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At least 73 records · Page 4Linked to original sources
PubMed1996

Glial cell line-derived neurotrophic factor: selective reduction of the intermolecular disulfide linkage and characterization of its disulfide structure.

Glial cell line-derived neurotrophic factor is a protein known to enhance the survival of dopaminergic neurons against several neurotoxins. It has been shown to have therapeutic potential in the treatment of Parkinson's disease and other neurodegenerative diseases. We have determined the inter- and intramolecular disulfide linkages of the dimeric molecule by a combination of direct peptide analysis and peptide analysis after either partial reduction or partial oxidation of the protein. Under an acidic condition, the interchain disulfide bond was selectively cleaved with tris(2-carboxyethyl)phosphine, revealing that Cys101 was involved in the intermolecular disulfide linkage. Three other disulfides, Cys68-Cys131, Cys72-Cys133, and Cys41-Cys102, were identified as intramolecular linkages. The determined disulfide structure is highly homologous to that of transforming growth factor beta 2. Since one intramolecular disulfide points through a ring consisting of eight amino acid residues based on the similarity with transforming growth factor beta 2, the disulfide-linked peptides were not purified by conventional methods. Only the peptides from an N-terminal region (residues -1 to 37) were liberated by proteolytic treatment with trypsin or endoproteinase Lys-C, resulting in a stable cystine-knot protein.

Amino Acid Sequence↗
PubMed2001

Flavor-protein binding: disulfide interchange reactions between ovalbumin and volatile disulfides.

The irreversible binding of selected sulfur-containing flavor compounds to proteins was investigated in aqueous solutions containing ovalbumin and a mixture of disulfides (diethyl, dipropyl, dibutyl, diallyl, and 2-furfuryl methyl) using solid-phase micro-extraction (SPME). In systems which had not been heated, the recovery of disulfides from the headspace above the protein at the native pH (6.7) was similar to that from an aqueous blank. However, significant losses were observed when the pH of the solution was increased to 8.0. When the protein was denatured by heating, much greater losses were observed and some free thiols were produced. In similar heat-denatured systems at pH 2.0, no losses of disulfides were observed. Disulfides containing allyl or furfuryl groups were more reactive than saturated alkyl disulfides. Interchange reactions between protein sulfhydryl groups and the disulfides are believed to be responsible for the loss of the disulfides.

Chromatography, Liquid↗
PubMed2004

Heat-induced redistribution of disulfide bonds in milk proteins. 2. Disulfide bonding patterns between bovine beta-lactoglobulin and kappa-casein.

Heat treatment of milk causes the heat-denaturable whey proteins to aggregate with kappa-casein (kappa-CN) via thiol-disulfide bond interchange reactions. The particular disulfide bonds that are important in the aggregates are uncertain, although Cys(121) of beta-lactoglobulin (beta-LG) has been implicated. The reaction at 60 degrees C between beta-LG A and an activated kappa-CN formed small disulfide-bonded aggregates. The tryptic peptides from this model system included a peptide with a disulfide bond between a Cys residue in the triple-Cys peptide [beta-LG(102-124)] and kappa-CN Cys(88) and others between kappa-CN Cys(88) or kappa-CN Cys(11) and beta-LG Cys(160). Only the latter two novel disulfide bonds were identified in heated (90 degrees C/20 min) milk. Application of computational search tools, notably MS2Assign and SearchXLinks, to the mass spectrometry (MS) and collision-induced dissociation (CID)-MS data was very valuable for identifying possible disulfide-bonded peptides. In two instances, peptides with measured masses of 4275.07 and 2312.07 were tentatively assigned to beta-LG(102-135):kappa-CN(11-13) and beta-LG A(61-69):kappa-CN(87-97), respectively. However, sequencing using the CID-MS data demonstrated that they were, in fact, beta-LG(1-40) and beta-LG(41-60), respectively. This study supports the notion that reversible intramolecular disulfide-bond interchange precedes the intermolecular interchange reactions.

Amino Acid Sequence↗
PubMed1977

Biologically oriented organic sulfur chemistry. 15. Organic disulfides and related substances. 41. Inhibition of the fungal pathogen Histoplasma capsulatum by some organic disulfides.

In an extension of promising inhibitory results in vitro against Histoplasma capsulatum, correlated earlier using substituent constants developed by regression analysis with 77 disulfides, one symmetrical and 14 unsymmetrical disulfides were prepared (3--17). About half were active in vitro against H. capsulatum (and one against Candida albicans). Groups that seemed most to lead to promising inhibition among the unsymmetrical disulfides were o-HO2CC6H4, (CH2)4SO2Na, Me2NC(S), p-ClC6H4, and perhaps p-CH3C6H4; the first two also might be used to increase solubility. Earlier inhibitory promise of the morpholino group did not materialize. None of the group 3--17 was significantly active in vivo. The unsymmetrical disulfides were prepared by reaction of thiols with sulfenyl chlorides or with acyclic or cyclic thiosulfonates. Two six-membered heterocyclic disulfides (5 and 6) were prepared by a novel cyclization, in which carbon disulfide reacted with an (N-alkylamino)ethyl Bunte salt, followed by ring closure; an explanation is suggested for formation of a thiazoline when the N-alkyl group is absent. One of the disulfides disproportionated with astonishing ease (31; 0.3--1 h at 25 degrees C).

Amphotericin B↗
PubMed2003

Attempt to map the receptor binding sites of human follicle-stimulating hormone using disulfide peptides of its beta-subunit indicates major involvement of the regions around disulfide bonds Cys28-Cys82 and Cys32-Cys84 in receptor binding of the hormone.

Follicle-stimulating hormone (FSH) is a heterodimeric glycoprotein hormone secreted by the anterior pituitary. It plays a very important role in folliculogenesis in females and is responsible for spermatogenesis in males. The alpha-subunit which is common within a species and the beta-subunit which is hormone-specific are held together by noncovalent association. This association is very essential for the biological activity of the hormone. Each of these subunits are highly cross-linked by disulfide bonds which appear to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. This study was initiated to delineate the role of the disulfide bonds of hFSH beta in receptor binding of the hormone. Five intermolecular and one intramolecular disulfide peptides corresponding to the disulfide bonds found in hFSH beta were synthesized and screened along with their linear counterparts, for their ability to competitively inhibit the radiolabelled [125I]hFSH from binding to the FSH receptor containing membranes from the testis of immature rats. The disulfide peptides Cys28-Cys82 and Cys32-Cys84 were found to be the most potent in inhibiting radiolabelled hFSH from binding to its receptor. The results suggest the involvement of the regions around disulfide bonds Cys28-Cys82 and Cys32-Cys84 in receptor binding of the hormone. The studies also suggest the involvement of beta L2 and beta L3 loop regions in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hFSH.

Amino Acid Sequence↗
PubMed1995

Sulfhydryl-disulfide modulation and the role of disulfide oxidoreductases in regulation of the catalytic activity of nitric oxide synthase in pulmonary artery endothelial cells.

The role of sulfhydryl groups (SH) and disulfide bonds as well as disulfide oxidoreductases in regulation of the catalytic activity of the membrane-bound constitutive isoform of nitric oxide (NO) synthase from porcine pulmonary artery endothelial cells (PAEC) was examined. Treatment of intact PAEC or a total membrane preparation isolated from PAEC with the SH alkylating agent N-ethylmaleimide (NEM) (10 to 50 microM) or with the intramolecular disulfide-forming agent diamide (20 to 100 microM) resulted in the reduction of NO synthase activity in a dose-dependent fashion. Similar loss of enzyme activity was observed when purified NO synthase from the membrane fraction of PAEC was incubated in the presence of NEM. The loss of membrane protein SH content from NEM- and diamide-treated preparations was associated with loss of NO synthase activity. In contrast, when intact PAEC or isolated total membranes derived from PAEC were treated with increasing concentrations (1 to 5 mM) of the disulfide-reducing agent dithiothreitol (DTT), but not oxidized DTT, NO synthase activity was increased by 20 to 85%. DTT reduction of native disulfides from NEM-treated preparations or of disulfides formed after diamide treatment of membranes reversed the inhibition of NO synthase activity. Similarly, enzymatic reduction by thioredoxin/thioredoxin reductase, but not by glutaredoxin, reversed the inhibition of membrane fraction and purified NO synthase isolated from diamide-treated cells. This enzyme-catalyzed disulfide reduction was > 1,000-fold more efficient than the DTT-induced reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1996

Glutathione-dependent pathways of refolding of RNase T1 by oxidation and disulfide isomerization: catalysis by protein disulfide isomerase.

Protein folding, associated with oxidation and isomerization of disulfide bonds, was studied using reduced and denatured RNase T1 (rd-RNase T1) and mixed disulfide between glutathione and reduced RNase T1 (GS-RNase T1) as starting materials. Folding was initiated by addition of free glutathione (GSH + GSSG) and was monitored by electrospray mass spectrometry (ES-MS) time-course analysis. This permitted both the identification and quantitation of the population of intermediates present during the refolding process. Refolding experiments were performed in the presence of different absolute concentrations of glutathione species while keeping the redox potential fixed, in order to evaluate the effect of the glutathione concentration on the distribution of the refolding intermediates. All the analyses indicate a pathway of sequential reactions in the formation of native RNase T1 which occurs via the reiteration of two steps: (i) formation of a species containing both mixed disulfides with glutathione and free protein thiols, and (ii) formation of an intramolecular disulfide via thiol-disulfide interchange reaction between them. Refolding of rd-RNase T1 and GS-RNase T1 was also performed in the presence of protein disulfide isomerase (PDI). Addition of PDI led to a catalysis of each individual reaction of the entire process without altering the refolding pathway. Refolding reactions carried out at different absolute concentrations of glutathione proved that GSH and/or GSSG participate directly in the reaction catalyzed by PDI. On the basis of these experiments and previous results on the refolding of RNase A [Torella, C., Ruoppolo, M., Marino, G., & Pucci, P. (1994) FEBS Lett. 352, 301-306], a hypothesis of a general pathway for folding of S--S containing proteins is proposed.

Animals↗
PubMed1996

Eukaryotic protein disulfide isomerase complements Escherichia coli dsbA mutants and increases the yield of a heterologous secreted protein with disulfide bonds.

Eukaryotic protein disulfide isomerase (PDI) is a 55-kDa enzyme with cysteine oxidoreductase, chaperone, and antichaperone activities that catalyzes disulfide formation and rearrangement in the eukaryotic endoplasmic reticulum. In Gram-negative bacteria, the formation of disulfide bonds in the periplasm is mediated by DsbA, a strong cysteine oxidase but an inefficient catalyst of disulfide bond isomerization with no known chaperone activity. We show that rat PDI (rPDI) secreted in the periplasmic space of Escherichia coli can catalyze the formation of disulfide bonds and complement several of the phenotypes of dsbA mutants. The function of rPDI was dependent on the dsbB gene, suggesting that the reoxidation of this eukaryotic enzyme involves direct interactions with bacterial redox proteins. Co-expression of rPDI increased the yield of bovine pancreatic trypsin inhibitor (BPTI) severalfold, an effect that was enhanced when reduced glutathione was added to the growth medium. Whereas PDI is thought to function primarily as an isomerase in the eukaryotic endoplasmic reticulum, rPDI failed to decrease the accumulation of two-disulfide folding intermediates of BPTI and thus did not appear to appreciably catalyze the rate-limiting step in the oxidative folding pathway of BPTI. These results demonstrate that expression of eukaryotic foldases in E. coli can be exploited to better understand their function in vivo and also to increase the yield of biotechnologically valuable proteins.

Animals↗
PubMed1989

Expression of disulfide-linked and non-disulfide-linked forms of the T cell receptor gamma/delta heterodimer in human intestinal intraepithelial lymphocytes.

Immunohistochemistry has been used to investigate disulfide- and non-disulfide-linked forms of the T cell receptor gamma/delta heterodimer (TcR gamma/delta) in blood and intestinal epithelium of normal human small intestine, intestine of patients with untreated coeliac disease (in whom T cells expressing TcR gamma/delta are disproportionately raised), intestine of patients with tropical malabsorption, and in the human fetus. In blood from adult volunteers, 90% of T cells expressing TcR gamma/delta use the disulfide-linked form. In contrast in the epithelium in normal small intestine, coeliac disease and tropical malabsorption, most of the T cells expressing TcR gamma/delta use the non-disulfide-linked form. This is especially prominent in untreated coeliac disease where the increase in TcR gamma/delta T cells is mainly restricted to those using the non-disulfide-linked form. In human fetal small intestinal epithelium, however, only cells using the disulfide-linked form are present. These variations in expression of different forms of TcR gamma/delta in the gut epithelium in different conditions suggests that antigen, or some as yet undefined factor may determine the frequency of each subpopulation.

Antibodies, Monoclonal↗
PubMed1986

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 mM 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; Ca2+ prevented this effect. Electrophoresis of single-chain Tsp prepared with 50 mM DTE in either EDTA or Ca2+ also revealed a Ca2+-stabilized intrachain disulfide bond. Ca2+ prevented the retention of Tsp on an activated thiol-Sepharose column, indicating protection of a thiol by Ca2+. Incubation at 37 degrees C for 60 min resulted in complexes with apparent mass much greater than 500 kDa. Formation of complexes was prevented by N-ethylmaleimide, by a temperature less than 25 degrees C, and by Ca2+ or Mg2+. 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 nM thrombin, complexes formed faster and included thrombin; Ca2+ only partially inhibited. The complex was very susceptible to dissociation by low concentrations (2.5 mM) of DTE. It is concluded that Tsp has a reactive thiol and an intrachain disulfide bond that are protected by Ca2+. When these groups are unprotected, there is intermolecular thiol-disulfide exchange.

Blood Platelets↗
PubMed1979

Disulfide-disulfide interchange catalyzed by a liver supernatant enzyme.

An enzyme widely distributed in rabbit tissues which catalyzes an interchange between N,N-di-dinitrophenyl-L-cystine and oxidized glutathione to form the mixed disulfide is described. D-Penicillamine disulfide can be substituted for oxidized glutathione and the mixed disulfide of cysteine and glutathione can serve as the sole substrate giving as one product of interchange, oxidized glutathione. The enzyme is very labile and only limited purification of it has been achieved. The activity increases with increasing pH above 6.6, the Km for N,N-di-dinitrophenyl-L-cystine is 0.2 mM and for oxidized glutathione 0.8 mM. The enzyme is inhibited by SH reagents with protection against iodoacetamide inactivation provided by N,N-di-dinitrophenyl-L-cystine. Evidence is presented that disulfide-disulfide interchange enzyme is a different activity from the previously described protein disulfide isomerase and thiol transferase.

Animals↗
PubMed1989

The position of the disulfide bonds in human plasma alpha 2 HS-glycoprotein and the repeating double disulfide bonds in the domain structure.

The positions of the inter- and intra-chain disulfide bonds of human plasma alpha 2 HS-glycoprotein were determined. alpha 2 HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)--Cys-14 (A-chain), Cys-71--Cys-82, Cys-96--Cys-114, Cys-128--Cys-131, Cys-190--Cys-201 and Cys-212--Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of alpha 2 HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the S--S bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two S--S bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second S--S loops and the end of domains A and B, and the positions of the ordered structures.

Amino Acid Sequence↗
PubMed2005

Brain PP2A is modified by thiol-disulfide exchange and intermolecular disulfide formation.

The regulation of protein phosphatase 2A (PP2A) activity by thiol-disulfide exchange and resulting formation of an intermolecular disulfide was examined following exposure of a rat brain soluble fraction to a biotinylated derivative of the model disulfide HPDP (HPDP-biotin) which would be expected to label reactive protein thiols with a disulfide-linked biotin. The results show that a low concentration (500 microM) of HPDP-biotin produced substantial inhibition of PP2A activity and promoted the binding of the catalytic subunit of PP2A to an immobilized avidin-affinity column. Both the inhibition of PP2A activity and the binding of PP2A to the avidin column were reversed by treatment with the disulfide reducing agent dithiothreitol (DTT). Furthermore, the specific activity of PP2A was up to 7-fold higher in the DTT-eluted fractions from the avidin-affinity column than in the soluble fraction. These findings demonstrate directly that PP2A is susceptible to reversible inhibitory modification by thiol-disulfide exchange and provide mechanistic support for the emerging view that PP2A is an oxidant-sensitive protein phosphatase.

Animals↗
PubMed1996

Synthesis and characterization of leiurotoxin I analogs lacking one disulfide bridge: evidence that disulfide pairing 3-21 is not required for full toxin activity.

Leiurotoxin I (Lei-NH2), a toxin isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus, is a blocker of the apamin-sensitive Ca(2+)-activated K+ channels. It is a 31-residue polypeptide cross-linked by three disulfide bridges which are presumably between Cys3-Cys21, Cys8-Cys26, and Cys12-Cys28. To investigate the role of these disulfides, analogs of Lei-NH2 lacking one disulfide bridge (i.e., [Abu3,21]Lei-NH2, [Abu8,26]Lei-NH2, and [Abu12,28]Lei-NH2) were chemically synthesized by selective replacement of each pair of half-cystines forming a bridge by two alpha-aminobutyrate (Abu) residues. The two disulfide pairings of the main folded form of the synthetic analogs were established by enzymatic proteolysis. They were as expected between Cys8-Cys26 and Cys12-Cys28 for [Abu3,21]Lei-NH2 but were unexpectedly between Cys3-Cys12 and Cys21-Cys28 for [Abu8,26]Lei-NH2 and between Cys3-Cys8 and Cys21-Cys26 for [Abu12,28]Lei-NH2. The synthetic peptides were tested in vitro for their capacity to compete with the binding of [125I]apamin to rat brain synaptosomes and in vivo for their neurotoxicity in mice. In both assays, [Abu3,21]Lei-NH2 exhibited full Lei-NH2-like activity whereas [Abu8,26]Lei-NH2 and [Abu12,28]-Lei-NH2 possessed only residual activities (< 2% native toxin activity). This suggests that disulfide bridge Cys3-Cys21 is not essential per se for high toxin activity. Circular dichroism (CD) spectroscopy of the three analogs showed that only [Abu3,21]Lei-NH2 exhibited a CD spectrum similar to that of Lei-NH2, suggesting they both adopt closely related conformations, in agreement with the pharmacological data. Structural models of the analogs were constructed on the basis of the disulfide pairing assignment and compared with that of Lei-NH2.

Amino Acid Sequence↗
PubMed1998

Consequence of the removal of evolutionary conserved disulfide bridges on the structure and function of charybdotoxin and evidence that particular cysteine spacings govern specific disulfide bond formation.

Scorpion toxins are miniglobular proteins containing a common structural motif formed by an alpha-helix on one face, an antiparallel beta-sheet on the opposite face, and three disulfide bonds making up most of its internal volume. We have investigated the role of these evolutionary conserved bonds by replacing each couple of bridged cysteine residues of the scorpion charybdotoxin by a pair of nonbridging L-alpha-aminobutyric acid (Aba) residues. Three analogues were obtained by solid-phase synthesis, Chab I, Chab II, and Chab III, containing the Aba residues in positions 7 and 28, 13 and 33, 17 and 35, respectively. Circular dichroism analysis showed that the purified Chab II acquired a conformation similar to that of charybdotoxin, while the Chab I and Chab III possess decreased nativelike characteristics. All analogues block single high-conductance Ca(2+)-activated K+ channels from rat skeletal muscle inserted into planar lipid bilayers, but with different potencies. Chab II is the most active analogue (KD = 8.0 x 10(-8) M), with a 9-fold lower affinity as compared to native charybdotoxin. Chab I and Chab III have, respectively, 180- and 580-fold lower affinity. Therefore, the removal of evolutionary conserved disulfide bridges does not prevent the toxin to adopt a functional and presumably nativelike structure. However, removal of one disulfide bond affects the yields of formation of correct pairing between the remaining cysteine residues, and only Chab I preserves the ability to form the native disulfide pairings with high efficiency. This is the only analogue to preserve particular spacings of three and one residue between the cysteines, which have been described to thermodynamically disfavor disulfide bond formation between the cysteines [Zhang R., and Snyder, G. H. (1989) J. Biol. Chem. 264, 18472-18479]. Therefore, we conclude that the position of the cysteine residues in the sequence of charybdotoxin, by disfavoring specific pairings and favoring others, may govern selective formation of specific disulfide bonds, thus, explaining the efficient folding properties of Chab I and of native charybdotoxin. The structural properties of the Chab analogues and the discovered role of the cysteine spacings have interesting implications in protein design and engineering.

Amino Acid Sequence↗
PubMed2002

The disulfide structure of denatured epidermal growth factor: preparation of scrambled disulfide isomers.

The conformational stability of human epidermal growth factor (EGF) and the structure of denatured EGF were investigated using the technique of disulfide scrambling. Under denaturing conditions and in the presence of a thiol catalyst, the native EGF denatures by shuffling its three native disulfide bonds and converts to a mixture of scrambled isomers. Analysis by HPLC reveals that the denatured EGF is composed of about 10 fractions of scrambled isomers. The heterogeneity varies under different denaturing conditions, with the heat-denatured samples exhibiting the highest degree of heterogeneity. The disulfide structures of eight major scrambled isomers of EGF were determined. The most predominant isomer adopts the bead-form structure with disulfide bonds bridged by three pairs of neighboring cysteines: Cys6-Cys14, Cys20-Cys31, and Cys33-Cys42. The denaturation curve of EGF is determined by the relative yield of the scrambled and native species of EGF. EGF is a highly stable molecule and can be effectively denatured only by guanidine chloride at a concentration of greater than 4-5 M. At 8 M urea, less than 16% of the native EGF was denatured. The unusual conformational stability of EGF was compared with that of eight different disulfide proteins that were similarly characterized by the method of disulfide scrambling.

Amino Acid Sequence↗
PubMed1998

The sulfonylurea-inhibited NADH oxidase activity of HeLa cell plasma membranes has properties of a protein disulfide-thiol oxidoreductase with protein disulfide-thiol interchange activity.

Plasma membrane vesicles of HeLa cells are characterized by a drug-responsive oxidation of NADH. The NADH oxidation takes place in an argon or nitrogen atmosphere and in samples purged of oxygen. Direct assay of protein thiols by reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB; Ellman's reagent), suggests that protein disulfides may be the natural electron acceptors for NADH oxidation by the plasma membrane vesicles. In the presence of NADH, protein disulfides of the membranes were reduced with a concomitant stoichiometric increase in protein thiols. The increase in protein thiols was inhibited in parallel to the inhibition of NADH oxidation by the antitumor sulfonylurea LY181984 with an EC50 of ca. 30 nM. LY 181984, with an EC50 of 30 nM, also inhibited a protein disulfide-thiol interchange activity based on the restoration of activity to inactive (scrambled) RNase and thiol oxidation. The findings suggest that thiol oxidation, NADH-dependent disulfide reduction (NADH oxidation), and protein disulfide-thiol interchange in the absence of NADH all may be manifestations of the same sulfonylurea binding protein of the HeLa plasma membrane. A surface location of the thiols involved was demonstrated using detergents and the impermeant thiol reagent p-chloromercuriphenylsulfonic acid (PCMPS). The surface location precludes a physiological role of the protein in NADH oxidation. Rather, it may carry out some other role more closely related to a function in growth, such as protein disulfide-thiol interchange coupled to cell enlargement.

Cell Membrane↗
PubMed2001

Stability of thiuram disulfides in patch test preparations and formation of asymmetric disulfides.

The thiuram mix used in patch testing originally contains 4 compounds. However, chemical analysis of the test preparation revealed that several new compounds are spontaneously formed during storage. The structures of these compounds have been determined and the rate of their formation has been studied in buffer solution at pH 7.4. After a few hours, a large amount of mixed disulfides are formed in solutions originally containing only symmetric disulfides. The impact on the test result of the formation of asymmetric disulfides has been investigated by testing on thiuram-sensitive volunteers with different preparations of mixed thiuram disulfides. In our study, the formation of new asymmetric thiuram disulfides from the original symmetric thiuram disulfides in the test preparation had no influence on the result of the patch testing. However, as the chemical analysis showed that the mix composition changes during the period when the preparations are used, and differs between suppliers, the question is raised as to whether it is acceptable to use test preparations with a composition that is different from that labelled on the protocol.

Allergens↗