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V Pigiet

Publications and source records attributed to V Pigiet.

At least 19 recordsLinked to original sources
PubMed1988

The effect of H2O2 upon thioredoxin-enriched lens epithelial cells.

Thioredoxin, a dithiol polypeptide, has been examined as a potential contributor to the recovery of lens epithelial cells from oxidative insult. It is reported that Escherichia coli thioredoxin can (a) effectively reduce lens-soluble protein disulfide bonds generated by H2O2, (b) restore to its initial activity H2O2-inactivated glyceraldehyde-3-phosphate dehydrogenase, (c) act as an effective source of reducing potential for lens methionine sulfoxide peptide reductase, and (d) act as a free radical quencher based on studies with a stable free radical system generated by ascorbic acid and 2,6-dimethoxy-p-benzoquinone. Thioredoxin is much more effective than dithiothreitol in restoring glyceraldehyde-3-phosphate dehydrogenase activity and as a cofactor for methionine sulfoxide peptide reductase. Upon incubation with epithelial cells, thioredoxin can be observed in the cell using rocket immunoelectrophoresis. These cells recover from H2O2 insult more rapidly than control cell preparations based upon 1) analyses of plasma membrane-related activities: leucine and 86Rb uptake and 2) analyses of parameters primarily related to the internal cell metabolism: ATP concentration and glyceraldehyde-3-phosphate dehydrogenase activity. Analysis of thioredoxin in cell preparations indicates that only about 9% is in the reduced state implying a low effective concentration of the polypeptide. The experiments suggest that low levels of thioredoxin may significantly increase the ability of lens epithelial cells to recover from exposure to H2O2.

Adenosine Triphosphate↗
PubMed1987

Protein disulfide crosslinking stabilizes a polyoma large T antigen-host protein complex on the nuclear matrix.

We have investigated the effects of intermolecular disulfide crosslinking and temperature-dependent insolubilization of nuclear proteins in vitro on the association of the polyoma large T antigen with the nuclear matrix in polyomavirus-infected mouse 3T6 cells. Nuclear matrices, prepared from polyomavirus-infected 3T6 cells by sequential extraction of isolated nuclei with 1% Triton X-100 (Triton wash), DNase I, and 2 M NaCl (high salt extract) at 4 degrees C, represented 18% of total nuclear protein. Incubation of nuclei with 1 mM sodium tetrathionate (NaTT) to induce disulfide crosslinks or at 37 degrees C to induce temperature-dependent insolubilization prior to extraction, transferred an additional 9-18% of the nuclear protein from the high salt extract to the nuclear matrix. This additional protein represented primarily an increased recovery of the same nuclear protein subset present in nuclear matrices prepared from untreated nuclei. Major constituents of chromatin including histones, hnRNP core proteins, and 98% of nuclear DNA were removed in the high salt extract following either incubation. Polyoma large T antigen was quantified in subcellular fractions by immunoblotting with rat anti-T ascites. Approximately 60-70% of the T antigen was retained in nuclei isolated in isotonic sucrose buffer at pH 7.2. Most (greater than 95%) of the T antigen retained in untreated nuclei was extracted by DNase-high salt treatment. Incubation at 37 degrees C or with NaTT transferred most (greater than 95%) of the T antigen to the nuclear matrix. T antigen solubilized from NaTT-treated matrices with 1% SDS sedimented on sucrose gradients as a large (50-S) complex. These complexes, isolated by immunoprecipitation with anti-T sera, were dissociated by reduction with 2-mercaptoethanol, and SDS-PAGE analysis revealed that T antigen was crosslinked in stoichiometric amounts to several host proteins: 150, 129, 72, and 70 kDa. These host proteins were not present in anti-T immunoprecipitates of solubilized nuclear matrices prepared from iodoacetamide-treated cells. Our results suggest that the majority of polyomavirus large T antigen in infected cells is localized to a specific subnuclear domain which is distinct from the bulk chromatin and is closely associated with the nuclear matrix.

Animals↗
PubMed1987

Association of thioredoxin with the inner membrane and adhesion sites in Escherichia coli.

The intracellular localization of thioredoxin in Escherichia coli was determined by immunoelectron microscopy and correlated to previous biochemical data which had suggested that thioredoxin resides at inner-outer membrane adhesion sites. Since a considerable amount of thioredoxin was lost during preparation of cells for electron microscopy, we immobilized the protein with the heterobifunctional photoactivatable cross-linker p-azidophenacylbromide before the cells were fixed with aldehyde and embedded in Lowicryl K4M. Thin sections were labeled with affinity-purified antithioredoxin antiserum and protein A-gold complexes. Densities of immunolabel in a designated membrane-associated area and in the rest of the cytoplasm were compared and the data were statistically evaluated. Wild-type strain W3110 and strain SK3981, an overproducer of thioredoxin, exhibited increased labeling at the inner membrane and its adjacent cytoplasmic area. In contrast, the more centrally located cytoplasm of both strains showed much lower label density. This label distribution did not change with cell growth or in the stationary phase. Immunolabel was often found at bridges between the inner and outer membranes; this result is consistent with a model which places at least a portion of the thioredoxin at membrane adhesion sites, corresponding to an osmotically sensitive cytoplasmic compartment bounded by a hybrid inner-outer membrane (C.A. Lunn and V. Pigiet, J. Biol. Chem. 257:11424-11430, 1982; C.A. Lunn and V. Pigiet, J. Biol. Chem. 261:832-838, 1986). Specific label was absent in the periplasmic space.

Azides↗
PubMed1987

Two phosphorylated subclasses of polyomavirus large T antigen that differ in their modes of association with the cell nucleus.

Two classes of polyomavirus large T antigen were distinguished, differing in their modes of association with the cell nucleus. A weakly associated class, the nucleoplasmic T antigen, representing 30 to 40% of the total, was solubilized when cells were lysed isotonic buffer at pH 7.2. A more tightly bound class retained in isolated nuclei, the retained T antigen, was extractable either at pH 9.0 or in 2 M NaCl. The retained T antigen contained an additional mole of phosphate, 4 mol of PO4 per mol of T antigen, compared with the nucleoplasmic T antigen (3 mol of PO4 per mol of T antigen). Limit digestion with staphylococcal V8 protease yielded equivalent amounts of five peptides ranging in size from 7.5 to 20 kilodaltons. Additional phosphorylation within a 12-kilodalton peptide accounted for most of the difference in phosphate content between retained and nucleoplasmic T-antigen classes.

Animals↗
PubMed1986

Determination of thiol proteins using monobromobimane labeling and high-performance liquid chromatographic analysis: application to Escherichia coli thioredoxin.

A highly sensitive and specific assay for Escherichia coli thioredoxin was developed using the thiol-specific reagent monobromobimane. Treatment of dithiothreitol-reduced thioredoxin with an excess of monobromobimane in Tris buffer (pH 8.0, 23 degrees C) for 30 min resulted in the formation of a stable derivative which was quantitated by reverse-phase high-performance liquid chromatography with fluorescence detection providing sensitivity in the low picomole range. This method was applied to the determination of intracellular levels of thioredoxin in E. coli. Cell extracts were heated, treated with dithiothreitol, reacted with monobromobimane, and desalted to give a solution which was analyzable for thioredoxin using the chromatographic procedure.

Bacterial Proteins↗
PubMed1986

The catalytic active site of thioredoxin: conformation and homology with bovine pancreatic trypsin inhibitor.

Rabbit polyclonal antibody was raised to a chemically synthesized nonapeptide (Trp-Ala-Glu-Trp-Cys-Gly-Pro-Cys-Lys) corresponding to the active-site sequence of Escherichia coli thioredoxin. The antiserum efficiently inhibited thioredoxin activity in the standard thioredoxin reductase/NADPH coupled assay. This inhibition was blocked by preincubation of the antiserum with the nonapeptide. Tight association of the E. coli thioredoxin to the active-site antibody required SDS denaturation. These results suggest that thioredoxin reductase (NADPH: oxidized-thioredoxin oxidoreductase, EC 1.6.4.5) alters the conformation of thioredoxin sufficiently to permit binding to the antibody. The antiserum bound to plant and liver thioredoxins. Bovine pancreatic trypsin inhibitor, whose active site (Gly-Pro-Cys-Lys) is homologous to that of thioredoxin, also competes for the active-site antibody. This result led to experiments showing that thioredoxin can inhibit the digestion of cytochrome c by trypsin. The ability of thioredoxin to act as a trypsin inhibitor analogue provides a rationale for thioredoxin's resistance to digestion by trypsin.

Amino Acid Sequence↗
PubMed1984

Amplification and purification of plasmid-encoded thioredoxin from Escherichia coli K12.

The thioredoxin gene (trxA) from Escherichia coli K12 has been cloned on a 3-kilobase pair PvuII fragment in a derivative of pBR325 (pBHK8). Thioredoxin protein production was amplified 150-200-fold in a strain containing pBHK8 (SK3981), with the greatest increase/cell observed after cultures reached stationary phase. A simple purification procedure, involving DEAE and AcA-54 column chromatography, yielded homogeneous protein with approximately 70% yield. The high amplification of thioredoxin in these cells (i.e. 10(6) copies/cell representing 40% of total cell protein) approaches the maximum yields seen in genetically constructed cloning vehicles (Bernard, H.U., and Helinski, D.R. (1980) in Genetic Engineering (Setlow, J. K., and Hollaender, A., eds) Vol. 2, pp. 133-167, Plenum Press, New York). This tremendous overproduction of thioredoxin protein is attributed to the high plasmid copy number observed in SK3981 (1700/cell). These results suggest a role for thioredoxin in plasmid DNA replication.

Bacterial Proteins↗
PubMed1983

Polyoma virus minichromosomes: poly ADP-ribosylation of associated chromatin proteins.

The host nuclear enzyme poly(ADP-ribose) polymerase has been shown to be associated with the replicative intermediate and mature forms of polyoma virus minichromosomes. Minichromosome-associated histones H2A and H2B as well as several nonhistone proteins were poly ADP-ribosylated by endogenous poly(ADP-ribose) polymerase. In addition, minichromosome fractions catalyzed the formation in vitro of dimers of endogenous histone H1 linked by poly(ADP-ribose). Poly ADP-ribosylated polyoma virus minichromosome chromatin labeled in vivo with [3H]thymidine could be retained and eluted from anti-poly(ADP-ribose) immunoglobulin G-Sepharose. Pulse-labeled replicative intermediate minichromosomes were retained better on the antibody columns than were mature minichromosomes labeled for 2.5 h. The possible role of poly ADP-ribosylation of viral nucleosomes during polyoma replication or transcription is discussed.

Chromatin↗
PubMed1982

Asynchronous bidirectional replication of polyoma virus DNA.

The structure of polyoma virus replicative intermediates isolated from infected 3T6 cells was analyzed by two-dimensional agarose gel electrophoresis (Sundin and Varshavsky, Cell 21:103-114, 1980) and quantitative electron microscopy (Krauss and Benbow, J. Virol. 38:815-825, 1981). DNA replication was initiated at a single site (ori) in essentially all of the replicative intermediates. Most of the early replicative intermediates were formed by unidirectional synthesis in the direction of early transcription. Most mid- and late replicative intermediates contained two replication forks which had traveled unequal distances from the origin. Asynchronous initiation of the two growing forks was postulated to account for these observations.

Animals↗
PubMed1982

Isolation and characterization of replication forks from discrete regions of the polyoma genome.

Synthesis of polyoma DNA in nuclei isolated from virus-infected 3T6 mouse fibroblasts leads to the selective labeling of replicative intermediates. Digestion of these replicative intermediates with the restriction endonuclease HpaII resulted in three highly labeled heterogeneous species in addition to the expected full-length fragments. These three species migrated more slowly in agarose than did any of the full-length restriction fragments and were shown to represent families of replication forks by criteria of sensitivity to S1 nuclease, kinetics of labeling both in vitro and in vivo, electron microscopy, and migration behavior during agarose gel electrophoresis. Subsequent digestion with other restriction enzymes showed that the two largest of the three fork bands originated from HpaII fragments 1 and 2. These fragments flank the putative terminus located 180 degrees relative to the origin. The third fork-containing band was less labeled and was derived from fragment 3, which is juxtaposed to the replication origin on the side corresponding to late transcription. A two-dimensional gel system revealed the presence of a fourth fork band, derived from fragment 4, that was obscured by full-length fragments 1 and 2 in the single-dimension electrophoresis. Resolution of the fork families revealed multiple discrete species within the major bands, implying the existence of stops or hesitations during replication of a given region of the genome. This conclusion is consistent with the presence of multiple species upon electrophoresis of the fork bands under denaturing conditions.

Animals↗
PubMed1981

Isolation and characterization of a protease-nicked thioredoxin.

An altered form of thioredoxin, composed of two peptides with molecular weights of 7 and 5 X 10(3) has been isolated from Escherichia coli B after chromatography on Ultrogel AcA54. The position of the clip site, determined by amino acid sequencing to lie between Pro(64) and Gly(65), is consistent with a cleavage within the hinge region connecting the two prominent folding domains of thioredoxin. The NH2-terminal domain containing the active site, and the carboxyl-terminal domain correspond to the 7 and 5 X 10(3) fragments, respectively. The two peptides combine to form a tight complex, T'12. At concentrations below 10(-6) M, the clipped species was not efficiently reduced by thioredoxin reductase, showing only half the activity of native thioredoxin at a protein concentration of 9 X 10(-8) M. The Km of the clipped species for thioredoxin reductase (2.9 X 10(-6) M) was nearly equivalent to that of native thioredoxin (2.2 X 10(-6) M), and the Vmax for both native and clipped thioredoxins were nearly identical. These data are taken to imply a reversible association of the fragments to produce a thioredoxin equivalent to the intact species in its interaction with thioredoxin reductase. Some evidence exists for an unfolding of the 7 X 10(3) fragment upon dissociation of the complex. A functional role for the two-domain structure of thioredoxin is proposed in which the carboxyl domain serves to stabilize the active site within the amino domain and confer the species specificity in the binding of thioredoxin to thioredoxin reductase.

Amino Acid Sequence↗
PubMed1981

Polyoma virus minichromosomes: a soluble in vitro replication system.

Polyoma virus minichromosomes were isolated from infected 3T6 cells by hypotonic extraction of isolated nuclei. The kinetics of in vitro DNA synthesis in the nuclear extract was similar to that observed with intact nuclei. The majority of the products of in vitro DNA synthesis sedimented with replicative intermediate (RI) minichromosomes and migrated as two bands (RI-a and RI-b) on 1.4% agarose gels. The kinetics of deoxynucleotide monophosphate incorporation into these species was consistent with the existence of several rate-limiting steps in in vitro replication by polyoma minichromosomes. Electron microscope analysis showed that the RI-a band consisted almost entirely of RI theta structures ranging from 46 to 87% replicated, with one-half of all theta structures 67 +/- 4% replicated. The RI-b material was more complex, consisting of sigma and alpha structures with tails ranging from 7 to 114% of polyoma genome length and, less frequently, of linked and multiple linked dimeric structures.

Bacterial Proteins↗
PubMed1981

Polyoma virus minichromosomes: associated enzyme activities.

Polyoma minichromosomes were isolated and fractionated on glycerol gradients as described by Gourlie et al. (J. Virol. 38:805-814, 1981). Specific assays for DNa polymerases alpha, beta, and gamma, DNA topoisomerase I, and RNase H were carried out on each fraction. The number of units of activity in each fraction was compared with the number of total polyoma and replicative intermediate DNA molecules in each fraction determined by quantitative electron microscopy (M. R. Krauss and R. M. Benbow, J. Virol. 38:815-825, 1981). DNA polymerase alpha cosedimented with polyoma replicative intermediate DNA molecules. DNA polymerase beta and DNA topoisomerase I activities sedimented with mature polyoma minichromosomes. Although the bulk of RNase H activity sedimented in the minichromosome region, the peak of activity was found one fraction behind the peak of mature minichromosomes. Virtually no DNA polymerase gamma activity cosedimented with polyoma minichromosomes.

Centrifugation, Density Gradient↗
PubMed1980

Association of polyoma T antigen and DNA with the nuclear matrix from lytically infected 3T6 cells.

Nuclear matrix prepared from mouse 3T6 cells lytically infected with polyoma virus retained significant amounts of the 100K T antigen and intact viral genomes. Bound T antigen was resistant to the extraction by high salt (2 M NaCl), detergent (1% Triton X-100) and exhaustive DNAase treatment. Only conditions sufficient to disrupt the integrity of the matrix itself solubilized the matrix T antigen. During the time period of 16-30 hr after infection, both the accumulation (in microgram) and the incorporation of 35S-methionine into T antigen increased steadily in cell extracts to a peak at 26 hr and then declined. In contrast, the amount of labeled T antigen retained by the matrix was relatively constant over the same time period. Matrix-bound T antigen was more highly phosphorylated and newly synthesized compared with the extractable T antigen. Viral DNA steadily accumulates in nuclei and on the matrix from 18 to 30 hr after infection. The fraction of viral DNA retained by the matrix was greatest early in infection (25% at 16 hr), declining to less than 10% by 24 hr. These data are consistent with the existence of a fixed (and limited) number of sites for T antigen (more highly phosphorylated) on the matrix and implicate the nuclear matrix as a site of viral DNA replication and possibly encapsidation.

Animals↗
PubMed1978

In vivo distribution of phosphothioredoxin and thioredoxin in Escherichia coli.

The phosphorylation state of thioredoxin was compared in intact cells and in crude extracts. In crude extracts, the extent of phosphorylation was 0.70 to 0.80 mol of phosphate per mol of thioredoxin, with approximately equal amounts of thioredoxin phosphorylated either on cysteinyl32 (formula: see text) or on cysteinyl35 (formula: see text). By comparison, the extent of thioredoxin phosphorylation in intact cells was nearly 1.0 with phosphate present almost exclusively on cysteine32. Nonphosphorylated thioredoxin was present as the reduced thiol form (formula: see text). These findings imply that (formula: see text) is the relevant in vivo species and that a mechanism is operative in crude extracts for transfer of phosphate from cysteine32 to cysteine35.

Bacterial Proteins↗
PubMed1978

Isolation and characterization of phosphothioredoxin from Excherichia coli.

Thioredoxin was isolated as a phosphoprotein from actively growing cultures of Escherichia coli. Labeling was performed in vivo by growing cells in the presence of 32P-labeled inorganic phosphate, and phosphothioredoxin was purified in one step by immunoabsorption to a thioredoxin antibody column. The stoichiometry of phosphate bound was 0.7 to 0.8 mol of phosphate/mol of thioredoxin. The phospho-amino acid linkage was identified as a thiol phosphate by several criteria: (a) the maximum lability of the phosphate bond was between pH 2.5 and 3.5 (t1/2 (37 degrees) = 200 h (pH 7 to 8); 0.4 h (pH 3.0); 200 h (pH 1.0)); (b) the phosphate linkage was very labile in the presence of iodine at neutral pH (t1/2 less than 1 min); and the phosphopeptide was identified as Cys32-Gly-Pro-Cys35-Lys, the same sequence previously implicated as the active site for disulfide-linked oxidation-reduction reactions. Phosphate was distributed on either cysteine, with 60% of the phosphate bound to cysteine32. Results are discussed in terms of the possible role of phosphothioredoxin as an intermediate in phosphotransferase reactions.

Amino Acids↗
PubMed1977

Incorporation of purine nucleoside 5'-[gamma-S]triphosphates as affinity probes for initiation of RNA synthesis in vitro.

Synthetic DNA templates were transcribed by Escherichia coli RNA polymerase using nucleoside 5'-[gamma-S]triphosphates as one of the nucleotide substrates. Substitution of the thiol analogues for the normal nucleotides had no effect on the rate of RNA synthesis. RNA synthesized with either adenosine 5'-[gamma-S]triphosphate or guanosine 5'-[gamma-S]triphosphate was isolated with high efficiency on mercury-agarose columns prepared by activation with low concentrations of cyanogen bromide. Sulfur was shown to be incorporated at the 5' end of RNA by identification of the tetraphosphate HSpppA32p liberated after alkaline hydrolysis of HS(A-32pU)n (alternating copolymer synthesized by the action of E. coli RNA polymerase on d(A-T)n-d(A-T)n with adenosine 5'-[gamma-S]triphosphate and uridine 5'-[alpha-32P]triphosphate as substrates). Transcripts elongated but not initiated with these thiol analogues did not bind to the affinity column. This technique provides an extremely sensitive assay for RNA synthesis initiation in vitro, since initiated transcripts containing radiolabel throught the entire transcript can be isolated.

Chromatography, Affinity↗