (TSHR) on the surface of thyrocytes is unique among the glycoprotein hormone receptors in comprising two subunits: an extracellular A-subunit, and a largely transmembrane and cytosolic B-subunit. Unlike its ligand TSH, whose subunits are encoded by two genes, the TSHR is expressed as a single polypeptide that subsequently undergoes intramolecular cleavage into disulfide-linked subunits. Cleavage is associated with removal of a C-peptide region, a mechanism similar in some respects to insulin cleavage into disulfide linked A- and B-subunits with loss of a C-peptide region. The potential pathophysiological importance of TSHR cleavage into A- and B-subunits is that some A-subunits are shed from the cell surface. Considerable experimental evidence supports the concept that A-subunit shedding in genetically susceptible individuals is a factor contributing to the induction and/or affinity maturation of pathogenic thyroid-stimulating autoantibodies, the direct cause of Graves' disease. The noncleaving gonadotropin receptors are not associated with autoantibodies that induce a "Graves' disease of the gonads." We also review herein current information on the location of the cleavage sites, the enzyme(s) responsible for cleavage, the mechanism by which A-subunits are shed, and the effects of cleavage on receptor signaling. PMID:26799472

  11. Sodium channel β subunits: emerging targets in channelopathies.

    PubMed

    O'Malley, Heather A; Isom, Lori L

    2015-01-01

    Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and β subunits. Although β subunits were originally termed auxiliary, we now know that they are multifunctional signaling molecules that play roles in both excitable and nonexcitable cell types and with or without the pore-forming α subunit present. β subunits function in VGSC and potassium channel modulation, cell adhesion, and gene regulation, with particularly important roles in brain development. Mutations in the genes encoding β subunits are linked to a number of diseases, including epilepsy, sudden death syndromes like SUDEP and SIDS, and cardiac arrhythmia. Although VGSC β subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target. PMID:25668026

  12. Sodium channel β subunits: emerging targets in channelopathies

    PubMed Central

    O’Malley, Heather A.; Isom, Lori L.

    2016-01-01

    Voltage-gated sodium channels (VGSCs) are responsible for initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and β subunits. Originally called “auxiliary,” we now know that β subunit proteins are multifunctional signaling molecules that play roles in both excitable and non-excitable cell types, and with or without the pore-forming α subunit present. β subunits function in VGSC and potassium channel modulation, cell adhesion, and gene regulation, with particularly important roles in brain development. Mutations in the genes encoding β subunits are linked to a number of diseases, including epilepsy, sudden death syndromes like SUDEP and SIDS, and cardiac arrhythmia. While VGSC β subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target. PMID:25668026

  13. Quantifying the cooperative subunit action in a multimeric membrane receptor

    PubMed Central

    Wongsamitkul, Nisa; Nache, Vasilica; Eick, Thomas; Hummert, Sabine; Schulz, Eckhard; Schmauder, Ralf; Schirmeyer, Jana; Zimmer, Thomas; Benndorf, Klaus

    2016-01-01

    In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits. We propose a method that permits quantification of ligand binding to and activation effects of the individual binding sites in a multimeric membrane receptor. The power of this method is demonstrated by gaining detailed insight into the subunit action in olfactory cyclic nucleotide-gated CNGA2 ion channels. PMID:26858151

  14. Regiospecificity of placental metabolism by cytochromes P450 and glutathione S-transferase.

    PubMed

    McRobie, D J; Glover, D D; Tracy, T S

    1996-01-01

    The placenta possesses the ability to metabolize numerous xenobiotics and endogenous steroids. However, it is unknown whether regional differences in these enzymatic reactions exist in the human placenta. To this end, we undertook a study of four regions of the placenta, the chorionic plate, maternal surface, placental margin and whole tissue, to assess the activities of cytochrome P450 1A1 and 19A1 (aromatase) and glutathione S-stransferase in these fractions. No differences in either P450 1A1 or glutathione S-transferase activities were noted among any of the placental fractions. However, with respect to P450 19A1 activity, the placental margin differed significantly from all other fractions (p < 0.05). This study demonstrates that whole tissue samples of the human placenta are adequate for placental cytochrome P450 and glutathione S-transferase metabolism studies. PMID:8938464

  15. Identification of a diazinon-metabolizing glutathione S-transferase in the silkworm, Bombyx mori.

    PubMed

    Yamamoto, Kohji; Yamada, Naotaka

    2016-01-01

    The glutathione S-transferase superfamily play key roles in the metabolism of numerous xenobiotics. We report herein the identification and characterization of a novel glutathione S-transferase in the silkworm, Bombyx mori. The enzyme (bmGSTu2) conjugates glutathione to 1-chloro-2,4-dinitrobenzene, as well as metabolizing diazinon, one of the organophosphate insecticides. Quantitative reverse transcription-polymerase chain reaction analysis of transcripts demonstrated that bmGSTu2 expression was induced 1.7-fold in a resistant strain of B. mori. Mutagenesis of putative amino acid residues in the glutathione-binding site revealed that Ile54, Glu66, Ser67, and Asn68 are crucial for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTu2 and into the detoxification of organophosphate insecticides. PMID:27440377

  16. Identification of a diazinon-metabolizing glutathione S-transferase in the silkworm, Bombyx mori

    PubMed Central

    Yamamoto, Kohji; Yamada, Naotaka

    2016-01-01

    The glutathione S-transferase superfamily play key roles in the metabolism of numerous xenobiotics. We report herein the identification and characterization of a novel glutathione S-transferase in the silkworm, Bombyx mori. The enzyme (bmGSTu2) conjugates glutathione to 1-chloro-2,4-dinitrobenzene, as well as metabolizing diazinon, one of the organophosphate insecticides. Quantitative reverse transcription–polymerase chain reaction analysis of transcripts demonstrated that bmGSTu2 expression was induced 1.7-fold in a resistant strain of B. mori. Mutagenesis of putative amino acid residues in the glutathione-binding site revealed that Ile54, Glu66, Ser67, and Asn68 are crucial for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTu2 and into the detoxification of organophosphate insecticides. PMID:27440377

  17. Glutathione and gamma-glutamyl transferases are involved in the formation of cadmium-glutathione complex.

    PubMed

    Adamis, Paula Daniela Braga; Mannarino, Sérgio Cantú; Eleutherio, Elis Cristina Araújo

    2009-05-01

    In a wild-type strain of Saccharomyces cerevisiae, cadmium induces the activities of both gamma-glutamyl transferase (gamma-GT) and glutathione transferase 2 (Gtt2). However, Gtt2 activity did not increase under gamma-GT or Ycf1 deficiencies, suggesting that the accumulation of glutathione-cadmium in the cytosol inhibits Gtt2. On the other hand, the balance between the cytoplasmic and vacuolar level of glutathione seems to regulate gamma-GT activity, since this enzyme was not activated in a gtt2 strain. Taken together, these results suggest that gamma-GT and Gtt2 work together to remove cadmium from the cytoplasm, a crucial mechanism for metal detoxification that is dependent on glutathione. PMID:19345220

  18. 2′,6′-Dihalostyrylanilines, Pyridines, and Pyrimidines for the Inhibition of the Catalytic Subunit of Methionine S-Adenosyltransferase-2

    PubMed Central

    2015-01-01

    Inhibition of the catalytic subunit of the heterodimeric methionine S-adenosyl transferase-2 (MAT2A) with fluorinated N,N-dialkylaminostilbenes (FIDAS agents) offers a potential avenue for the treatment of liver and colorectal cancers where upregulation of this enzyme occurs. A study of structure–activity relationships led to the identification of the most active compounds as those with (1) either a 2,6-difluorostyryl or 2-chloro-6-fluorostyryl subunit, (2) either an N-methylamino or N,N-dimethylamino group attached in a para orientation relative to the 2,6-dihalostyryl subunit, and (3) either an N-methylaniline or a 2-(N,N-dimethylamino)pyridine ring. These modifications led to FIDAS agents that were active in the low nanomolar range, that formed water-soluble hydrochloride salts, and that possessed the desired property of not inhibiting the human hERG potassium ion channel at concentrations at which the FIDAS agents inhibit MAT2A. The active FIDAS agents may inhibit cancer cells through alterations of methylation reactions essential for cancer cell survival and growth. PMID:24950374

  19. Glutathione transferase activity and formation of macromolecular adducts in two cases of acute methyl bromide poisoning.

    PubMed Central

    Garnier, R; Rambourg-Schepens, M O; Müller, A; Hallier, E

    1996-01-01

    OBJECTIVES: To determine the activity of glutathione transferase and to measure the S-methylcysteine adducts in blood proteins, after acute inhalation exposure to methyl bromide. To examine the influence of the polymorphism of glutathione-S-transferase theta (GSTT1) on the neurotoxicity of methyl bromide. METHODS: Two workers acutely exposed to methyl bromide with inadequate respiratory protective devices were poisoned. Seven weeks after the accident, blood samples were drawn from both patients, for measurement of glutathione transferase activity in erythrocytes (conjugator status--that is, GSTT1 phenotype) and measurement of binding products of methyl bromide with blood proteins. Conjugator status was determined by a standard procedure. The binding product of methyl bromide, S-methylcysteine, was measured in globin and albumin. RESULTS: Duration and intensity of exposure were identical for both patients as they worked together with the same protective devices and with similar physical effort. However, one patient had very severe poisoning, whereas the other only developed mild neurotoxic symptoms. The first patient was a "conjugator" with normal glutathone transferase activity, whereas this activity was undetectable in the erythrocytes of the second patient, who consequently had higher concentrations of S-methylcysteine adduct in albumin (149 v 91 nmol/g protein) and in globin (77 v 30 nmol/g protein). CONCLUSIONS: Methyl bromide is genotoxic and neurotoxic. Its genotoxicity seems to be the consequence of the alkylating activity of the parent compound, and conjugation to glutathione has a protective effect. The data presented here suggest a different mechanism for methyl bromide neurotoxicity which could be related to the transformation of methylglutathione into toxic metabolites such as methanethiol and formaldehyde. If such metabolites are the ultimate toxic species, N-acetylcysteine treatment could have a toxifying rather than a detoxifying effect. PMID:8704864

  20. A comparison of erythrocyte glutathione S-transferase activity from human foetuses and adults.

    PubMed Central

    Strange, R C; Johnston, J D; Coghill, D R; Hume, R

    1980-01-01

    Glutathione S-transferase activity was measured in partially purified haemolysates of erythrocytes from human foetuses and adults. Enzyme activity was present in erythrocytes obtained between 12 and 40 weeks of gestation. The catalytic properties of the enzyme from foetal cells were similar to those of the enzyme from adult erythrocytes, indicating that probably only one form of the erythrocytes enzyme exists throughout foetal and adult life. PMID:7396875

  1. Homology between O-linked GlcNAc transferases and proteins of the glycogen phosphorylase superfamily.

    PubMed

    Wrabl, J O; Grishin, N V

    2001-11-30

    The O-linked GlcNAc transferases (OGTs) are a recently characterized group of largely eukaryotic enzymes that add a single beta-N-acetylglucosamine moiety to specific serine or threonine hydroxyls. In humans, this process may be part of a sugar regulation mechanism or cellular signaling pathway that is involved in many important diseases, such as diabetes, cancer, and neurodegeneration. However, no structural information about the human OGT exists, except for the identification of tetratricopeptide repeats (TPR) at the N terminus. The locations of substrate binding sites are unknown and the structural basis for this enzyme's function is not clear. Here, remote homology is reported between the OGTs and a large group of diverse sugar processing enzymes, including proteins with known structure such as glycogen phosphorylase, UDP-GlcNAc 2-epimerase, and the glycosyl transferase MurG. This relationship, in conjunction with amino acid similarity spanning the entire length of the sequence, implies that the fold of the human OGT consists of two Rossmann-like domains C-terminal to the TPR region. A conserved motif in the second Rossmann domain points to the UDP-GlcNAc donor binding site. This conclusion is supported by a combination of statistically significant PSI-BLAST hits, consensus secondary structure predictions, and a fold recognition hit to MurG. Additionally, iterative PSI-BLAST database searches reveal that proteins homologous to the OGTs form a large and diverse superfamily that is termed GPGTF (glycogen phosphorylase/glycosyl transferase). Up to one-third of the 51 functional families in the CAZY database, a glycosyl transferase classification scheme based on catalytic residue and sequence homology considerations, can be unified through this common predicted fold. GPGTF homologs constitute a substantial fraction of known proteins: 0.4% of all non-redundant sequences and about 1% of proteins in the Escherichia coli genome are found to belong to the GPGTF

  2. Imidazopyridine and Pyrazolopiperidine Derivatives as Novel Inhibitors of Serine Palmitoyl Transferase.

    PubMed

    Genin, Michael J; Gonzalez Valcarcel, Isabel C; Holloway, William G; Lamar, Jason; Mosior, Marian; Hawkins, Eric; Estridge, Thomas; Weidner, Jeffrey; Seng, Thomas; Yurek, David; Adams, Lisa A; Weller, Jennifer; Reynolds, Vincent L; Brozinick, Joseph T

    2016-06-23

    To develop novel treatments for type 2 diabetes and dyslipidemia, we pursued inhibitors of serine palmitoyl transferase (SPT). To this end compounds 1 and 2 were developed as potent SPT inhibitors in vitro. 1 and 2 reduce plasma ceramides in rodents, have a slight trend toward enhanced insulin sensitization in DIO mice, and reduce triglycerides and raise HDL in cholesterol/cholic acid fed rats. Unfortunately these molecules cause a gastric enteropathy after chronic dosing in rats. PMID:27213958

  3. Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

    PubMed Central

    Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

    2014-01-01

    Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

  4. Characterization of affinity-purified isoforms of Acinetobacter calcoaceticus Y1 glutathione transferases.

    PubMed

    Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

    2014-01-01

    Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

  5. Subfunctionality of Hydride Transferases of the Old Yellow Enzyme Family of Flavoproteins of Pseudomonas putida▿

    PubMed Central

    van Dillewijn, Pieter; Wittich, Rolf-Michael; Caballero, Antonio; Ramos, Juan-Luis

    2008-01-01

    To investigate potential complementary activities of multiple enzymes belonging to the same family within a single microorganism, we chose a set of Old Yellow Enzyme (OYE) homologs of Pseudomonas putida. The physiological function of these enzymes is not well established; however, an activity associated with OYE family members from different microorganisms is their ability to reduce nitroaromatic compounds. Using an in silico approach, we identified six OYE homologs in P. putida KT2440. Each gene was subcloned into an expression vector, and each corresponding gene product was purified to homogeneity prior to in vitro analysis for its catalytic activity against 2,4,6-trinitrotoluene (TNT). One of the enzymes, called XenD, lacked in vitro activity, whereas the other five enzymes demonstrated type I hydride transferase activity and reduced the nitro groups of TNT to hydroxylaminodinitrotoluene derivatives. XenB has the additional ability to reduce the aromatic ring of TNT to produce Meisenheimer complexes, defined as type II hydride transferase activity. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite; the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo. PMID:18791012

  6. Compilation of small ribosomal subunit RNA structures.

    PubMed Central

    Neefs, J M; Van de Peer, Y; De Rijk, P; Chapelle, S; De Wachter, R

    1993-01-01

    The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk. PMID:8332525

  7. Dynamic regulation of β1 subunit trafficking controls vascular contractility

    PubMed Central

    Leo, M. Dennis; Bannister, John P.; Narayanan, Damodaran; Nair, Anitha; Grubbs, Jordan E.; Gabrick, Kyle S.; Boop, Frederick A.; Jaggar, Jonathan H.

    2014-01-01

    Ion channels composed of pore-forming and auxiliary subunits control physiological functions in virtually all cell types. A conventional view is that channels assemble with their auxiliary subunits before anterograde plasma membrane trafficking of the protein complex. Whether the multisubunit composition of surface channels is fixed following protein synthesis or flexible and open to acute and, potentially, rapid modulation to control activity and cellular excitability is unclear. Arterial smooth muscle cells (myocytes) express large-conductance Ca2+-activated potassium (BK) channel α and auxiliary β1 subunits that are functionally significant modulators of arterial contractility. Here, we show that native BKα subunits are primarily (∼95%) plasma membrane-localized in human and rat arterial myocytes. In contrast, only a small fraction (∼10%) of total β1 subunits are located at the cell surface. Immunofluorescence resonance energy transfer microscopy demonstrated that intracellular β1 subunits are stored within Rab11A-postive recycling endosomes. Nitric oxide (NO), acting via cGMP-dependent protein kinase, and cAMP-dependent pathways stimulated rapid (≤1 min) anterograde trafficking of β1 subunit-containing recycling endosomes, which increased surface β1 almost threefold. These β1 subunits associated with surface-resident BKα proteins, elevating channel Ca2+ sensitivity and activity. Our data also show that rapid β1 subunit anterograde trafficking is the primary mechanism by which NO activates myocyte BK channels and induces vasodilation. In summary, we show that rapid β1 subunit surface trafficking controls functional BK channel activity in arterial myocytes and vascular contractility. Conceivably, regulated auxiliary subunit trafficking may control ion channel activity in a wide variety of cell types. PMID:24464482

  8. Crystallographic trapping of the glutamyl-CoA thioester intermediate of family I CoA transferases

    SciTech Connect

    Rangarajan,E.; Li, Y.; Ajamian, E.; Iannuzzi, P.; Kernaghan, S.; Fraser, M.; Cygler, M.; Matte, A.

    2005-01-01

    Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 Angstrom resolution, respectively. YdiF is organized into tetramers, with each monomer having an open {alpha}/{beta} structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent {gamma}-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.

  9. Probing the leucyl/phenylalanyl tRNA protein transferase active site with tRNA substrate analogues.

    PubMed

    Fung, Angela Wai Shan; Ebhardt, H Alexander; Krishnakumar, Kollappillil S; Moore, Jack; Xu, Zhizhong; Strazewski, Peter; Fahlman, Richard P

    2014-07-01

    Aminoacyl-tRNA protein transferases post-translationally conjugate an amino acid from an aminoacyl-tRNA onto the N-terminus of a target polypeptide. The eubacterial aminoacyl-tRNA protein transferase, L/F transferase, utilizes both leucyl-tRNA(Leu) and phenylalanyl-tRNA(Phe) as substrates. X-ray crystal structures with substrate analogues, the minimal substrate phenylalanyl adenosine (rA-Phe) and inhibitor puromycin, have been used to characterize tRNA recognition by L/F transferase. However analyses of these two X-ray crystal structures reveal significant differences in binding. Through structural analyses, mutagenesis, and enzymatic activity assays, we rationalize and demonstrate that the substrate analogues bind to L/F transferase with similar binding affinities using a series of different interactions by the various chemical groups of the analogues. Our data also demonstrates that enlarging the hydrophobic pocket of L/F transferase selectively enhances puromycin inhibition and may aid in the development of improved inhibitors for this class of enzymes. PMID:24521222

  10. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    PubMed Central

    Tandrup Schmidt, Signe; Foged, Camilla; Smith Korsholm, Karen; Rades, Thomas; Christensen, Dennis

    2016-01-01

    The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs) concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI). Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs), which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the specific PRR

  11. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators.

    PubMed

    Tandrup Schmidt, Signe; Foged, Camilla; Korsholm, Karen Smith; Rades, Thomas; Christensen, Dennis

    2016-01-01

    The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs) concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI). Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs), which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the specific PRR

  12. The light subunit of system bo,+ is fully functional in the absence of the heavy subunit

    PubMed Central

    Reig, Núria; Chillarón, Josep; Bartoccioni, Paola; Fernández, Esperanza; Bendahan, Annie; Zorzano, Antonio; Kanner, Baruch; Palacín, Manuel; Bertran, Joan

    2002-01-01

    The heteromeric amino acid transporters are composed of a type II glycoprotein and a non-glycosylated polytopic membrane protein. System bo,+ exchanges dibasic for neutral amino acids. It is composed of rBAT and bo,+AT, the latter being the polytopic membrane subunit. Mutations in either of them cause malfunction of the system, leading to cystinuria. bo,+AT-reconstituted systems from HeLa or MDCK cells catalysed transport of arginine that was totally dependent on the presence of one of the bo,+ substrates inside the liposomes. rBAT was essential for the cell surface expression of bo,+AT, but it was not required for reconstituted bo,+AT transport activity. No system bo,+ transport was detected in liposomes derived from cells expressing rBAT alone. The reconstituted bo,+AT showed kinetic asymmetry. Expressing the cystinuria-specific mutant A354T of bo,+AT in HeLa cells together with rBAT resulted in defective arginine uptake in whole cells, which was paralleled by the reconstituted bo,+AT activity. Thus, subunit bo,+AT by itself is sufficient to catalyse transmembrane amino acid exchange. The polytopic subunits may also be the catalytic part in other heteromeric transporters. PMID:12234930

  13. Epitopes from two soybean glycinin subunits antigenic in pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Glycinin is a seed storage protein in soybean (Glycine max) that is allergenic in pigs. Glycinin is a hexamer composed of subunits consisting of a basic and acidic portion joined by disulfide bridges. There are 5 glycinin subunits designated Gy1-Gy5. Results: Twenty seven out of 30 pi...

  14. Proteopedia Entry: The Large Ribosomal Subunit of "Haloarcula Marismortui"

    ERIC Educational Resources Information Center

    Decatur, Wayne A.

    2010-01-01

    This article presents a "Proteopedia" page that shows the refined version of the structure of the "Haloarcula" large ribosomal subunit as solved by the laboratories of Thomas Steitz and Peter Moore. The landmark structure is of great impact as it is the first atomic-resolution structure of the highly conserved ribosomal subunit which harbors…

  15. The Development and Institutionalization of Subunit Power in Organizations.

    ERIC Educational Resources Information Center

    Boeker, Warren

    1989-01-01

    Examines the effects of founding events on the evolution of subunit importance in the semiconductor industry from 1958 to 1985. Distributions of power and subunit importance represent not only influences of current conditions, but also vestiges of earlier events, including the institution's founding. Includes 55 references. (MLH)

  16. Geranyl diphosphate synthase large subunit, and methods of use

    DOEpatents

    Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

    2001-10-16

    A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

  17. Modulation of Kv4.3 current by accessory subunits.

    PubMed

    Deschênes, Isabelle; Tomaselli, Gordon F

    2002-09-25

    Kv4.3 encodes the pore-forming subunit of the cardiac transient outward potassium current (I(to)). hKv4.3-encoded current does not fully replicate cardiac I(to), suggesting a functionally significant role for accessory subunits. KChIP2 associates with Kv4.3 and modifies hKv4.3-encoded currents but does not replicate native I(to). We examined the effect of several ancillary subunits expressed in the heart on hKv4.3-encoded currents. Remarkably, the ancillary subunits Kvbeta(3), minK, MiRP-1, the Na channel beta(1) and KChIP2 increased the density and modified the gating of hKv4.3 current. hKv4.3 promiscuously assembles with ancillary subunits in vitro, functionally modifying the encoded currents; however, the physiological significance is uncertain. PMID:12297301

  18. Immunolabeling of Gamma-glutamyl transferase 5 in Normal Human Tissues Reveals Expression and Localization Differs from Gamma-glutamyl transferase 1

    PubMed Central

    Hanigan, Marie H.; Gillies, Elizabeth M.; Wickham, Stephanie; Wakeham, Nancy; Wirsig-Wiechmann, Celeste R.

    2014-01-01

    Gamma-glutamyl transferase (GGT5) was discovered due to its ability to convert leukotriene C4 (LTC4, a glutathione S-conjugate) to LTD4 and may have an important role in the immune system. However, it was not known which cells express the enzyme in humans. We have developed a sensitive and specific antibody that can be used to detect human GGT5 on western blots and in fixed tissue sections. We localized GGT5 expression in normal human tissues. We observed GGT5 expressed by macrophages present in many tissues, including tissue-fixed macrophages such as Kupffer cells in the liver and dust cells in the lung. GGT5 was expressed in some of the same tissues that have been shown to express gamma-glutamyl transferase (GGT1), the only other enzymatically active protein in this family. But, the two enzymes were often expressed by different cell types within the tissue. For example, GGT5 was expressed by the interstitial cells of the kidney; whereas, GGT1 is expressed on the apical surface of the renal proximal tubules. Other tissues with GGT5-positive cells included: adrenal gland, salivary gland, pituitary, thymus, spleen, liver, bone marrow, small intestine, stomach, testis, prostate and placenta. GGT5 and GGT1 are cell surface enzymes. The different pattern of expression results in their access to different extracellular fluids and therefore different substrates. GGT5 has access to substrates in blood and intercellular fluids, while GGT1 has access primarily to fluids in ducts and glands throughout the body. These data provide new insights into the different functions of these two related enzymes. PMID:25377544

  19. Method for the detection of a polypeptide subunit in the presence of a quaternary protein containing the subunit

    SciTech Connect

    Wands, J.R.; Ozturk, M.; Bellet, D.

    1990-06-12

    This patent describes a method for the determination of a free protein subunit of hCG in a sample containing intact quaternary hCG. It comprises: contacting the sample with a first monoclonal antibody which is bound to a carrier, wherein the first monoclonal antibody binds epitopic determinants bindable only on the free protein subunit; incubating the components for a period of time and under conditions sufficient to form an immune complex between the free protein subunit, the first monoclonal antibody, and the carrier; separating the carrier from the sample; adding to the carrier a detectably labeled second monoclonal antibody, wherein the second monoclonal antibody binds epitopic determinants bindable on both the free protein subunit and the intact quaternary hCG; separating the carrier from the liquid phase; and determining the detectably labeled second monoclonal antibody in the carrier or in the liquid phase, which is a measure of the amount of the free protein subunit in the sample.

  20. Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis

    PubMed Central

    Sato, Mitsunari; Yoshida, Yasuo; Nagano, Keiji; Hasegawa, Yoshiaki; Takebe, Jun; Yoshimura, Fuminobu

    2016-01-01

    Butyryl-CoA:acetate CoA transferase, which produces butyrate and acetyl-CoA from butyryl-CoA and acetate, is responsible for the final step of butyrate production in bacteria. This study demonstrates that in the periodontopathogenic bacterium Porphyromonas gingivalis this reaction is not catalyzed by PGN_1171, previously annotated as butyryl-CoA:acetate CoA transferase, but by three distinct CoA transferases, PGN_0725, PGN_1341, and PGN_1888. Gas chromatography/mass spectrometry (GC-MS) and spectrophotometric analyses were performed using crude enzyme extracts from deletion mutant strains and purified recombinant proteins. The experiments revealed that, in the presence of acetate, PGN_0725 preferentially utilized butyryl-CoA rather than propionyl-CoA. By contrast, this preference was reversed in PGN_1888. The only butyryl-CoA:acetate CoA transferase activity was observed in PGN_1341. Double reciprocal plots revealed that all the reactions catalyzed by these enzymes follow a ternary-complex mechanism, in contrast to previously characterized CoA transferases. GC-MS analysis to determine the concentrations of short chain fatty acids (SCFAs) in culture supernatants of P. gingivalis wild type and mutant strains revealed that PGN_0725 and PGN_1888 play a major role in the production of butyrate and propionate, respectively. Interestingly, a triple deletion mutant lacking PGN_0725, PGN_1341, and PGN_1888 produced low levels of SCFAs, suggesting that the microorganism contains CoA transferase(s) in addition to these three enzymes. Growth rates of the mutant strains were mostly slower than that of the wild type, indicating that many carbon compounds produced in the SCFA synthesis appear to be important for the biological activity of this microorganism. PMID:27486457

  1. Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis.

    PubMed

    Sato, Mitsunari; Yoshida, Yasuo; Nagano, Keiji; Hasegawa, Yoshiaki; Takebe, Jun; Yoshimura, Fuminobu

    2016-01-01

    Butyryl-CoA:acetate CoA transferase, which produces butyrate and acetyl-CoA from butyryl-CoA and acetate, is responsible for the final step of butyrate production in bacteria. This study demonstrates that in the periodontopathogenic bacterium Porphyromonas gingivalis this reaction is not catalyzed by PGN_1171, previously annotated as butyryl-CoA:acetate CoA transferase, but by three distinct CoA transferases, PGN_0725, PGN_1341, and PGN_1888. Gas chromatography/mass spectrometry (GC-MS) and spectrophotometric analyses were performed using crude enzyme extracts from deletion mutant strains and purified recombinant proteins. The experiments revealed that, in the presence of acetate, PGN_0725 preferentially utilized butyryl-CoA rather than propionyl-CoA. By contrast, this preference was reversed in PGN_1888. The only butyryl-CoA:acetate CoA transferase activity was observed in PGN_1341. Double reciprocal plots revealed that all the reactions catalyzed by these enzymes follow a ternary-complex mechanism, in contrast to previously characterized CoA transferases. GC-MS analysis to determine the concentrations of short chain fatty acids (SCFAs) in culture supernatants of P. gingivalis wild type and mutant strains revealed that PGN_0725 and PGN_1888 play a major role in the production of butyrate and propionate, respectively. Interestingly, a triple deletion mutant lacking PGN_0725, PGN_1341, and PGN_1888 produced low levels of SCFAs, suggesting that the microorganism contains CoA transferase(s) in addition to these three enzymes. Growth rates of the mutant strains were mostly slower than that of the wild type, indicating that many carbon compounds produced in the SCFA synthesis appear to be important for the biological activity of this microorganism. PMID:27486457

  2. Gel-based chemical cross-linking analysis of 20S proteasome subunit-subunit interactions in breast cancer.

    PubMed

    Song, Hai; Xiong, Hua; Che, Jing; Xi, Qing-Song; Huang, Liu; Xiong, Hui-Hua; Zhang, Peng

    2016-08-01

    The ubiquitin-proteasome system plays a pivotal role in breast tumorigenesis by controlling transcription factors, thus promoting cell cycle growth, and degradation of tumor suppressor proteins. However, breast cancer patients have failed to benefit from proteasome inhibitor treatment partially due to proteasome heterogeneity, which is poorly understood in malignant breast neoplasm. Chemical crosslinking is an increasingly important tool for mapping protein three-dimensional structures and proteinprotein interactions. In the present study, two cross-linkers, bis (sulfosuccinimidyl) suberate (BS(3)) and its water-insoluble analog disuccinimidyl suberate (DSS), were used to map the subunit-subunit interactions in 20S proteasome core particle (CP) from MDA-MB-231 cells. Different types of gel electrophoresis technologies were used. In combination with chemical cross-linking and mass spectrometry, we applied these gel electrophoresis technologies to the study of the noncovalent interactions among 20S proteasome subunits. Firstly, the CP subunit isoforms were profiled. Subsequently, using native/SDSPAGE, it was observed that 0.5 mmol/L BS(3) was a relatively optimal cross-linking concentration for CP subunit-subunit interaction study. 2-DE analysis of the cross-linked CP revealed that α1 might preinteract with α2, and α3 might pre-interact with α4. Moreover, there were different subtypes of α1α2 and α3α4 due to proteasome heterogeneity. There was no significant difference in cross-linking pattern for CP subunits between BS(3) and DSS. Taken together, the gel-based characterization in combination with chemical cross-linking could serve as a tool for the study of subunit interactions within a multi-subunit protein complex. The heterogeneity of 20S proteasome subunit observed in breast cancer cells may provide some key information for proteasome inhibition strategy. PMID:27465334

  3. Light is essential for degradation of ribulose-1,5-bisphosphate carboxylase-oxygenase large subunit during sudden death syndrome development in soybean.

    PubMed

    Ji, J; Scott, M P; Bhattacharyya, M K

    2006-09-01

    FUSARIUM SOLANI f. sp. GLYCINES (Fsg) has been reported to produce at least two phytotoxins. Cell-free FSG culture filtrates containing phytotoxins have been shown to develop foliar sudden death syndrome (SDS) in soybean. We have investigated the changes in protein profiles of diseased leaves caused by cell-free FSG culture filtrates prepared from FSG isolates. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) was conducted to investigate the protein profiles of diseased and healthy leaves. An approximately 55 kDa protein was found to be absent in diseased leaves. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometric analyses and a database search revealed that the missing protein is the ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit, which is involved in carbon assimilation and photorespiration. This result was confirmed by Western blot experiments. We have shown that light is essential for disappearance of the Rubisco large subunit initiated by cell-free FSG culture filtrates. The disappearance of the protein is fairly rapid and occurs within 24 h, presumably due to degradation. Cell-free, FSG culture-induced degradation of the Rubisco large subunit was accompanied by accumulation of reactive oxygen species under light conditions. Terminal deoxynucleotidyl transferase-mediated nick end labelling experiments suggested that programmed cell death was initiated in leaves of seedlings fed with cell-free FSG culture filtrates. These results suggest that, in the presence of light, FSG culture filtrates containing phytotoxins cause degradation of the Rubisco large subunit and accumulation of free radicals and, thereby, initiate programmed cell death leading to foliar SDS development in soybean. PMID:16821191

  4. Stargazin is an AMPA receptor auxiliary subunit.

    PubMed

    Vandenberghe, Wim; Nicoll, Roger A; Bredt, David S

    2005-01-11

    AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors mediate fast excitatory synaptic transmission in brain and underlie aspects of synaptic plasticity. Numerous AMPA receptor-binding proteins have been implicated in AMPA receptor trafficking and anchoring. However, the relative contributions of these proteins to the composition of native AMPA receptor complexes in brain remain uncertain. Here, we use blue native gel electrophoresis to analyze the composition of native AMPA receptor complexes in cerebellar extracts. We identify two receptor populations: a functional form that contains the transmembrane AMPA receptor-regulatory protein stargazin and an apo-form that lacks stargazin. Limited proteolysis confirms assembly of stargazin with a large proportion of native AMPA receptors. In contrast, other AMPA receptor-interacting proteins, such as synapse-associated protein 97, glutamate receptor-interacting protein 1, protein kinase Calpha binding protein, N-ethylmaleimide-sensitive fusion protein, AP2, and protein 4.1N, do not show significant association with AMPA receptor complexes on native gels. These data identify stargazin as an auxiliary subunit for a neurotransmitter-gated ion channel. PMID:15630087

  5. MIF protein are theta-class glutathione S-transferase homologs.

    PubMed Central

    Blocki, F. A.; Ellis, L. B.; Wackett, L. P.

    1993-01-01

    MIF proteins are mammalian polypeptides of approximately 13,000 molecular weight. This class includes human macrophage migration inhibitory factor (MIF), a rat liver protein that has glutathione S-transferase (GST) activity (TRANSMIF), and the mouse delayed early response gene 6 (DER6) protein. MIF proteins were previously linked to GSTs by demonstrating transferase activity and observing N-terminal sequence homology with a mu-class GST (Blocki, F.A., Schlievert, P.M., & Wackett, L.P., 1992, Nature 360, 269-270). In this study, MIF proteins are shown to be structurally related to the theta class of GSTs. This is established in three ways. First, unique primary sequence patterns are developed for each of the GST gene classes. The patterns identify the three MIF proteins as theta-like transferase homologs. Second, pattern analysis indicates that GST members of the theta class contain a serine residue in place of the N-terminal tyrosine that is implicated in glutathione deprotonation and activation in GSTs of known structure (Liu, S., et al., 1992, J. Biol. Chem. 267, 4296-4299). The MIF proteins contain a threonine at this position. Third, polyclonal antibodies raised against recombinant human MIF cross-react on Western blots with rat theta GST but not with alpha and mu GSTs. That MIF proteins have glutathione-binding ability may provide a common structural key toward understanding the varied functions of this widely distributed emerging gene family. Because theta is thought to be the most ancient evolutionary GST class, MIF proteins may have diverged early in evolution but retained a glutathione-binding domain. PMID:8298459

  6. Prediction of function for the polyprenyl transferase subgroup in the isoprenoid synthase superfamily

    PubMed Central

    Wallrapp, Frank H.; Pan, Jian-Jung; Ramamoorthy, Gurusankar; Almonacid, Daniel E.; Hillerich, Brandan S.; Seidel, Ronald; Patskovsky, Yury; Babbitt, Patricia C.; Almo, Steven C.; Jacobson, Matthew P.; Poulter, C. Dale

    2013-01-01

    The number of available protein sequences has increased exponentially with the advent of high-throughput genomic sequencing, creating a significant challenge for functional annotation. Here, we describe a large-scale study on assigning function to unknown members of the trans-polyprenyl transferase (E-PTS) subgroup in the isoprenoid synthase superfamily, which provides substrates for the biosynthesis of the more than 55,000 isoprenoid metabolites. Although the mechanism for determining the product chain length for these enzymes is known, there is no simple relationship between function and primary sequence, so that assigning function is challenging. We addressed this challenge through large-scale bioinformatics analysis of >5,000 putative polyprenyl transferases; experimental characterization of the chain-length specificity of 79 diverse members of this group; determination of 27 structures of 19 of these enzymes, including seven cocrystallized with substrate analogs or products; and the development and successful application of a computational approach to predict function that leverages available structural data through homology modeling and docking of possible products into the active site. The crystallographic structures and computational structural models of the enzyme–ligand complexes elucidate the structural basis of specificity. As a result of this study, the percentage of E-PTS sequences similar to functionally annotated ones (BLAST e-value ≤ 1e−70) increased from 40.6 to 68.8%, and the percentage of sequences similar to available crystal structures increased from 28.9 to 47.4%. The high accuracy of our blind prediction of newly characterized enzymes indicates the potential to predict function to the complete polyprenyl transferase subgroup of the isoprenoid synthase superfamily computationally. PMID:23493556

  7. The DinB Superfamily Includes Novel Mycothiol, Bacillithiol and Glutathione S-transferases

    PubMed Central

    Newton, Gerald L.; Leung, Stephan S.; Wakabayashi, Judy I.; Rawat, Mamta; Fahey, Robert C.

    2011-01-01

    The superfamily of glutathione S-transferases has been the subject of extensive study but Actinobacteria produce mycothiol (MSH) in place of glutathione and no mycothiol S-transferase (MST) has been identified. Using mycothiol and monochlorobimane as substrates a MST activity was detected in extracts of Mycobacterium smegmatis and purified sufficiently to allow identification of MSMEG_0887, a member the DUF664 family of the DinB superfamily, as the MST. The identity of the M. smegmatis and homologous Mycobacterium tuberculosis (Rv0443) enzymes was confirmed by cloning and the expressed proteins were found to be active with MSH but not bacillithiol (BSH) or glutathione (GSH). Bacillus subtilis YfiT is another member of the DinB superfamily but this bacterium produces BSH. The YfiT protein was shown to have S-transferase activity with monochlorobimane when assayed with BSH but not with MSH or GSH. Enterococcus faecalis EF_3021 shares some homology with MSMEG_0887 but this organism produces GSH but not MSH or BSH. Cloned and expressed EF_0321 was active with monochlorobimane and GSH but not with MSH or BSH. MDMPI_2 is another member of the DinB superfamily and has been previously shown to have mycothiol-dependent maleylpyruvate isomerase activity. Three of the eight families of the DinB superfamily include proteins shown to catalyze thiol-dependent metabolic or detoxification activities. Since more than two-thirds of the sequences assigned to the DinB superfamily are members of these families it seems likely that such activity is dominant in the DinB superfamily. PMID:22059487

  8. RNA polymerase II subunit composition, stoichiometry, and phosphorylation.

    PubMed Central

    Kolodziej, P A; Woychik, N; Liao, S M; Young, R A

    1990-01-01

    RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme. Images PMID:2183013

  9. Prokaryotic and eukaryotic RNA polymerases have homologous core subunits.

    PubMed Central

    Sweetser, D; Nonet, M; Young, R A

    1987-01-01

    Eukaryotic RNA polymerases are complex aggregates whose component subunits are functionally ill-defined. The gene that encodes the 140,000-dalton subunit of Saccharomyces cerevisiae RNA polymerase II was isolated and studied in detail to obtain clues to the protein's function. This gene, RPB2, exists in a single copy in the haploid genome. Disruption of the gene is lethal to the yeast cell. RPB2 encodes a protein of 138,750 daltons, which contains sequences implicated in binding purine nucleotides and zinc ions and exhibits striking sequence homology with the beta subunit of Escherichia coli RNA polymerase. These observations suggest that the yeast and the E. coli subunit have similar roles in RNA synthesis, as the beta subunit contains binding sites for nucleotide substrates and a portion of the catalytic site for RNA synthesis. The subunit homologies reported here, and those observed previously with the largest RNA polymerase subunit, indicate that components of the prokaryotic RNA polymerase "core" enzyme have counterparts in eukaryotic RNA polymerases. PMID:3547406

  10. Autocatalytic processing of m-AAA protease subunits in mitochondria.

    PubMed

    Koppen, Mirko; Bonn, Florian; Ehses, Sarah; Langer, Thomas

    2009-10-01

    m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individual subunits is only poorly understood. Here, we have examined the processing of nuclear-encoded m-AAA protease subunits upon import into mitochondria and demonstrate autocatalytic processing of Afg3l1 and Afg3l2. The mitochondrial processing peptidase MPP generates an intermediate form of Afg3l2 that is matured autocatalytically. Afg3l1 or Afg3l2 are also required for maturation of newly imported paraplegin subunits after their cleavage by MPP. Our results establish that mammalian m-AAA proteases can act as processing enzymes in vivo and reveal overlapping activities of Afg3l1 and Afg3l2. These findings might be of relevance for the pathogenesis of neurodegenerative disorders associated with mutations in different m-AAA protease subunits. PMID:19656850

  11. Both subunits of ADP-glucose pyrophosphorylase are regulatory.

    PubMed

    Cross, Joanna M; Clancy, Maureen; Shaw, Janine R; Greene, Thomas W; Schmidt, Robert R; Okita, Thomas W; Hannah, L Curtis

    2004-05-01

    The allosteric enzyme ADP-Glc pyrophosphorylase (AGPase) catalyzes the synthesis of ADP-Glc, a rate-limiting step in starch synthesis. Plant AGPases are heterotetramers, most of which are activated by 3-phosphoglyceric acid (3-PGA) and inhibited by phosphate. The objectives of these studies were to test a hypothesis concerning the relative roles of the two subunits and to identify regions in the subunits important in allosteric regulation. We exploited an Escherichia coli expression system and mosaic AGPases composed of potato (Solanum tuberosum) tuber and maize (Zea mays) endosperm subunit fragments to pursue this objective. Whereas potato and maize subunits have long been separated by speciation and evolution, they are sufficiently similar to form active mosaic enzymes. Potato tuber and maize endosperm AGPases exhibit radically different allosteric properties. Hence, comparing the kinetic properties of the mosaics to those of the maize endosperm and potato tuber AGPases has enabled us to identify regions important in regulation. The data herein conclusively show that both subunits are involved in the allosteric regulation of AGPase. Alterations in the small subunit condition drastically different allosteric properties. In addition, extent of 3-PGA activation and extent of 3-PGA affinity were found to be separate entities, mapping to different regions in both subunits. PMID:15122037

  12. Photoactivation of hypericin down-regulates glutathione S-transferase activity in nasopharyngeal cancer cells.

    PubMed

    Du, H Y; Olivo, M; Tan, B K H; Bay, B H

    2004-04-30

    Photodynamic therapy (PDT) is a new modality of treatment for cancer. Hypericin is a photosensitizer, which is known to generate reactive oxygen species upon activation with light. We observed that photoactivated hypericin induces the generation of reactive oxygen intermediates in nasopharyngeal cancer (NPC) cells in vitro. There was also significant reduction of Glutathione S-transferase (GST) activity in HK1 and CNE-2 NPC cells and in tumor tissues from the NPC/HK1 murine tumor model by hypericin-mediated PDT. As antioxidants protect cells against phototoxicity, down-regulation of GST activity would potentiate the efficacy of hypericin-PDT treatment. PMID:15072826

  13. Induction of glutathione-S-transferase activity by antioxidants in hepatocyte culture.

    PubMed

    Chen, L H; Shiau, C C

    1989-01-01

    Twelve male Sprague-Dawley rats were used for the study. Six rats were injected with benzo(a)pyrene (BP); the other six rats served as the control. Twenty-four hours after injection, hepatocytes were isolated and cultured. The cultured plates were divided into 5 groups and treated with absolute ethanol (control), butylated hydroxytoluene, vitamin E, ascorbic acid or vitamin Elascorbic acid. After 48 hours, the hepatocytes were harvested for enzyme activation determination. With both control and BP-injected rats, each antioxidant treatment significantly increased glutathione-S-transferase activity. The results suggest that antioxidants may have a detoxifying effect against BP-induced carcinogenesis. PMID:2817788

  14. The Phosphopantetheinyl Transferases: Catalysis of a Posttranslational Modification Crucial for Life

    PubMed Central

    Beld, Joris; Sonnenschein, Eva C.; Vickery, Christopher R.; Noel, Joseph P.; Burkart, Michael D.

    2014-01-01

    Although holo-acyl carrier protein synthase, AcpS, a phosphopantetheinyl transferase (PPTase), was characterized in the 1960s, it was not until the publication of the landmark paper by Lambalot et al. in 1996 that PPTases garnered wide-spread attention being classified as a distinct enzyme superfamily. In the past two decades an increasing number of papers has been published on PPTases ranging from identification, characterization, structure determination, mutagenesis, inhibition, and engineering in synthetic biology. In this review, we comprehensively discuss all current knowledge on this class of enzymes that post-translationally install a 4′-phosphopantetheine arm on various carrier proteins. PMID:24292120

  15. Fucosylation of xyloglucan: localization of the transferase in dictyosomes of pea stem cells. [Pisum sativum

    SciTech Connect

    Camirand, A.; Brummell, D.; MacLachlan, G.

    1987-07-01

    Microsomal membranes from elongating regions of etiolated Pisum sativum stems were separated by rate-zonal centrifugation on Renografin gradients. The transfer of labeled fucose and xylose from GDP-(/sup 14/C) fucose and UDP-(/sup 14/C)xylose to xyloglucan occurred mainly in dictyosome-enriched fractions. No transferase activity was detected in secretory vesicle fractions. Pulse-chase experiments using pea stem slices incubated with (/sup 3/H)fucose suggest that xyloglucan chains are fucosylated and their structure completed within the dictyosomes, before being transported to the cell wall by secretory vesicles.

  16. Glutathion S-transferase activity and DDT-susceptibility of Malaysian mosquitos.

    PubMed

    Lee, H L; Chong, W L

    1995-03-01

    Comparative DDT-susceptibility status and glutathion s-transferase (GST) activity of Malaysian Anopheles maculatus, Culex quinquefasciatus and Aedes aegypti was investigated to ascertain the role of this enzyme in DDT resistance. The standardised WHO dose-mortality bioassay tests were used to determine DDT susceptibility in these mosquitos, whilst GST microassay (Brogdon and Barber, 1990) was conducted to measure the activity of this enzyme in mosquito homogenate. It appeared that DDT susceptibility status of Malaysian mosquitos was not correlated with GST activity. PMID:8525405

  17. Proteomic Analysis of Glycine Receptor β Subunit (GlyRβ)-interacting Proteins

    PubMed Central

    del Pino, Isabel; Koch, Dennis; Schemm, Rudolf; Qualmann, Britta; Betz, Heinrich; Paarmann, Ingo

    2014-01-01

    Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord and brainstem. They are clustered at inhibitory postsynapses via a tight interaction of their β subunits (GlyRβ) with the scaffolding protein gephyrin. In an attempt to isolate additional proteins interacting with GlyRβ, we performed pulldown experiments with rat brain extracts using a glutathione S-transferase fusion protein encompassing amino acids 378–455 of the large intracellular loop of GlyRβ as bait. This identified syndapin I (SdpI) as a novel interaction partner of GlyRβ that coimmunoprecipitates with native GlyRs from brainstem extracts. Both SdpI and SdpII bound efficiently to the intracellular loop of GlyRβ in vitro and colocalized with GlyRβ upon coexpression in COS-7 cells. The SdpI-binding site was mapped to a proline-rich sequence of 22 amino acids within the intracellular loop of GlyRβ. Deletion and point mutation analysis disclosed that SdpI binding to GlyRβ is Src homology 3 domain-dependent. In cultured rat spinal cord neurons, SdpI immunoreactivity was found to partially colocalize with marker proteins of inhibitory and excitatory synapses. When SdpI was acutely knocked down in cultured spinal cord neurons by viral miRNA expression, postsynaptic GlyR clusters were significantly reduced in both size and number. Similar changes in GlyR cluster properties were found in spinal cultures from SdpI-deficient mice. Our results are consistent with a role of SdpI in the trafficking and/or cytoskeletal anchoring of synaptic GlyRs. PMID:24509844

  18. Mutations in GABAA receptor subunits associated with genetic epilepsies.

    PubMed

    Macdonald, Robert L; Kang, Jing-Qiong; Gallagher, Martin J

    2010-06-01

    Mutations in inhibitory GABAA receptor subunit genes (GABRA1, GABRB3, GABRG2 and GABRD) have been associated with genetic epilepsy syndromes including childhood absence epilepsy (CAE), juvenile myoclonic epilepsy (JME), pure febrile seizures (FS), generalized epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome (DS)/severe myoclonic epilepsy in infancy (SMEI). These mutations are found in both translated and untranslated gene regions and have been shown to affect the GABAA receptors by altering receptor function and/or by impairing receptor biogenesis by multiple mechanisms including reducing subunit mRNA transcription or stability, impairing subunit folding, stability, or oligomerization and by inhibiting receptor trafficking. PMID:20308251

  19. Induction of Glutathione S-Transferase in Biofilms and Germinating Spores of Mucor hiemalis Strain EH5 from Cold Sulfidic Spring Waters▿

    PubMed Central

    Hoque, Enamul; Pflugmacher, Stephan; Fritscher, Johannes; Wolf, Manfred

    2007-01-01

    The occurrence and activation of glutathione S-transferase (GST) and the GST activities in biofilms in cold sulfidic spring waters were compared to the occurrence and activation of GST and the GST activities of the aquatic fungal strains EH5 and EH7 of Mucor hiemalis isolated for the first time from such waters. Using fluorescently labeled polyclonal anti-GST antibodies and GST activity measurements, we demonstrated that a high level of GST occurred in situ in natural biofilms and pure cultures of strain EH5. Measurement of microsomal and cytosolic soluble GST activities using different xenobiotic substrates, including 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, 1,2-epoxy-3-(4-nitrophenoxy)propane, 1-iodo-2,4-dinitrobenzene, and fluorodifen, showed that the overall biotransforming abilities of biofilms were at least sixfold greater than that of strain EH5 alone. Increasing the level of sodium thiosulfate (STS) in the medium stimulated the microsomal and cytosolic GST activities with CDNB of strain EH5 about 44- and 94-fold, respectively, compared to the activities in the control. The induction of microsomal GST activity with fluorodifen by STS was strongly linear, but the initial strong linear increase in cytosolic GST activity with fluorodifen showed saturation-like effects at STS concentrations higher than approximately 1 mM. Using laser scanning confocal and conventional fluorescence microscopy, abundant fluorescently labeled GST proteins were identified in germinating sporangiospores of strain EH5 after activation by STS. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of at least two main GSTs (∼27.8- and ∼25.6-kDa subunits) in the cytosol of EH5, whereas the major 27.8-kDa subunit was the only GST in microsomes. We suggest that differential cellular GST expression takes place in strain EH5 depending on spore and hyphal development. Our results may

  20. An ethylene-responsive enhancer element is involved in the senescence-related expression of the carnation glutathione-S-transferase (GST1) gene.

    PubMed

    Itzhaki, H; Maxson, J M; Woodson, W R

    1994-09-13

    The increased production of ethylene during carnation petal senescence regulates the transcription of the GST1 gene encoding a subunit of glutathione-S-transferase. We have investigated the molecular basis for this ethylene-responsive transcription by examining the cis elements and trans-acting factors involved in the expression of the GST1 gene. Transient expression assays following delivery of GST1 5' flanking DNA fused to a beta-glucuronidase receptor gene were used to functionally define sequences responsible for ethylene-responsive expression. Deletion analysis of the 5' flanking sequences of GST1 identified a single positive regulatory element of 197 bp between -667 and -470 necessary for ethylene-responsive expression. The sequences within this ethylene-responsive region were further localized to 126 bp between -596 and -470. The ethylene-responsive element (ERE) within this region conferred ethylene-regulated expression upon a minimal cauliflower mosaic virus-35S TATA-box promoter in an orientation-independent manner. Gel electrophoresis mobility-shift assays and DNase I footprinting were used to identify proteins that bind to sequences within the ERE. Nuclear proteins from carnation petals were shown to specifically interact with the 126-bp ERE and the presence and binding of these proteins were independent of ethylene or petal senescence. DNase I footprinting defined DNA sequences between -510 and -488 within the ERE specifically protected by bound protein. An 8-bp sequence (ATTTCAAA) within the protected region shares significant homology with promoter sequences required for ethylene responsiveness from the tomato fruit-ripening E4 gene. PMID:8090746

  1. Microsomal glutathione S-transferase A1-1 with glutathione peroxidase activity from sheep liver: molecular cloning, expression and characterization.

    PubMed Central

    Prabhu, K S; Reddy, P V; Gumpricht, E; Hildenbrandt, G R; Scholz, R W; Sordillo, L M; Reddy, C C

    2001-01-01

    A 25 kDa subunit of glutathione S-transferase (GST) from sheep liver microsomes (microsomal GSTA1-1) with a significant selenium-independent glutathione peroxidase activity has been isolated and characterized. Several analytical criteria, including EDTA stripping, protease protection assay and extraction with alkaline Na(2)CO(3), indicate that the microsomal GSTA1-1 is associated with the inner microsomal membrane. The specific cDNA nucleotide sequence reveals that the enzyme is made up of 222 amino acid residues and shares approx. 73-83% sequence similarity to Alpha-class GSTs from different species. The molecular mass, as determined by electrospray mass ionization, is 25611.3 Da. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, Guthenberg and DePierre (1982) Eur. J. Biochem. 128, 243-248]. The microsomal GSTA1-1 differs from the sheep liver cytosolic GSTs, reported previously from this laboratory, in its substrate specificity profile and molecular mass [Reddy, Burgess, Gong, Massaro and Tu (1983) Arch. Biochem. Biophys. 224, 87-101]. In addition to catalysing the conjugation of 4-hydroxynonenal with GSH, the enzyme also exhibits significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides, such as linoleic and arachidonic acid hydroperoxides, as well as phosphatidylcholine hydroperoxide, but not with H(2)O(2). Thus the microsomal GSTA1-1 isoenzyme might have an important role in the protection of biological membranes against oxidative damage. PMID:11716762

  2. Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag.

    PubMed Central

    Kaplan, W.; Hüsler, P.; Klump, H.; Erhardt, J.; Sluis-Cremer, N.; Dirr, H.

    1997-01-01

    A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is used widely as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an additional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible formation of significant amounts of 160-kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, differential scanning microcalorimetry, and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the unfolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration, with a delta G degree (H2O) = 26.0 +/- 1.7 kcal/mol. The strong relationship observed between the m-value and the size of the protein indicates that the amount of protein surface area exposed to solvent upon unfolding is the major structural determinant for the dependence of the protein's free energy of unfolding on urea concentration. Thermograms obtained by differential scanning microcalorimetry also fitted a two-state unfolding transition model with values of delta Cp = 7,440 J/mol per K, delta H = 950.4 kJ/mol, and delta S = 1,484 J/mol. PMID:9041642

  3. Characterisation of the Candida albicans Phosphopantetheinyl Transferase Ppt2 as a Potential Antifungal Drug Target

    PubMed Central

    Dobb, Katharine S.; Kaye, Sarah J.; Beckmann, Nicola; Thain, John L.; Stateva, Lubomira; Birch, Mike; Oliver, Jason D.

    2015-01-01

    Antifungal drugs acting via new mechanisms of action are urgently needed to combat the increasing numbers of severe fungal infections caused by pathogens such as Candida albicans. The phosphopantetheinyl transferase of Aspergillus fumigatus, encoded by the essential gene pptB, has previously been identified as a potential antifungal target. This study investigated the function of its orthologue in C. albicans, PPT2/C1_09480W by placing one allele under the control of the regulatable MET3 promoter, and deleting the remaining allele. The phenotypes of this conditional null mutant showed that, as in A. fumigatus, the gene PPT2 is essential for growth in C. albicans, thus fulfilling one aspect of an efficient antifungal target. The catalytic activity of Ppt2 as a phosphopantetheinyl transferase and the acyl carrier protein Acp1 as a substrate were demonstrated in a fluorescence transfer assay, using recombinant Ppt2 and Acp1 produced and purified from E.coli. A fluorescence polarisation assay amenable to high-throughput screening was also developed. Therefore we have identified Ppt2 as a broad-spectrum novel antifungal target and developed tools to identify inhibitors as potentially new antifungal compounds. PMID:26606674

  4. Structural and thermodynamic properties of kappa class glutathione transferase from Camelus dromedarius.

    PubMed

    Malik, Ajamaluddin; Fouad, Dalia; Labrou, Nikolaos E; Al-Senaidy, Abdulrahman M; Ismael, Mohamed A; Saeed, Hesham M; Ataya, Farid S

    2016-07-01

    The Arabian camel, Camelus dromedarius is naturally adapted to extreme desert climate and has evolved protective mechanisms to limit oxidative stress. The mitochondrial kappa class glutathione transferase enzyme is a member of GST supergene family that represents an important enzyme group in cellular Phase II detoxification machinery and is involved in the protection against oxidative stress and xenobiotics. In the present study, C. dromedarius kappa class glutathione transferase (CdGSTK1-1) was cloned, expressed in E. coli BL21, purified and its structural, thermodynamic and unfolding pathway was investigated. The results showed that CdGSTK1-1 has unique trimeric structure, exhibits low thermostability and a complex equilibrium unfolding profile. It unfolds through three folding states with formation of thinly populated intermediate species. The melting points (Tm) of the first unfolding transition was 40.3±0.2°C and Tm of the second unfolding transition was 49.1±0.1°C. The van't Hoff enthalpy of the first and second transition were 298.7±13.2 and 616.5±2.4kJ/mol, respectively. Moreover, intrinsic fluorescence and near-UV CD studies indicates that substrate binding does not leads to major conformational changes in CdGSTK1-1. PMID:27044344

  5. Expression of glutathione transferases in corneal cell lines, corneal tissues and a human cornea construct.

    PubMed

    Kölln, Christian; Reichl, Stephan

    2016-06-15

    Glutathione transferase (GST) expression and activity were examined in a three-dimensional human cornea construct and were compared to those of excised animal corneas. The objective of this study was to characterize phase II enzyme expression in the cornea construct with respect to its utility as an alternative to animal cornea models. The expression of the GSTO1-1 and GSTP1-1 enzymes was investigated using immunofluorescence staining and western blotting. The level of total glutathione transferase activity was determined using 1-chloro-2,4- dinitrobenzene as the substrate. Furthermore, the levels of GSTO1-1 and GSTP1-1 activity were examined using S-(4-nitrophenacyl)glutathione and ethacrynic acid, respectively, as the specific substrates. The expression and activity levels of these enzymes were examined in the epithelium, stroma and endothelium, the three main cellular layers of the cornea. In summary, the investigated enzymes were detected at both the protein and functional levels in the cornea construct and the excised animal corneas. However, the enzymatic activity levels of the human cornea construct were lower than those of the animal corneas. PMID:27113863

  6. Functional Diversification of Fungal Glutathione Transferases from the Ure2p Class

    PubMed Central

    Thuillier, Anne; Ngadin, Andrew A.; Thion, Cécile; Billard, Patrick; Jacquot, Jean-Pierre; Gelhaye, Eric; Morel, Mélanie

    2011-01-01

    The glutathione-S-transferase (GST) proteins represent an extended family involved in detoxification processes. They are divided into various classes with high diversity in various organisms. The Ure2p class is especially expanded in saprophytic fungi compared to other fungi. This class is subdivided into two subclasses named Ure2pA and Ure2pB, which have rapidly diversified among fungal phyla. We have focused our analysis on Basidiomycetes and used Phanerochaete chrysosporium as a model to correlate the sequence diversity with the functional diversity of these glutathione transferases. The results show that among the nine isoforms found in P. chrysosporium, two belonging to Ure2pA subclass are exclusively expressed at the transcriptional level in presence of polycyclic aromatic compounds. Moreover, we have highlighted differential catalytic activities and substrate specificities between Ure2pA and Ure2pB isoforms. This diversity of sequence and function suggests that fungal Ure2p sequences have evolved rapidly in response to environmental constraints. PMID:22164343

  7. Glutathione Transferases Superfamily: Cold-Inducible Expression of Distinct GST Genes in Brassica oleracea.

    PubMed

    Vijayakumar, Harshavardhanan; Thamilarasan, Senthil Kumar; Shanmugam, Ashokraj; Natarajan, Sathishkumar; Jung, Hee-Jeong; Park, Jong-In; Kim, HyeRan; Chung, Mi-Young; Nou, Ill-Sup

    2016-01-01

    Plants, as sessile organisms, can suffer serious growth and developmental consequences under cold stress conditions. Glutathione transferases (GSTs, EC 2.5.1.18) are ubiquitous and multifunctional conjugating proteins, which play a major role in stress responses by preventing oxidative damage by reactive oxygen species (ROS). Currently, understanding of their function(s) during different biochemical and signaling pathways under cold stress condition remain unclear. In this study, using combined computational strategy, we identified 65 Brassica oleracea glutathione transferases (BoGST) and characterized them based on evolutionary analysis into 11 classes. Inter-species and intra-species duplication was evident between BoGSTs and Arabidopsis GSTs. Based on localization analyses, we propose possible pathways in which GST genes are involved during cold stress. Further, expression analysis of the predicted putative functions for GST genes were investigated in two cold contrasting genotypes (cold tolerance and susceptible) under cold condition, most of these genes were highly expressed at 6 h and 1 h in the cold tolerant (CT) and cold susceptible (CS) lines, respectively. Overall, BoGSTU19, BoGSTU24, BoGSTF10 are candidate genes highly expressed in B. oleracea. Further investigation of GST superfamily in B. oleracea will aid in understanding complex mechanism underlying cold tolerance in plants. PMID:27472324

  8. Nucleoside Diphosphate Sugar-Starch Glucosyl Transferase Activity of wx Starch Granules 1

    PubMed Central

    Nelson, Oliver E.; Chourey, Prem S.; Chang, Ming Tu

    1978-01-01

    Starch granule preparations from the endosperm tissue of all waxy maize (Zea mays L.) mutants tested have low and approximately equal capability to incorporate glucose from adenosine diphosphate glucose into starch. As the substrate concentration is reduced, however, the activity of waxy preparations relative to nonmutant increases until, at the lowest substrate concentration utilized (0.1 μM), the activity of the waxy preparations is nearly equal to that of the nonmutant preparation. The apparent Km (adenosine diphosphate glucose) for starch granule preparations from wx-C/wx-C/wx-C endosperms was 7.1 × 10−5 M, which is compared to 3 × 10−3 M for preparations from nonwaxy endosperms. Starch granule preparations from three other waxy mutants of independent mutational origin have levels of enzymic activity approximately equal to wx-C at a given substrate concentration giving rise to similar apparent Km estimates. We conclude that there is in maize endosperm starch granules a second starch granule-bound glycosyl transferase, whose presence is revealed when mutation eliminates activity of the more active glucosyl transferase catalyzing the same reaction. PMID:16660522

  9. Glutathione Transferases Superfamily: Cold-Inducible Expression of Distinct GST Genes in Brassica oleracea

    PubMed Central

    Vijayakumar, Harshavardhanan; Thamilarasan, Senthil Kumar; Shanmugam, Ashokraj; Natarajan, Sathishkumar; Jung, Hee-Jeong; Park, Jong-In; Kim, HyeRan; Chung, Mi-Young; Nou, Ill-Sup

    2016-01-01

    Plants, as sessile organisms, can suffer serious growth and developmental consequences under cold stress conditions. Glutathione transferases (GSTs, EC 2.5.1.18) are ubiquitous and multifunctional conjugating proteins, which play a major role in stress responses by preventing oxidative damage by reactive oxygen species (ROS). Currently, understanding of their function(s) during different biochemical and signaling pathways under cold stress condition remain unclear. In this study, using combined computational strategy, we identified 65 Brassica oleracea glutathione transferases (BoGST) and characterized them based on evolutionary analysis into 11 classes. Inter-species and intra-species duplication was evident between BoGSTs and Arabidopsis GSTs. Based on localization analyses, we propose possible pathways in which GST genes are involved during cold stress. Further, expression analysis of the predicted putative functions for GST genes were investigated in two cold contrasting genotypes (cold tolerance and susceptible) under cold condition, most of these genes were highly expressed at 6 h and 1 h in the cold tolerant (CT) and cold susceptible (CS) lines, respectively. Overall, BoGSTU19, BoGSTU24, BoGSTF10 are candidate genes highly expressed in B. oleracea. Further investigation of GST superfamily in B. oleracea will aid in understanding complex mechanism underlying cold tolerance in plants. PMID:27472324

  10. Acyl carrier protein-specific 4'-phosphopantetheinyl transferase activates 10-formyltetrahydrofolate dehydrogenase.

    PubMed

    Strickland, Kyle C; Hoeferlin, L Alexis; Oleinik, Natalia V; Krupenko, Natalia I; Krupenko, Sergey A

    2010-01-15

    4'-Phosphopantetheinyl transferases (PPTs) catalyze the transfer of 4'-phosphopantetheine (4-PP) from coenzyme A to a conserved serine residue of their protein substrates. In humans, the number of pathways utilizing the 4-PP post-translational modification is limited and may only require a single broad specificity PPT for all phosphopantetheinylation reactions. Recently, we have shown that one of the enzymes of folate metabolism, 10-formyltetrahydrofolate dehydrogenase (FDH), requires a 4-PP prosthetic group for catalysis. This moiety acts as a swinging arm to couple the activities of the two catalytic domains of FDH and allows the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. In the current study, we demonstrate that the broad specificity human PPT converts apo-FDH to holoenzyme and thus activates FDH catalysis. Silencing PPT by small interfering RNA in A549 cells prevents FDH modification, indicating the lack of alternative enzymes capable of accomplishing this transferase reaction. Interestingly, PPT-silenced cells demonstrate significantly reduced proliferation and undergo strong G(1) arrest, suggesting that the enzymatic function of PPT is essential and nonredundant. Our study identifies human PPT as the FDH-modifying enzyme and supports the hypothesis that mammals utilize a single enzyme for all phosphopantetheinylation reactions. PMID:19933275