Dithioerythritol Research Articles

The mitochondrial oxoglutarate carrier: sulfhydryl reagents bind to cysteine-184, and this interaction is enhanced by substrate binding.

The interaction of sulfhydryl reagents with the oxoglutarate carrier (OGC) of bovine heart mitochondria was investigated in proteoliposomes reconstituted from purified carrier and lipids. Incubation of the proteoliposomes with maleimides or mercurials led to inhibition of the oxoglutarate carrier protein. The inhibition of oxoglutarate transport by mercurials was removed by dithioerythritol (DTE), whereas inhibition by maleimides was not. Preincubation of the proteoliposomes with mercurials protected the carrier protein against inactivation by the fluorescent sulfhydryl reagent N-(1-pyrenyl)maleimide (PM) and decreased the fluorescence associated with the carrier, indicating that mercurials bind to the same cysteine which is modified by PM. The presence of the substrates oxoglutarate and malate increased the binding of PM to the reconstituted carrier as well as the degree of inhibition of the reconstituted transport activity caused by PM, other maleimides, and mercurials. This result is consistent with the assumption that substrate binding causes a change in the tertiary structure of the carrier protein. The primary sequence of the oxoglutarate carrier contains three cysteines (Cys-184, Cys-221, and Cys-224). We provide evidence that PM labels only Cys-184, whereas Cys-221 and Cys-224 are linked by a disulfide bridge.

Immunoglobulin A subclass measurement in serum and saliva: sensitivity of detection of dimeric IgA2 in ELISA depends on the antibody used

The existence of two IgA subclasses in humans has been reliably shown by biochemical, immunochemical and genetic means. IgA is unique among immunoglobulins in the regular occurrence of both monomeric and polymeric forms. In order to be able to study the relationship between monomeric and polymeric IgA1 and IgA2 concentrations in the circulation and mucosal compartment i.e. secretions, it is essential that the methods used are not biased by the molecular size of the IgA under investigation. We validated IgA and IgA subclass measurements in serum and saliva by sandwich enzyme-linked immunosorbent assay (ELISA). Coating reagents were specific mAbs against IgA (clone 4E8), IgA1 (clone 69-11.4) or IgA2 (clone 16-512-H5 and clone IF8.58). Pooled normal human serum and purified dimeric IgA1 (d-IgA1) or IgA2 (d-IgA2) myeloma proteins were used to standardize the assays. Polymeric and monomeric forms of IgA in sera from volunteers and patients with myelomatosis were assayed in fractions separated by high performance liquid chromatography (HPLC). Dithioerythritol (DTE) was used to determine the influence of the quarternary stucture of IgA on its detection by mAbs. We found that mAbs 4E8, 69-11.4 and 16-512-H5 reliably measured d-IgA, d-IgA1 and d-IgA2 respectively, independent of the standard employed. Clone IF8.58 underestimated the concentration of d-IgA2 (correction factor ±2) with increased sensitivity in the presence of DTE. This difference is probably explained by the composition of the immunogen against which the mAb was raised. We conclude that no reliable conclusions can be made concerning the subclass ratio in biological fluids unless the monoclonal antibodies used have been appropriately validated.

Isolation and characterization of five fibrin(ogen)olytic enzymes from the venom of Philodryas olfersii (green snake)

M. T. Assakura, A. P. Reichl and F. R. Mandelbaum. Isolation and characterization of five fibrin(ogen)olytic enzymes from the venom of Philodryas olfersii (green snake). Toxicon 32, 819–831, 1994.—Five distinct fibrin(ogen)olytic proteinases PofibC 1, C 2, C 3, H and S were isolated by gel filtration and ion-exchange chromatographies. PofibC 1, C 2, C 3 and H are metalloproteinases inhibited by ethylenediamine tetracetic acid (EDTA) or 1,10-phenanthroline. Only PofibH had hemorrhagic activity. PofibS is a serine proteinase, inhibited by phenylmethylsulfonyl fluoride (PMSF) or Torresea cearensis trypsin inhibitor (TCTI). All five enzymes were inhibited by dithiothreitol (DTT) or dithioerythritol (DTE). PofibC 1 and C 2 presented the same mol. wt of 47,000 and are acidic proteins of p I 6.2. PofibC 3 is a basic proteinase of p I 8.5 and mol. wt 45,000. The hemorrhagic proteinase PofibH had a mol. wt of 58,000 and p I of 4.6 and PofibS had a mol. wt of 36,000 and p I of 4.5. The five proteinases degraded fibrin and fibrinogen. PofibC 1, C 2, C 3 and H degraded preferentially Aα-chains while PofibS cleaved concomitantly Aα and Bβ-chains of fibrinogen. None of these enzymes cleaved the γ-chain of fibrinogen. When correlated with the thrombin delay time, the most active was PofibS, while PofibH and PofibC 1 showed almost no activity. The proteinases also differed in the peptide cleavage of B-chain of insulin. Philodryas olfersii venom promoted in vivo a loss of the circulant plasma fibrinogen, as was observed in experiments with rats.

POSITIVE PRETRANSPLANT CROSSMATCHES PREDICT EARLY GRAFT LOSS IN LIVER ALLOGRAFT RECIPIENTS

We evaluated the influence of donor-recipient HLA compatibility and recipient pretransplant antidonor sensitization on liver allograft recipient survival. The overall graft survival results for 67 cyclosporine-prednisone treated liver allograft recipients at 3, 6, and 12 months posttransplant were 86%, 83%, and 83%, respectively. No significant differences were observed when comparing the one-year survivals of 81% vs. 85% for men and women or 80% vs. 85% for adult and pediatric patients. Similarly, no differences were observed when comparing one-year graft survivals for well vs. poorly matched recipients of 77% vs. 83% for recipients with < or = 2 HLA A, B vs. > 2 HLA A, B mismatches (MMs) and 82% vs. 82% for recipients with 0-1 HLA-DR MMs vs. 2 HLA-DR MMs, respectively. Pretransplant transfusion history and race also did not influence survival. Standard NIH (long-incubation) and anti-human globulin (AHG) crossmatches were performed. The 12% of recipients (8/67) displaying a positive NIH crossmatch experienced significantly poorer 3-, 6-, and 12-month survivals of 62% vs. 89%, 62% vs. 86%, and 62% vs. 86% (all P < 0.05), respectively, than the 59 NIH-crossmatch negative recipients. Similarly, the 16% (11/67) of recipients displaying a positive AHG crossmatch had significantly poorer 3-, 6-, and 12-month survivals of 63% vs. 91%, 54% vs. 89%, and 54% vs. 89% (all P < 0.05) respectively, than the 56 AHG crossmatch-negative recipients. NIH and AHG crossmatch-positive sera were treated with dithioerythritol (DTE) to establish whether reactivity was due to IgM or IgG immunoglobulin. One-year graft survivals of 65% vs. 30% (P < 0.05) were observed when the crossmatch-positive sera reactivities were due to IgM vs. IgG immunoglobulin. While graft survivals were improved when positive crossmatch serum reactivity was due to IgM, these survivals were still significantly poorer than when the crossmatches were completely negative (86% vs. 60%, P < 0.05 for NIH-negative vs. NIH-positive, but DTE-negative, and 88% vs. 77%, P < 0.05 for AHG-negative vs. AHG-positive, but DTE-negative). Therefore, an NIH- or AHG-positive crossmatch, due either to IgM or IgG reactivity, results in poor early (3- and 6-months) liver allograft survival. Crossmatch-positive recipients experienced significantly (P < 0.05) more rejections and more steroid-resistant rejections (P < 0.05) than crossmatch-negative recipients.(ABSTRACT TRUNCATED AT 400 WORDS)

Erythrocyte membrane barrier function: role of bilayer polarity and skeletal proteins.

Conference Article| November 01 1992 Erythrocyte membrane barrier function: role of bilayer polarity and skeletal proteins Bernhard Deuticke; Bernhard Deuticke * 1Institut für Physiologie, Rheinisch-Westfälische Technische Hochschule, D-5100 Aachen, Germany *Prof. Dr. B. Deuticke, Institut für Physiologie, Klinikum, Pauwelsstr. 30, D-5100 Aachen, Germany Search for other works by this author on: This Site PubMed Google Scholar Sabine Klonk; Sabine Klonk 1Institut für Physiologie, Rheinisch-Westfälische Technische Hochschule, D-5100 Aachen, Germany Search for other works by this author on: This Site PubMed Google Scholar Cees W. M. Haest Cees W. M. Haest 1Institut für Physiologie, Rheinisch-Westfälische Technische Hochschule, D-5100 Aachen, Germany Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1992) 20 (4): 769–773. https://doi.org/10.1042/bst0200769 Article history Received: July 14 1992 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Bernhard Deuticke, Sabine Klonk, Cees W. M. Haest; Erythrocyte membrane barrier function: role of bilayer polarity and skeletal proteins. Biochem Soc Trans 1 November 1992; 20 (4): 769–773. doi: https://doi.org/10.1042/bst0200769 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: DTE, dithioerythritol, NEM, N-ethylmaleimide, MTPP+, methyltriphenylphosphonium ion This content is only available as a PDF. © 1992 Biochemical Society1992 Article PDF first page preview Close Modal You do not currently have access to this content.

A quantitative investigation of proton— and lead(II)—dithioerythritol complex equilibria under physiological conditions

Dithioerythritol (DTE) is frequently employed as a reducing agent or a protective reagent for thiol groups in biological assays. Owing to its known inhibiting properties in enzyme catalysis reactions, lead is also commonly used in such experiments, and often simultaneously with DTE. Given the potential affinity of these two reactants, their measured individual effects may well depend on their interactions in the medium used. Any quantitative assessment of these interactions necessitates, however, that the complex equilibria between lead and DTE be investigated beforehand. To test this hypothesis, the formation constants of lead(II) complexes with DTE under physiological conditions (37°, NaCl 0.15M) have been calculated from the results of glass electrode potentiometry, with the help of the MINIQUAD and ESTA computer programs. The p K values for dissociation of DTE have been found equal to 8.926 ± 0.003 and 9.840 ± 0.003. The following lead—DTE species have been characterized: ML (12.774 ± 0.037), MLH −1 (2.858 ± 0.037), M 2LH −1 (13.349 ± 0.025) and M 6L 5 (86.586 ± 0.099); the log *β-values are given in the parentheses. Appropriate computer simulations effectively show that the interactions of the two reactants are indeed quite significant within the concentration ranges commonly used in in vitro biological assays. They should thus be taken into account in interpretation of the effects observed.

Xanthine Oxidase/Dehydrogenase Activity in Intact Cultured Cells (In Situ Analysis)

The measured ratio of xanthine oxidase activity to the total activity of xanthine oxidase and dehydrogenase showed higher values in intact cells than when similar cells were homogenized. The total activity was the same for both systems. The xanthine oxidase ratio was 90, 60, 50, 50, 60% in V79, RIF/Ha3, SCC7, KHT intact cells and freshly extracted murine peritoneal macrophages respectively while the corresponding ratios measured were 25, 40, 38, 35, 22% when the cells were lysed by homogenization. Superoxide radical O2-. production by addition of xanthine to intact or homogenized cells to activate intracellular xanthine oxidase was higher in intact than homogenized cells. Homogenization of cells and tissues in the presence of dithioerythritol (DTE) can evidently lead to a considerable under-estimation of the xanthine oxidase ratio. The effect of hypoxia on cells has also been examined.

Oxidative inhibition of red blood cell ATPases by glyceraldehyde

Glyceraldehyde and other simple monosaccharides autoxidize under physiological conditions, forming dicarbonyl compounds and hydrogen peroxide via intermediate free radicals. These products may have deleterious effects on cell components. In this paper we study the effect of glyceraldehyde autoxidation on red-cell ATPase activities. The autoxidation of glyceraldehyde in imidazole-glycylglycine buffer, measured by oxygen consumption, depends on the buffer concentration and decreases in the presence of superoxide dismutase and catalase. The addition of DETAPAC inhibits the autoxidation almost completely. When human red-blood-cell membranes are incubated with glyceraldehyde, the red-blood-cell ATPase activities decrease significantly. The addition of DETAPAC, GSH and DTE (dithioerythritol) protects the enzyme from inactivation, but superoxide dismutase and catalase have no effect. Methylglyoxal (a dicarbonyl which is analogous to hydroxypyruvaldehyde derived from glyceraldehyde autoxidation) proved to have a powerful inhibitory action on ATPase activities. The addition of DTE completely protects the enzyme from inactivation, suggesting that the sulphydryl groups of the active site of the enzyme are the critical targets for dicarbonyl compounds.

AHG AND DTE/AHG PROCEDURE IDENTIFICATION OF CROSSMATCH-APPROPRIATE DONOR-RECIPIENT PAIRINGS THAT RESULT IN IMPROVED GRAFT SURVIVAL

We compared our standard NIH (extended incubation) crossmatch (XM) with antihuman globulin (AHG) and flow cytometry crossmatches (FCXM) and correlated the results with primary cadaveric and retransplant graft survivals. In addition, we treated the XM sera with the reducing reagent dithioerythritol (DTE) to discriminate IgM from IgG immunoglobulin reactivity. For the 166 CsA-Pred-treated primary cadaveric renal allograft recipients the 1-year graft survival rate following an NIH-NEG XM was 81%. NIH-XM-NEG recipients who were also AHG-XM-NEG displayed an 82% 1-year graft survival as well. In contrast, NIH-NEG, but AHG-POS XM primary CAD recipients displayed a significantly reduced graft survival rate of 67%. Treatment of AHG-POS XM sera with DTE-delineated DTE/AHG-NEG and POS crossmatches associated with significantly different graft survivals of 83% and 0%, respectively, for these primary recipients. Flow cytometry XM results did not improve on the AHG-NEG or DTE/AHG-NEG XM primary graft survivals. These results were seen whether testing pre-Tx or historical (Hx) sera. For Re-Tx recipients an AHG-NEG XM resulted in significantly improved graft survival compared with the NIH-XM-NEG results. The overall 1-year graft survival rate for the 70 Re-Tx recipients studied was 64% (following a NEG pre-Tx NIH-XM). Re-Tx recipients with an AHG-NEG XM displayed an improved graft survival compared with NIH-XM-NEG recipients (77% vs. 64%, P less than 0.05) and with AHG-POS recipients (77% vs. 36%, P less than 0.01). However, treatment of Re-Tx, AHG-POS sera with DTE resulted in comparably poor graft survival rates of 31% and 50% for DTE/AHG-NEG and POS crossmatches, respectively. A FCXM did not improve on the results of Re-Tx graft survival following an AHG-NEG XM. These results were obtained whether testing pre-Tx or Hx sera. HLA matching, PRA, and the time the first Tx was lost did not influence the Re-Tx graft survival outcome following an AHG-NEG XM. Therefore, successful primary cadaveric renal allograft survival can be accomplished following either an AHG-NEG XM or an AHG-DTE-NEG XM. Re-Tx graft survival is significantly improved following an AHG-NEG XM. Re-Tx recipients with an AHG-POS XM who are either DTE/AHG-POS or -NEG display reduced graft survivals compared with AHG-NEG Re-Tx recipients.

Determination of Ascorbic Acid in Blood Plasma or Serum and in Seminal Plasma Using a Simplified Sample Preparation and High-Performance Liquid Chromatography Coupled with Uv Detection

Abstract A simplified method of sample preparation and high-performance liquid chromatography procedure using UV detection is described for the determination of ascorbic acid (AA) in blood plasma or serum and seminal plasma. Within two hours from collection samples are treated with dithioerythritol (DTE) and then stored under Argon at -80°C. Prior to analysis, protein precipitation is initiated with the addition of cold methanol. AA elution is carried out on a C18 reverse phase column using dodecyltrimethylammonium bromide as an ion-pairing agent. the detection is accomplished by measuring ultraviolet absorption at 265 nm. the analysis time for sample is 10.5 min, the retention time of AA being 9.7 min. Within- and between-day coefficients of variation are 2.9% and 4.9% for blood serum, 1.0 % and 2.3 % for seminal plasma. Mean analytical recovery of 102.5 ± 3.7% was found analyzing a serum pool after addition of a standard amount of AA. AA levels are stable for at least 43 days under the described storage...

Technetium-99m labeled monoclonal antibodies: Evaluation of reducing agents

We have evaluated five compounds, stannous chloride (SnCl 2), 2-mercaptoethanol (2-ME), dithiothreitol (DTT), dithioerythritol (DTE), and ascorbic acid (AA) to reduce monoclonal antibody MoAb (disulfide groups and compared their efficacy for labeling MoAbs with 99mTc. The reduction of 99mTc with dithionite at pH 11 was nearly quantitative. The use of AA, at a molar ratio of 3500:1, for three IgG and three IgM antibodies examined, gave a labeling efficiency greater than 95%. Hence no purification was needed. The immunospecificity of AA preparations determined by specific antigen assay was 84 ± 1% for an IgM and 82.6 ± 1.1% for an IgG, highest among all agents tested. The stability of the tracer was evaluated by challenging the product with such 99mTc avid agents as cysteine, DTPA, and human serum albumin. By HPLC analysis, no 99mTc was transchelated using chelating agent to protein molar ratios as high as 500:1. In two separate groups of five mice each, the liver uptake at 4 h post injection averaged 6.8 ± 2.9% per gram for 125I-TNT-1 (IgG) and 6 ± 5.1% per gram for the same MoAb labeled with 99mTc using AA. The AA technique promises to label antibodies with 99mTc and perhaps with 186Re, by a simple “kit” procedure.

Molecular forms in human serum of enzymes synthesizing DNA precursors and DNA

Both thymidine kinase (TK) and DNA polymerase (DNAp) are present in measurable amounts in human serum. Even though the use of TK as a clinical marker is rapidly increasing there has been no attempt to characterize the serum TK in a wider extent, i.e.; with respect to Mw or other biochemical parameters. Therefore sera with high TK or DNAp activities derived from patients with cytomegalovirus (CMV) infection, B12-deficiency and leukaemia were fractionated by gel exclusion chromatography. The TK activity eluted as two peaks, one major TK activity with an apparent molecular weight (Mw) or 730 kD and one minor TK activity corresponding to a Mw of 58 kD. The amount of TK activity at 58 kD varied between 7 and 23% of total activity, depending on the serum fractionated. The DNAp activity in sera from patients with malignant disease and B12 deficiency eluted as a single peak corresponding to a Mw of 240 kD. A DNAp with a different Mw (greater than 1000 kD) was recovered from 1 of 3 investigated immunosuppressed patients with CMV infection. A similar pattern of enzyme forms was observed when sera were separated by glycerol gradient centrifugation. The effect of high salt and various reaction solution components on the enzymes were studied. The only condition found that affected the molecular forms of TK was the state of reduction. Incubation of sera with high concentrations of dithioerythritol (DTE) (400 mM) prior to separation transferred all serum TK to the 58 kD form, it also converted most of the serum DNAp from the 240 kD form to a smaller form (56 kD) without affecting the total recovery of enzymatic activity. The reaction product from both TK forms was exclusively monophosphate and none of the TK forms could efficiently utilize cytidine triphosphate as phosphate donor. The substrate kinetics of the small serum TK fraction was identical with those of an enzyme with similar size purified from proliferating HeLa cells, indicating that both serum TK activities are forms of TK 1, the proliferation associated cellular isozyme.

Oxidation of NADH by vanadium compounds in the presence of thiols

The nonenzymatic oxidation of NADH was studied spectrophotometrically in the presence of two vanadium compounds, sodium orthovanadate and vanadyl sulfate. At physiological pH 7.4, in 25 m m sodium phosphate buffer, addition of the synthetic thiol, dithioerythritol (DTE) results in a marked increase of NADH oxidation in the presence of sodium orthovanadate, but not in the presence of vanadyl sulfate. Other reductants, such as dithiothreitol and cysteine, can also increase NADH oxidation, whereas glutathione and ascorbate cannot. In all reactions, superoxide dismutase and catalase completely inhibit the vanadium-stimulated oxidation of NADH. Inhibition occurs in a concentration-dependent manner, and the boiled enzymes do not inhibit the thiol reaction. The hydroxyl radical scavenger, thiourea, inhibits the reaction, whereas urea cannot. ESR studies show that the ability of the thiol to reduce vanadate can be correlated with the degree of NADH oxidation. Using spin trapping techniques, hydroxyl radicals are detected during the course of the reaction. Addition of hydrogen peroxide to vanadyl in the presence of DTE greatly increases NADH oxidation; however, no NADH oxidation occurs when hydrogen peroxide is added to vanadyl and ascorbic acid. These results provide a partial explanation for the ability of vanadium compounds to both decrease cellular reducing equivalents and promote lipid peroxidation.

The inhibition of catalase by glutathione

Reduced glutathione (GSH) inhibited catalase activity in a dose-dependent manner. DL-dithiothreitol (DL-DTT) and dithioerythritol (DTE) also inhibited catalase activity. The inhibition of catalase by GSH and DL-DTT could be reduced by NADPH. Polyacrilamide gel electrophoresis demonstrated the inhibition was partially reversible. The inhibition of catalase by GSH appeared to be partly due to superoxide radicals, since it was inhibited by active manganese superoxide dismutase, but not by heat-inactivated enzyme. Other chemical species also appear to take part in the inhibition, but they could not be identified.

Interaction of phenylisothiocyanates with the mitochondrial phosphate carrier. I. Covalent modification and inhibition of phosphate transport

The effects of phenylisothiocyanate (PITC) and of the polar analogue p-sulfophenylisothiocyanate ( p-sulfoPITC) on the phosphate carrier of bovine heart mitochondria have been investigated. Incubation of mitochondria with the two phenylisothiocyanates leads to inhibition of the phosphate carrier protein. The inhibition of phosphate transport by PITC is unaffected by the addition of dithioerythritol (DTE) or by variation of the pH. The inhibition by p-sulfoPITC is in part removed by DTE; the remaining inactivation of the phosphate carrier, which can be attributed to the reaction with NH 2 groups, is temperature and pH-dependent. Inhibition of phosphate transport by both p-sulfoPITC and PITC depends on the time of incubation and the concentration of the inhibitor. Preincubation with mersalyl protects the carrier protein against the inactivation by p-sulfoPITC but not against PITC. Other SH reagents tested do not show any protective effect. It can thus be concluded that two types of lysine residues are essential for the activity of the phosphate carrier. Lysine(s) of the former type are located at the surface of the membrane and are topologically related to the functional SH groups of the protein. Lysine residue(s) of the latter type are buried in the hydrophobic phase of the membrane.

Stimulation of synaptosomal ATP-dependent Ca 2+-uptake by N-ethylmaleimide

Treatment of rat brain synaptosomal lysate with N-ethylmaleimide (NEM) was found to stimulate ATP-dependent Ca 2+-uptake. This Ca 2+-uptake stimulation was blocked by dithioerythritol (DTE), mitochondrial inhibitors oligomycin and sodium azide, but not by vanadate, an inhibitor of plasma membrane Ca 2+ pump. Maximal stimulation of Ca 2+-uptake was observed at a NEM/protein ratio of 0.1 μmole/0.5–1.0 mg. On fractionation, it was found that NEM did not affect synaptic plasma membrane Ca 2+-uptake, but almost doubled it in synaptic mitochondria.

Chemical reduction of disulfides

This chapter discusses the chemical reduction of disulfides. Disulfides are easily and specifically reduced by thiols, which are the most used reagents for this purpose. However, the excess of the thiol used as a reductant has to be removed before it is possible to assay the newly generated SH groups. The methods discussed for the reduction of disulfides by thiols are dithiothreitol (DTT) and dithioerythritol (DTE); and 2-mercaptoethanol. DTT and DTE have also been extensively used to reduce soluble nonprotein disulfides such as cystine and mixed homocysteine-cysteine disulfides. Mercaptoethanol, the simplest water-soluble organic thiol, is “the poor man's DTT,”—that is, mole for mole it is 150-175 times less expensive. Mercaptoethanol is much used especially in conjunction with SDS-gel electrophoresis. Direct chemical reduction is achieved with sodium borohydride or sodium amalgam whereas more specialized reagents include the phosphines and phosphorothioates. For cleavage generating half thiol only, there is sulfitolysis and cyanolysis. Less well controllable and defined is alkaline hydrolysis.

Thiol-disulfide exchange by thrombospondin: Evidence for a thiol and a disulfide bond protected by calcium

Thrombospondin (Tsp), a protein secreted by activated platelets, forms disulfide-linked complexes with thrombin [ K. J. Danishefsky, R. J. Alexander and T. C. Detwiler (1984) Biochemistry 23, 4984]. Thiols and disulfide bonds of Tsp were analyzed, and a search was made for other Tsp covalent complexes. Platelets in 1 m m EDTA were activated with ionophore A23187, and the secreted proteins were analyzed by gel electrophoresis in sodium dodecyl sulfate. One millimolar dithioerythritol (DTE) decreased the electrophoretic mobility of Tsp, indicating reduction of an intrachain disulfide bond; Ca 2+ prevented this effect. Electrophoresis of single-chain Tsp prepared with 50 m m DTE in either EDTA or Ca 2+ also revealed a Ca 2+-stabilized intrachain disulfide bond. Ca 2+ prevented the retention of Tsp on an activated thiol-Sepharose column, indicating protection of a thiol by Ca 2+. Incubation at 37 °C for 60 min resulted in complexes with apparent mass ⪢500 kDa. Formation of complexes was prevented by N-ethylmaleimide, by a temperature < 25 °C, and by Ca 2+ or Mg 2+. From pH 6 to 9, complexes formed better at lower pH. Two-dimensional (nonreduced/reduced) electrophoresis revealed Tsp but no other constituents of the complexes. With 10 n m thrombin, complexes formed faster and included thrombin; Ca 2+ only partially inhibited. The complex was very susceptible to dissociation by low concentrations (2.5 m m) of DTE. It is concluded that Tsp has a reactive thiol and an intrachain disulfide bond that are protected by Ca 2+. When these groups are unprotected, there is intermolecular thioldisulfide exchange.

Cellular oxygen utilization and hypoxia: interaction of dithiols with cellular electron transfer systems.

DL-Dithiothreitol (DTT) better known as Cleland’s reagent, and dithioerythritol (DTE) are isomers of 2,3-dihydroxy-l-4-dithiobutane which were originally used by Cleland (1964) as antioxidants (1). They have found wide spread use in chemistry, biochemistry, biology and medicine, principally as reductants for disulfides as well as protectors of sulfhydryls against oxidants. DTT has found application as a chemical that protects microbes (2) and mammalian cells (3,4) against the damaging effects of ionizing radiation. It is this latter property that has been of interest to us. The radioprotection conferred upon cells by DTT may be due to its stimulation of cellular oxygen utilization. Increases in oxygen consumption may produce hypoxic cells in multicellular systems (5) as well as create intracellular hypoxia (4,5). The lowering of oxygen concentration results in protection against radiation damage due to the well known oxygen effect (6). In addition to effects on oxygen utilization, dithiols also react spontaneously with oxygen to produce superoxide and peroxide. This reaction is enhanced in the presence of copper and iron that are normally found in the growth medium of cultured cells. Depletion of oxygen by this spontaneous reaction will also cause hypoxia and radiation protection (7).KeywordsOxygen ConsumptionLipoic AcidPyridine NucleotideOxygen UtilizationEhrlich Ascites Tumor CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Effects of heavy metal cations, sulfhydryl reagents and other chemical agents on striatal D 2 dopamine receptors

To investigate aspects of the biochemical nature of the membrane-bound D 2 dopamine receptor, rat striatal homogenates were pretreated with heavy metal cations and a variety of other chemical agents, and their effects on D 2 receptor density were subsequently determined using a standard [ 3H]spiperone binding assay. Preincubation of striatal membranes in the presence of 3 mM Mn 2+, Fe 2+, Co 2+, EDTA, or ascorbate enhanced subsequently measured stereospecific binding of [ 3H]spiperone compared to control (e.g. control: B max = 140 mfoles/mg protein, K D = 0.21 nM; Mn 2+-treated: B max = 253 fmoles/mg protein, K D = 0.20 NM). Another group of metal cations, is that Zn 2+, Cd 2+, Cu 2+, Hg 2+ and CH 3Hg +, all of which have significant −SH reactivity, as well as the −SH alkylating agent N-ethylmaleimide (NEM), caused a decrease in the specific binding sites. Pretreatment with 3mM Cd 2+ or Cu 2+ resulted in a 40–60% reduction in the subesequently measured stereospecific binding of [ 3H]-spiperone, whereas 1 mM Hg 2+ or 3 mM NEM completely abolished specific [ 3Hspiperone binding. The effect of Hg 2+ could not be reversed by washing the membranes, nor by further incubation of the membranes in the presence of excess EDTA or 2,3-dimercapto-1-propanesulfonic acid (DMPS). Further incubation in the presence of 3 mM dithioerythritol (DTE) resulted in the regeneration of about 40% of lost sites. Agents which enhanced receptor density, such as Mn 2+ or EDTA, could not antagonize the effect of Hg 2+, nor could the mercury-chelating agent DMPS, when added to crude homogenates prior to Hg 2+. Ascorbate protected 25–35% of specific binding sites by virtue of its ability to reduce Hg 2+ to insoluble Hg +. Only 3 mM DTE afforded afforded complete protection against 1 mM Hg 2+. Prior formation of the spiperone/receptor complex also demonstrated considerable ability to protect receptors from destruction by Hg 2+. Preincubation of striatal membranes in the presence of 0.5 mM spiperone protected about 80% of sites from the subsequent addition of 1 mM Hg 2+. A major conclusion of these studies is that one or more free -SH groups on or adjacent to the active site may be a requirement for specific antagonist binding to the membrane-bound D 2 receptor. Occlusion of these -SH groups by sulfhydryl reagents results in partial to complete abolition of subsequently measured specific 3H-antagonist binding. Only agents which can regenerate free -SH groups, such as DTE, are able to induce any recovery in specific binding sites. Other substances, such as Mn 2+, EDTA and ascorbate which, on their own, cause an enhanced density of specific antagonist binding sites are generally ineffective in preventing the action of sulfhydryl binding agents. Their enhancing effect is probably due to the protection of receptors from normal degradation during incubation and to an improved recovery of receptor sites after centrifugation procedures.