Disulphide-reducing Agent Research Articles

An ERp57-mediated disulphide exchange promotes the interaction between Burkholderia cenocepacia and epithelial respiratory cells.

Previous studies have demonstrated that extracellular glutathione reduces the ability of the Cystic Fibrosis pathogen Burkholderia cenocepacia to infect primary or immortalized epithelial respiratory cells. We report here that the adhesion and invasion ability of B. cenocepacia is limited also by thiol-oxidizing and disulphide-reducing agents and by protein disulfide isomerase (PDI) inhibitors. PDI inhibitors also reduce the proinflammatory response elicited by cells in response to Burkholderia. These findings indicate that a membrane-associated PDI catalyzes thiol/disulphide exchange reactions which favor bacterial infection. The combined use of selective PDI inhibitors, RNA silencing and specific antibodies identified ERp57 as a major PDI involved in the interaction between B. cenocepacia and epithelial cells. This study contributes to the elucidation of the Burkholderia pathogenic mechanisms by showing that this microorganism exploits a membrane-associated host protein to infect epithelial cells and identifies ERp57 as a putative pharmacological target for the treatment of Burkholderia lung infections.

Fermentation modifies protein/protein and protein/starch interactions in sorghum dough

A study was made on the molecular and structural changes occurring in the protein and starch components of sorghum flour when fermented to prepare typical non-malted Sudanese foods. Protein solubility and SDS-PAGE studies indicated that water-soluble proteins are the main target of hydrolysis during fermentation. Proteolysis products are taken up for bacterial growth. Kafirins are among the proteins left intact by proteolytic events in the fermentation step. Upon cooking in boiling water kafirins are converted into protein aggregates almost insoluble even in the presence of 8 M urea and of disulphide-reducing agents. Viscoamylographic and microstructural studies indicate that fermentation leads to the release of starch granules from very compact structures in the original sorghum flour, in which proteolysis-sensitive, water-soluble proteins form an essential part of the outermost shell of large structures, where starch granules are embedded into a kafirin-rich protein matrix that is not affected by proteolytic events during fermentation.

Thiol reagents and nitric oxide modulate the gating of BKCa channels from the guinea-pig taenia caeci.

1. The site of the direct modulation of the gating of BKCa channels by the nitric oxide donor s-nitroso-l-cysteine (NOCys) was examined in excised membrane patches of the guinea-pig taenia caeci by the use of various thiol (sulphydryl)-specific reagents, including N-ethylmaleimide (NEM) and three charged methanethiosulphonate (MTS) reagents, namely positively charged 2-aminoethyl MTS hydrobromide (MTSEA) and [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium (2-sulphonatoethyl) MTS (MTSES), which all specifically convert sulphydryls to a disulphide. 2. At 10 micro mol/L, NOCys transiently increased the probability of opening (N.Po) of the BKCa channels (at 0 mV) after a delay of 1-2 min. 3. Disulphide-reducing agents, such as dithiothreitol (10 micro mol/L), increased N.Po in a manner that was reversed by the sulphide-oxidizing agent thimerosal (10 micro mol/L). Both positively charged MTSET (2.5 mmol/L) and negatively charged MTSES (2.5 mmol/L) rapidly increased N.Po. However, only the MTSES-evoked increase in N.Po remained after a prolonged washout period. 4. The specific alkylating agent of cysteine thiols NEM (1 mmol/L) and the positively charged, but membrane permeable, MTSEA (2.5 mmol/L) decreased N.Po (at 0 mV). 5. Pre-exposure of excised membrane patches to NEM or MTSES prevented the excitatory actions of NOCys (10 micro mol/L). 6. We conclude that MTSES and NOCys must modify thiols on cysteine residues within basic regions of the channel protein that would electrostatically exclude MTSEA and MTSET. A consensus sequence of various mammalian alpha-subunits of the BKCa channel reveals two pairs of cysteine residues surrounded by basic amino acids that could be the site of action for NOCys and MTSES.

Thiol oxidation by 2,2'-dithiodipyridine causes a reversible increase in cytoplasmic free Ca2+ concentration in pancreatic beta-cells. Role for inositol 1,4,5-trisphosphate-sensitive Ca2+ stores.

2,2'-Dithiodipyridine (2,2'-DTDP), a reactive disulphide that mobilizes Ca2+ from ryanodine-sensitive Ca2+ stores in muscle, induced a biphasic increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells loaded with fura 2. This increase consisted of an early transient followed by a second, slower, rise. The [Ca2+]i transient was dependent on extracellular Ca2+ and disappeared on treatment with nimodipine. The reactive disulphide caused plasma membrane depolarization, as studied by the perforated-patch configuration of the patch-clamp technique. Hence membrane depolarization and opening of the L-type voltage-gated Ca2+ channels were responsible for the first transient in [Ca2+]i. The second slower increase in [Ca2+]i was prolonged but readily reversed by the disulphide-reducing agent 1,4-dithiothreitol. This increase in [Ca2+]i was not decreased by nimodipine or by omission of extracellular Ca2+, but was eliminated when the Ins(1,4,5)P3-sensitive Ca2+ pool was first depleted by carbachol. Ryanodine or its beta-alanyl analogue did not release Ca2+ from intracellular stores, and a high concentration of ryanodine did not inhibit Ca2+ release by 2,2'-DTDP. The disulphide compound suppressed glucose metabolism and decreased the mitochondrial inner-membrane potential. We conclude that thiol oxidation by 2,2'-DTDP affects Ca2+ homeostasis in beta-cells by multiple mechanisms. However, unlike the situation in muscle, in beta-cells 2,2'-DTDP releases Ca2+ from intracellular pools by mechanisms that do not involve activation of ryanodine receptors. Instead, in these cells the Ins(1,4,5)P3-sensitive intracellular Ca2+ store comprises an alternative target for the Ca(2+)-mobilizing action of the reactive disulphide compound.

Overexpression, purification and characterization of Dictyostelium calcineurin A

The catalytic subunit of Ca2+/calmodulin-dependent protein phosphatase (calcineurin A) was overexpressed about 50-fold in Dictyostelium discoideum cells transformed with a vector containing the cDNA for D. discoideum calcineurin A under control of the actin-6 promoter. In crude lysates from the overexpressing cell line, high Ca2+/calmodulin-stimulated phosphatase activity was detected. Calcineurin A was purified by anion exchange chromatography and calmodulin-Sepharose affinity chromatography, and the enzymatic activity of the isolated protein was characterized. Its phosphatase activity was strictly dependent on the addition of divalent metal ions such as Mg2+ or Mn2+. Disulphide-reducing agents increased the activity more than 10-fold. Ca2+/calmodulin stimulated the activity by a factor of 2.5-5. Despite the high extra Ca2+/calmodulin-dependent phosphatase activity, the overexpressing cell line showed no phenotypic aberrations.

DNA and chromatin structure: Influence of incubation on the chromatin condensation and nuclear stability of human spermatozoa by flow cytometry

Flow cytometry analysis was used for the accurate and objective evaluation of sperm chromatin condensation and chromatin stability of sperm nuclei. It was also possible to determine the influence of incubation on sperm chromatin. Different types of spermatozoa were studied: unprocessed spermatozoa at 1 and 45 min after ejaculation, after swim-up (migrated), spermatozoa incubated for 6 h in non-capacitating conditions (aged), or in B2 medium (capacitated) or B2 medium followed 1 h later with A23187 (reacted). All types of spermatozoa were analysed before and after treatment with various decondensation agents: sodium dodecyl sulphate (SDS), SDS plus EDTA and SDS plus disulphide-reducing agent [dithiotreitol (DTT)]. Sperm nuclei were enzymatically isolated and stained with propidium iodide. Three flow cytometric parameters were then measured: forward light scatter (cellular size), side light scatter (cellular complexity) and fluorescence (uptake of propidium iodide). Fluorescence was the most suitable parameter to study the degree of condensation and resistance to decondensation of DNA in the spermatozoa. Unprocessed spermatozoa 1 min after ejaculation underwent decondensation by all assessed treatments (anionic detergent, chelating or disulphide-reducing agents). Unprocessed spermatozoa 45 min after ejaculation and migrated spermatozoa did not undergo decondensation with SDS treatment, but decondensation occurred after treatment with SDS+EDTA or SDS+DTT. Spermatozoa incubated for 6 h under both non-capacitating (aged spermatozoa) and capacitating conditions (capacitated spermatozoa) and reacted spermatozoa were decondensed only after treatment with SDS+DTT.(ABSTRACT TRUNCATED AT 250 WORDS)

Fusion between Newcastle disease virus and erythrocyte ghosts using octadecyl Rhodamine B fluorescence assay produces dequenching curves that fit the sum of two exponentials.

The kinetics of fusion between Newcastle disease virus and erythrocyte ghosts has been investigated with the octadecyl Rhodamine B chloride assay [Hoekstra, De Boer, Klappe, and Wilschut (1984) Biochemistry 23, 5675-5681], and the data from the dequenching curves were fitted by non-linear regression to currently used kinetic models. We used direct computer-assisted fitting of the dequenching curves to the mathematical equations. Discrimination between models was performed by statistical analysis of different fits. The experimental data fit the exponential model previously published [Nir, Klappe, and Hoekstra (1986) Biochemistry 25, 2155-2161] but we describe for the first time that the best fit was achieved for the sum of two exponential terms: A1[1-exp(-k1t)]+A2[1-exp(-k2t)]. The first exponential term represents a fast reaction and the second a slow dequenching reaction. These findings reveal the existence of two independent, but simultaneous, processes during the fusion assay. In order to challenge the model and to understand the meaning of both equation, fusion experiments were carried out under different conditions well known to affect viral fusion (changes in pH, temperature and ghost concentration, and the presence of disulphide-reducing agents or inhibitors of viral neuraminidase activity), and the same computer fitting scheme was followed. The first exponential equation represents the viral protein-dependent fusion process itself, because it is affected by the assay conditions. The second exponential equation accounts for a nonspecific reaction, because it is completely independent of the assay conditions and hence of the viral proteins. An interpretation of this second process is discussed in terms of probe transfer between vesicles.

Dissociation and reassociation of trichocyst proteins: biochemical and ultrastructural studies.

Trichocysts, the crystalline exocytotic organelles in Paramecium tetraurelia, are composed of small, acidic proteins existing primarily as disulphide-linked dimers. We have disaggregated trichocyst proteins with heat, simultaneously observing the changes in morphology and protein composition. The tip matrix was most heat-labile; its subunits progressively broke away from the distal end. During this process, breakdown of the cylindrical shaft began. Shafts first became flattened and torn lengthwise, yielding smaller, interconnected pieces still having the crystalline arrangement of their 5 nm thick fibres. Ultimately this pattern became disordered, and discrete fibrils of the same thickness disengaged from the meshwork. In freeze-etched preparations these fibrils were composed of thinner filaments in side-by-side association. Disaggregation of the tip sheath began from the distal end before shaft dissociation was complete. Trichocysts broke down to thin fibrils, but probably not to monomeric subunits. At least three proteins were preferentially released in the initial phase of dissociation. Disulphide-reducing agent present during heating increased the rate of dissociation without altering the sequence of morphological changes or the order of release of individual proteins. The rate and extent of heat-induced dissociation were strongly dependent on pH and cation concentration. The stabilizing effects of low pH and of cations were additive. A cooled suspension of fully dissociated trichocysts reassociated into sedimentable aggregates with discernible filamentous order, but without the crystalline structure of intact trichocysts. Reassociation was dependent upon time, temperature and protein concentration. All but one of the trichocyst proteins re-entered the sedimentable aggregate during reassociation. Reassociation was faster and more complete at pH 6 than at pH 8 and was stimulated by Ca2+, Mg2+ and La3+. Trichocyst proteins dissociated in the presence of dithiothreitol did not reassociate, even after removal of the reducing agent. Trichocysts from mutants defective to varying degrees in trichocyst formation were subjected to similar experimental protocols. Heat-dissociated trichocysts of the mutants scc6 and ptA1 reassociated at rates similar to those of wild-type; ftA3 showed slower reassociation, and tam38 showed little or no reassociation. Reassociation of wild-type trichocyst proteins was blocked by the addition of an equal amount of tam38 trichocyst proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Destruction and reconstitution of the activity of hydrogenase from Chromatium vinosum

The specific activity and the EPR spectra of purified hydrogenase from Chromatium vinosum vary from preparation to preparation. It has been found that both properties are dependent on the redox state of specific redox groups, probably thiols, in the enzyme. In defect enzyme molecules the proposed thiol groups are oxidized to an -S-S- bridge. As a result, the activity of the enzyme in the assay with viologens is minimal and nickel and the iron-sulphur cluster in the oxidized enzyme are present as two non-interacting S = 1 2 systems, Ni(III) and a [3Fe-4S] 1+ cluster. In intact enzyme the -S-S- bridge does not exist. Such enzyme molecules are 15- (H 2-production reaction) or 65- (H 2-uptake reaction) times as active as defect molecules. Intact oxidized enzyme can accomodate a spin-coupled Ni(III)/[4Fe-4S] 3+ pair. It can be converted to defect molecules by partial reduction with phenazine methosulphate plus ascorbate in air at 40–50°C. The reverse transition is induced by anaerobic incubation with a disulphide-reducing agent at 50°C.

Graves' immunoglobulins protect the human TSH receptor: further evidence for TSH receptor antibodies in Graves' disease.

We have investigated whether membrane-bound TSH and Graves' immunoglobulins (Ig) were able to inhibit the action of the disulphide-reducing agent dithiothreitol (DTT) which reduced the binding of 125I-bTSH to thyroid membranes. Human thyroid (20,000 g fraction) was incubated with increasing concentrations of cold bTSH, washed and resuspended in 10 mmol/lDTT for 60 min at 37 degrees C. After the DTT was removed, 70-80% of the receptor-bound bTSH dissociated with 2 mol/l NaCl. In the absence of cold bTSH, 80% of the binding of receptor-purified 125I-bTSH was inhibited after membrane exposure to DTT but in the presence of bTSH-occupied receptor sites there was a dose related protection of the receptors, with 100% protection following incubation with 30 mU/ml bTSH. Like bTSH, immunoglobulin fractions from Graves' sera caused a time-dependent reduction in 125I-bTSH binding-inhibition after incubation and washing of thyroid membranes prior to ligand binding. Approximately 25-40% of this reduction was reversible by exposure to 2 mol/l NaCl. After pre-binding normal Ig to thyroid membranes, and subsequent incubation with DTT, there was no protection of TSH receptors. However, each of the Graves' Ig examined (n = 4) was able to provide protection of the TSH receptor binding sites in proportion to their derived TSH receptor occupancy. Receptor-bound bTSH was, therefore, able to protect the human TSH binding site from disulphide reduction. In addition, Graves' Ig, but not normal Ig, contained antibodies which were able to protect the TSH receptor binding sites in the same way as bTSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of zinc and other metal ions on the stability and activity of Escherichia coli alkaline phosphatase

Measurement of the ultraviolet circular dichroism of apo-(alkaline phosphatase) in urea solutions showed substantial denaturation in 3m-urea. A zinc-deficient mutant alkaline phosphatase behaved similarly. The stability of the enzyme in 6m-urea was followed as a function of its zinc content and was found to be dependent on the first two of the four zinc atoms bound by apoenzyme. Phosphatase activity was mostly dependent on a second pair of zinc atoms. Mn(2+), Co(2+), Cu(2+) or Cd(2+) also restored structural stability. Sedimentation-velocity and -equilibrium experiments revealed that dissociation of the dimer accompanied apoenzyme denaturation in urea concentrations of 1m or higher, without treatment with disulphide-reducing agent.