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PubMed2005

A structural disulfide of yeast protein-disulfide isomerase destabilizes the active site disulfide of the N-terminal thioredoxin domain.

Protein-disulfide isomerase (PDI) is an essential catalyst of disulfide formation and isomerization in the eukaryotic endoplasmic reticulum. PDI has two active sites at either end of the molecule, each containing two cysteines that facilitate thiol-disulfide exchange. In addition to its four catalytic cysteines, PDI possesses two non-active site cysteines whose location and separation distance varies by organism. In higher eukaryotes, the non-active site cysteines are located in the C-terminal half of the protein sequence and are separated by 30 amino acids. In contrast, the internal cysteines of PDI from lower eukaryotes are located near the N-terminal active site and are much closer together in sequence. The function of these cysteines and the significance of their unique location in yeast PDI have been unclear. Previous data (Xiao, R., Wilkinson, B., Solovyov, A., Winther, J. R., Holmgren, A., Lundstrom-Ljung, J., and Gilbert, H. F. (2004) J. Biol. Chem. 279, 49780-49786) suggest that the internal cysteines exist as a disulfide in the endoplasmic reticulum of Saccharomyces cerevisiae. By coupling mass spectrometry with a gel-shift technique that allows us to measure the redox potentials of the PDI active sites in the presence and absence of the non-active site cysteines, we find that the non-active site cysteines form a disulfide that is stable even in a very reducing environment and demonstrate that this disulfide exists to destabilize the N-terminal active site disulfide, making it a better oxidant by 18-fold. Consistent with this finding, we show that mutating the non-active site cysteines to alanines disrupts both the oxidase and isomerase activities of PDI in vitro.

Amino Acid Sequence↗
PubMed1991

Copper(II) (3,5-diisopropylsalicylate)2 oxidizes thiols to symmetrical disulfides and oxidatively converts mixtures of 5-thio-2-nitrobenzoic acid and nonsymmetrical 5-thio-2-nitrobenzoic acid disulfides to symmetrical disulfides.

L-cysteine, D-penicillamine, and L-glutathione were oxidized to symmetrical disulfides in the presence of Cu(II)(3,5-DIPS)2 and air-oxygen at physiologic pH, 7.3. Air-oxygen caused the oxidation of thiol reduced copper, Cu(I), to Cu(II), as evidenced by expected spectrophotometric changes in these reaction mixtures. L-cysteine, D-penicillamine, and L-glutathione formed mixed disulfides and TNB with the addition of DTNB to solutions of these thiols. The observed order of reactivity for these thiols with DTNB was: L-cysteine greater than D-penicillamine greater than L-glutathione. Surprisingly, Cu(II)(3,5-DIPS)2 converted these mixed disulfides to their symmetrical disulfides and DTNB, and although the initial conversion rate was rapid, complete conversion required more than two hours. These observations suggest caution with regard to the spectrophotometric determination of thiols immediately after the addition of Ellman's reagent. These results also clarify an earlier report concerning the oxidation of thiols by Cu(II)(o-phenanthroline)2 and offer caution with regard to the determination of thiols using DTNB in the presence of copper complexes. Spectrophotometric data are provided in support of the suggestion that analysis of plasma or cellular samples for thiols be done in the absence of copper(II) complexes to avoid false negative results.

Cysteine↗
PubMed1977

Identification of a coenzyme A--glutathione disulfide (DSI), a modified coenzyme A disulfide (DSII), and a NADPH-dependent coenzyme A--glutathione disulfide reductase in E. coli.

The nucleotides DSI and DSII induced during a slowdown in growth of E. coli have been characterized using chemical and biochemical analysis and by enzymic and alkaline fragmentation. DSI consists a coenzyme A and glutathione joined by a disulfide linkage. DSI could be isolated either containing Fe(III) with an A250:260 ratio of 1.05 or not containing iron with an A250:260 of 0.87. DSII (isolated in 10% the yield of DSI) is a coenzyme A disulfide dimer that also contains two molecules of glutamic acid. DSI was a substrate for NADPH-dependent CoAS-SG reductase (EC 1.6.4.6) which was present in crude extracts of E. coli. The specific activity of CoAS-SG reductase increased during growth from early log phase into stationary phase and during a shift from aerobic to anaerobic growth.

Coenzyme A↗
PubMed1996

Catalysis of disulfide isomerization in thrombospondin 1 by protein disulfide isomerase.

Thrombospondin 1 is a multidomain glycoprotein from platelets and most cells that participates in diverse biological processes. The structure and some functional properties of thrombospondin 1 are regulated by disulfide interchange in the Ca(2+)-binding repeats and C-globular domain. The recent identification of the enzyme, protein disulfide isomerase, on the platelet surface suggested that protein disulfide isomerase may catalyze disulfide isomerization in platelet thrombospondin 1. Protein disulfide isomerase was found to form disulfide-linked complexes with thrombospondin 1, which is consistent with protein disulfide isomerase-mediated rearrangement of disulfide bonds in thrombospondin 1. To quantitate disulfide interchange in thrombospondin 1, perturbation of the enzyme inhibitory properties of platelet thrombospondin 1 were measured, specifically changes in the apparent dissociation constant for inhibition of neutrophil cathepsin G by thrombospondin 1. The inhibition constant increased > or = 10-14-fold following incubation of either Ca(2+)-replete or Ca(2+)-depleted thrombospondin 1 with protein disulfide isomerase and reduced glutathione. The rate of protein disulfide isomerase-catalyzed disulfide interchange in thrombospondin 1 increased linearly with protein disulfide isomerase concentration and the K(m) for reduced glutathione was 0.4 +/- 0.2 mM. Disulfide isomerization in both platelet and fibroblast thrombospondin 1 was probed by measuring perturbation in epitopes for two anti-thrombospondin 1 monoclonal antibodies. Antibody D4.6 binds to the C-terminal Ca(2+)-binding domains which are involved in disulfide interchange, whereas antibody HB8432 binds toward the N-terminus of the thrombospondin 1 subunit. In accordance with the location of these epitopes, incubation of platelet thrombospondin 1 or fibroblast thrombospondin 1 with protein disulfide isomerase and reduced glutathione resulted in 2-fold enhancement of binding of D4.6, whereas binding of HB8432 did not significantly change. In summary, protein disulfide isomerase catalyzes disulfide interchange in thrombospondin 1 which alters binding of neutrophil cathepsin G and antibody D4.6 to thrombospondin 1.

Blood Platelets↗
PubMed1993

The reactive and destabilizing disulfide bond of DsbA, a protein required for protein disulfide bond formation in vivo.

The protein DsbA facilitates disulfide bond formation in the periplasm of Escherichia coli. It has only two cysteine residues that are separated in the sequence by two other residues and are shown to form a disulfide bond reversibly. Chemical modification studies demonstrate that only one of the cysteine residues has an accessible thiol group in the reduced protein. Equilibrium and kinetic characterization of thiol-disulfide exchange between DsbA and glutathione showed that the DsbA disulfide bond was 10(3)-fold more reactive than a normal protein disulfide. Similarly, the mixed disulfide between the accessible cysteine residue and glutathione was 10(4)-fold more reactive than normal. The overall equilibrium constant for DsbA disulfide bond formation from GSSG was only 8 x 10(-5) M. These properties indicate that disulfide-bonded DsbA is a potent oxidant and ideally suited for generating protein disulfide bonds. Disulfide bonds generally increase the stabilities of folded proteins, when the folded conformation reciprocally stabilizes the disulfide bonds. In contrast, the disulfide bond of DsbA was so unstable in the folded state that its stability increased by 4.5 +/- 0.1 kcal.mol-1 when the protein unfolded. This implies that the disulfide bond destabilizes the folded conformation of DsbA. This was confirmed by demonstrating that the reduced protein was 3.6 +/- 1.4 kcal.mol-1 more stable than that with the disulfide bond.

Amino Acid Sequence↗
PubMed1994

Dissecting the mechanism of protein disulfide isomerase: catalysis of disulfide bond formation in a model peptide.

As a model for understanding how protein disulfide isomerase (PDI) catalyzes disulfide bond formation in proteins, its action on a 28-residue disordered peptide containing only two cysteine residues has been examined. Disulfide formation in the peptide using the chemical reaction with small molecule thiol/disulfide reagents, such as oxidized and reduced glutathione or cystamine and cysteamine, occurs in two steps, via two alternative intermediate mixed disulfides between the reagent and either peptide cysteine residue. All thiol/disulfide forms of the peptide could be trapped and quantified, so the rates of their interconversion could be measured. Catalytic amounts of PDI increased the rates of these reactions. All rate enhancements were independent of the concentration of the peptide, indicating that it bound to PDI with an apparent Km of less than 3 microM. In the presence of glutathione, PDI accelerated the formation of both single mixed disulfide species, plus their subsequent rearrangement to form the peptide disulfide bond, but not interchange of the mixed disulfide glutathione between the two cysteine residues. In contrast, PDI did not catalyze the reaction of the reagent cystamine with the reduced peptide to form the mixed disulfide, nor the interchange of this mixed disulfide between cysteine residues, but did catalyze the subsequent intramolecular step of peptide disulfide bond formation to a similar extent as with the glutathione mixed disulfide. These effects on the two steps involving the mixed disulfides with glutathione or cystamine accounted for much of the overall catalytic effect of PDI on disulfide bond formation in the peptide, indicating that direct transfer of disulfide bonds from PDI to the peptide occurred less frequently. These findings demonstrate the utility of using such peptides as PDI substrates and have implications for the mechanism of action of PDI.

Amino Acid Sequence↗
PubMed1999

Disulfide bridges in interleukin-8 probed using non-natural disulfide analogues: dissociation of roles in structure from function.

The structural and functional roles of the two disulfide bridges in interleukin-8 (IL-8) were addressed using IL-8 analogues with covalently modified disulfide bridges. The analogues were prepared using chemical synthesis by replacement of a cysteine for either homocysteine, penicillamine, or selenocysteine and on folding resulted in a covalently modified disulfide. Deletion of either of the two disulfide bridges by replacement of either cysteine pair with alanine resulted in loss of both structure and function. In contrast, all of the analogues with modified disulfide bridges had native tertiary fold as determined by nuclear magnetic resonance spectroscopic methods. Their structural similarity provided a rational basis for assessing the functional effects of the changes to the disulfide. Modification to the disulfide bridge between cysteines 9 and 50 had only a modest effect on IL-8 function. In contrast, alterations to the 7-34 disulfide bridge resulted in a dramatic reduction in biological potency. Thus, although both disulfide bridges are required for maintenance of the native tertiary fold, their role in determining IL-8 activity is distinct. We propose that 7-34 disulfide has a direct role in determining receptor binding and activation, whereas the 9-50 was not directly involved. The synthesis of non-natural disulfide analogues is a novel general approach to structure-activity relationships of disulfide bridges. The demonstration that the participation of disulfide bridges in function can be dissociated from their effects on the stability of the tertiary structure suggests that this method will lead to increased understanding of the roles of disulfide bridges in proteins.

Antigens, CD↗
PubMed1982

Role of disulfides in the subunit structure of the insulin receptor. Reduction of class I disulfides does not impair transmembrane signalling.

The native insulin receptor affinity-labeled by covalent cross-linking to 125I-insulin has been proposed to consist of two alpha receptor subunits and two beta-receptor subunits all disulfide-linked as a Mr = 350,000 (beta-S-S-alpha)-S-S-(alpha-S-S-beta) receptor complex (Massague, J., Pilch, P. F., and Czech, M. P. (1981) J. Biol. Chem. 256, 3181-3190). We denote the disulfide bonds linking the two symmetrical (alpha-S-S-beta) receptor halves as class I insulin receptor disulfides, whereas the disulfide bonds linking one alpha receptor subunit to one beta receptor subunit are termed class II disulfides. In the present studies, dithiothreitol added directly to intact rat fat cells or to membranes prepared from rat fat cell, rat liver, or human placenta, reduced the class I disulfides, but not the class II disulfides, of the insulin receptor. Reduction of class I insulin receptor disulfides did not prevent binding of insulin to the insulin receptor. After treatment with dithiothreitol, the number of apparent "high affinity" insulin binding sites was increased in rat fat cells or fat cell membranes but was decreased in rat liver and human placenta membranes. Reduction of class I insulin receptor disulfides did not alter the sensitivity to insulin of glucose oxidation in intact rat fat cells. The resistance of class II disulfides to reduction by dithiothreitol was not affected by solubilization of the membrane structure by Triton X-100. Class II insulin receptor disulfides were fully reduced by dithiothreitol only after denaturation of the insulin receptor by sodium dodecyl sulfate. Class I insulin receptor disulfides were partially reoxidized by an incubation mixture consisting of reduced and oxidized glutathione. When solubilized insulin receptor containing reduced class I disulfides was exposed to ethylene glycol bis(succinimidyl succinate), the receptor subunits were internally cross-linked and migrated like intact receptor complexes on nonreduced dodecyl sulfate gels. We conclude that the (alpha-S-S-beta) receptor halves in the partially reduced receptor remain associated by noncovalent interactions and that this receptor structure without class I disulfides remains biologically competent.

Adipose Tissue↗
PubMed1996

S-glutathiolated hepatocyte proteins and insulin disulfides as substrates for reduction by glutaredoxin, thioredoxin, protein disulfide isomerase, and glutathione.

The disulfide-reducing activities of glutaredoxin, thioredoxin, protein disulfide isomerase, glutathione, and cysteine were directly compared with a mixture of hepatocyte 35S-glutathiolated proteins as the substrate. Dethiolation of individual 35S-labeled protein bands from the mixture of hepatocyte proteins was analyzed by SDS-PAGE. All of the 35S-labeled protein bands could be completely dethiolated by glutaredoxin, thioredoxin, protein disulfide isomerase, glutathione, or cysteine. On a molar basis glutaredoxin was over 10 times more effective than either thioredoxin or protein disulfide isomerase. Dethiolation rates of individual proteins varied in minor ways. For example, glutaredoxin dethiolated the 15-, 30-, and 48-kDa protein bands 3 to 4 times faster than the 27-, 28-, and 77-kDa bands. Glutaredoxins from pig liver or from bovine heart had the same specificity and similar activity. The rate of dethiolation by glutathione alone was low compared to the glutaredoxin-catalyzed process, but all 35S-labeled protein bands could be reduced by glutathione, cysteine, or dithiothreitol. Glutathione was clearly more effective than cysteine when these two thiols were compared on the basis of the concentration of thiolate anion available at neutral pH. Therefore, glutathione is a more specific reductant of S-glutathiolated proteins than is cysteine but it is much less effective than glutaredoxin. Since glutaredoxin activity in cells is 10 times higher than the concentration used in these experiments, ample activity is available to account for substantial rates of dethiolation in vivo. Thioredoxin is quite inefficient as a reductant of S-glutathiolated proteins, but it was reasoned that it might first reduce glutaredoxin, which then could reduce the S-glutathiolated protein. A combination of thioredoxin and glutaredoxin was effective. It is proposed that glutaredoxin is the principal agent responsible for protein dethiolation in vivo. The effectiveness of glutaredoxin, thioredoxin, and protein disulfide isomerase as reductants for protein disulfide bonds was examined with insulin as the substrate. Protein disulfide isomerase was very effective and thioredoxin was nearly as effective. Human thioredoxin was similar to Escherichia coli thioredoxin in reactivity and specificity. Glutaredoxin did not facilitate insulin reduction at equal concentrations. Thus, protein disulfide isomerase and thioredoxin are more effective than glutaredoxin as reductants of insulin protein disulfides. The apparent reduction potential of pig liver glutaredoxin (-0.159 +/- 0.004 V) was determined by measuring the amount of reduced glutaredoxin in equilibrium with mixtures of glutathione and glutathione disulfide. Glutaredoxin was a weaker reductant than E. coli thioredoxin (-0.260 V) and was similar to protein disulfide isomerase (-0.11 to -0.19 V). The role of these proteins as disulfide reductants is not determined solely by thermodynamic considerations. A glutathione binding site at the dithiol region of glutaredoxin may be of primary importance for its function in protein dethiolation, while a different specific peptide binding site in thioredoxin may be more suited to certain protein disulfide structures.

Animals↗
PubMed2006

Protein disulfides and protein disulfide oxidoreductases in hyperthermophiles.

Disulfide bonds are required for the stability and function of a large number of proteins. Recently, the results from genome analysis have suggested an important role for disulfide bonds concerning the structural stabilization of intracellular proteins from hyperthermophilic Archaea and Bacteria, contrary to the conventional view that structural disulfide bonds are rare in proteins from Archaea. A specific protein, known as protein disulfide oxidoreductase (PDO) is recognized as a potential key player in intracellular disulfide-shuffling in hyperthermophiles. The structure of this protein shows a combination of two thioredoxin-related units with low sequence identity which together, in tandem-like manner, form a closed protein domain. Each of these units contains a distinct CXXC active site motif. Due to their estimated conformational energies, both sites are likely to have different redox properties. The observed structural and functional characteristics suggest a relation to eukaryotic protein disulfide isomerase. Functional studies have revealed that both the archaeal and bacterial forms of this protein show oxidative and reductive activity and are able to isomerize protein disulfides. The physiological substrates and reduction systems, however, are to date unknown. The variety of active site disulfides found in PDOs from hyperthermophiles is puzzling. Nevertheless, the catalytic function of any PDO is expected to be correlated with the redox properties of its active site disulfides CXXC and with the distinct nature of its redox environment. The residues around the two active sites form two grooves on the protein surface. In analogy to a similar groove in thioredoxin, both grooves are suggested to constitute the substrate binding sites of PDO. The direct neighbourhood of the grooves and the different redox properties of both sites may favour sequential reactions in protein disulfide shuffling, like reduction followed by oxidation. A model for peptide binding by PDO is proposed to be derived from the analysis of crystal packing contacts mimicking substrate binding interactions. It is assumed, that PDO enzymes in hyperthermophilic Archaea and Bacteria may be part of a complex system involved in the maintenance of protein disulfide bonds. The regulation of disulfide bond formation may be dependent on a distinct interplay of thermodynamic and kinetic effects, including functional asymmetry and substrate-mediated protection of the active sites, in analogy to the situation in protein disulfide isomerase. Numerous questions related to the function of PDO enzymes in hyperthermophiles remain unanswered to date, but can probably successfully be studied by a number of approaches, such as first-line genetic and in vivo studies.

Archaea↗
PubMed2000

Development of disulfide peptide mapping and determination of disulfide structure of recombinant human osteoprotegerin chimera produced in Escherichia coli.

Recombinant human osteoprotegerin chimera is a 90-kDa protein containing a human IgG Fc domain fused to human osteoprotegerin. The molecule is a dimer linked by two intermolecular disulfide bonds and contains eleven intramolecular disulfide bonds per monomer. A cysteine-rich region in osteoprotegerin contains nine disulfide bridges homologous to the cysteine-rich signature structure of the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In this report, we have developed peptide mapping procedures suitable to generate disulfide-containing peptides for disulfide structure assignment of the fusion molecule. The methods employed included proteolytic digestion using endoproteinases Glu-C and Lys-C in combination followed by LC-MS analyses. Disulfide linkages of peptide fragments containing a single disulfide bond were assigned by sequence analysis via detection of (phenylthiohydantoinyl) cystine and/or by MS analysis. Disulfide bonds of a large, core fragment containing three peptide sequences linked by four disulfides were assigned after generation of smaller disulfide-linked peptides by a secondary thermolysin digestion. Disulfide structures of peptide fragments containing two disulfide bonds were assigned using matrix-assisted laser desorption ionization mass spectrometry with postsource decay. Both the inter- and intramolecular disulfide linkages of the chimeric dimer were confirmed.

Amino Acid Sequence↗
PubMed1992

Disulfide bond-coupled folding of bovine pancreatic trypsin inhibitor derivatives missing one or two disulfide bonds.

The disulfide bond-coupled folding and unfolding mechanism (at pH 8.7, 25 degrees C in the presence of oxidized and reduced dithiothreitol) was determined for a bovine pancreatic trypsin inhibitor mutant in which cysteines 30 and 51 were replaced with alanines so that only two disulfides, between cysteines 14 and 38 and cysteines 5 and 55, remain. Similar studies were made on a chemically-modified derivative of the mutant retaining only the 5-55 disulfide. The preferred unfolding mechanism for the Ala30/Ala51 mutant begins with reduction of the 14-38 disulfide. An intramolecular rearrangement via thiol-disulfide exchange, involving the 5-55 disulfide and cysteines 14 and/or 38, then occurs. At least five of six possible one-disulfide bond species accumulate during unfolding. Finally, the disulfide of one or more of the one-disulfide bond intermediates (excluding that with the 5-55 disulfide) is reduced giving unfolded protein. The folding mechanism seems to be the reverse of the unfolding mechanism; the observed folding and unfolding reactions are consistent with a single kinetic scheme. The rate constant for the rate-limiting intramolecular folding step--rearrangements of other one-disulfide bond species to the 5-55 disulfide intermediate--seems to depend primarily on the number of amino acids separating cysteines 5 and 55 in the unfolded chain. The energetics and kinetics of the mutant's folding mechanism are compared to those of wild-type protein [Creighton, T. E., & Goldenberg, D. P. (1984) J. Mol. Biol. 179, 497] and a mutant missing the 14-38 disulfide [Goldenberg, D. P. (1988) Biochemistry 27, 2481]. The most striking effects are destabilization of the native structure and a large increase in the rate of unfolding.

Alanine↗
PubMed2001

Detection of disulfide bonds in bovine brain tubulin and their role in protein folding and microtubule assembly in vitro: a novel disulfide detection approach.

Cysteine residues in tubulin are actively involved in regulating ligand interactions and microtubule formation both in vivo and in vitro. These cysteine residues are sensitive reporters in determining the conformation of tubulin. Although some of the cysteines are critical in modulating drug binding and microtubule assembly, it is not clear how many of these normally exist as disulfides. The controversy regarding the disulfide bonds led us to develop a disulfide detection assay to reexamine the presence of the disulfide linkages in purified alphabeta tubulin and explore their possible biological functions in vitro. The accessible cysteine residues in alphabeta tubulin were alkylated with an excess of iodoacetamide to prevent artifactual generation of disulfide linkages in tubulin. After removal of excess iodoacetamide, tubulin was unfolded in 8 M urea. Half of the unfolded tubulin was treated with dithiothreitol to reduce any disulfide bonds present. The aliquots were then treated with iodo[(14)C]acetamide and the incorporation of radioactivity was measured. We also used the same approach to detect the disulfide linkages in the tubulin in a whole-cell extract. We found in both cases that the samples which were not treated with dithiothreitol had little or no incorporation of iodo[(14)C]acetamide, while the others that were treated with dithiothreitol had significant amounts of (14)C incorporation into tubulin. Moreover, the reduction of the disulfide linkages in tubulin resulted in inhibition of microtubule assembly (29-54%) and markedly affected refolding of the tubulin from both an intermediate and a completely unfolded state. All these data therefore suggest that tubulin has intrachain disulfide bonds in the alpha- and beta-subunits and that these disulfides assist in correct refolding of tubulin from the intermediate unfolded state or help to recover the hydrophobic domains from the completely unfolded state. These disulfides also regulate microtubule assembly and the stability of tubulin in vitro. Our results suggest that tubulin disulfides may play a role in tubulin folding and that thiol-disulfide exchange in tubulin could be a key regulator in microtubule assembly and dynamics of tubulin in vivo.

Ammonium Sulfate↗
PubMed1997

Disulfide exchange folding of disulfide mutants of insulin-like growth factor I in vitro.

We have previously concluded that insulin-like growth factor-I (IGF-I) is thermodynamically unable to quantitatively form its disulfide bonds under reversible redox conditions in vitro. From detailed analyses it was hypothesized that the 47-52 disulfide is energetically unfavorable in the native IGF-I structure [Hober et al. (1992) Biochemistry 31, 1749-1756]. In this paper, this hypothesis has been tested by refolding of IGF-I mutant proteins lacking either the 47-52 or 6-48 disulfide bond. The disulfide exchange folding equilibrium behavior of these mutated IGF-I variants were examined in a glutathione redox buffer. The mutant protein IGF-I(C47A,C52A) was demonstrated to form both remaining native disulfide bonds. In contrast, IGF-I(C6A,C48A) was unable to quantitatively form both of its disulfides and was shown to accumulate a one disulfide variant lacking the 47-52 disulfide bond. These folding data corroborate the hypothesis that the 47-52 disulfide bond of IGF-I is energetically unfavorable also in the absence of the 6-48 disulfide bond. The two IGF-I variants were purified in oxidized forms where both native disulfides are formed. Both variants were suggested to be structurally perturbed compared with the native molecule as determined by circular dichroism spectroscopy. Further, binding affinities to the IGF binding protein 1 and a soluble IGF type I receptor, respectively, were severely lowered in both disulfide mutant proteins compared to the native IGF-I molecule. Interestingly, the binding affinity toward the IGF type I receptor is higher for IGF-I(C6A,C48A) than for IGF-I(C47A,C52A) while the binding affinity to IGFBP-1 is higher for IGF-I(C47A,C52A) than for IGF-I(C6A,C48A). Thus, the structural changes due to removal of the 6-48 or 47-52 disulfide bonds, respectively, yield structural changes in different regions of the IGF-I molecule reflected in the different binding activities.

Alanine↗
PubMedDate not supplied

Disulfide-linked and non-disulfide-linked gamma/delta T-cell antigen receptors: differential expression on T-cell lines and clones derived from normal donors and patients with primary immunodeficiency disorders.

We studied the expression of gamma delta T cell receptors (TCR) on T-cell lines and clones derived from peripheral blood lymphocytes (PBL) from certain patients with primary immunodeficiency disorders and normal donors. Immunoprecipitation with the anti-Leu 4, anti-gamma-chain and/or anti-delta-chain monoclonal antibodies followed by SDS-PAGE analysis revealed that 7 of 13 (54%) T-cell lines and clones developed from PBL of patients with primary immunodeficiency disorders expressed non-disulfide-linked gamma delta TCR, utilizing either the C gamma 2abc or the C gamma 2bc gamma-chain constant region gene segment. 5 of 13 (38%) T-cell lines/clones expressed disulfide-linked gamma delta TCR, whereas an additional T-cell line was comprised of T cells expressing either disulfide-linked (C gamma 1) or non-disulfide-linked (C gamma 2bc) gamma delta TCR. T-cell lines and clones developed from four of light patients with primary immunodeficiency disorders exhibited exclusively non-disulfide-linked gamma delta TCR utilizing either the C gamma 2abc or the C gamma 2bc gamma-chain segment. T-cell lines derived from a fifth patient exhibited primarily non-disulfide-linked gamma delta TCR, bringing to five of eight the numbers of patients that expressed exclusively or primarily non-disulfide-linked gamma delta TCR. T-cell lines/clones derived from the remaining three patients exhibited exclusively disulfide-linked gamma delta TCR. The age of these patients varied over a wide range and there was not an association between their age and the type of gamma delta TCR expressed on T-cell lines derived from their PBL. In contrast, to these findings 14 of 16 (87.5%) T-cell clones derived from PBL of normal donors expressed disulfide-linked gamma delta TCR, whereas only 2 of 16 (12.5% expressed non-disulfide linked gamma delta TCR. Among the T-cell clones from normal donors which express disulfide-linked gamma delta TCR two different types were identified. Those exhibiting under reducing conditions on SDS-PAGE two completely resolved polypeptide chains in the range of 37 kD to 44 kD, and those exhibiting under the same conditions indistinguishable overlapping gamma- and delta- chains in the range of 40-42 kD. Several T-cell lines and clones from normal donors or patients with primary immunodeficiency that expressed either disulfide- or non-disulfide-linked gamma delta TCR were delta TCS1+, demonstrating that the delta TCS1 determinant is expressed on both types of gamma delta TCR.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, CD↗
PubMed1996

Determination and metabolism of dithiol chelating agents. XVII. In humans, sodium 2,3-dimercapto-1-propanesulfonate is bound to plasma albumin via mixed disulfide formation and is found in the urine as cyclic polymeric disulfides.

The binding of 2,3-dimercapto-1-propanesulfonate (DMPS) in plasma was determined in three healthy young adults after a single 300-mg p.o. dose. By 5 hr after DMPS administration, 62.5% of the total plasma DMPS was bound to proteins. The remainder consisted of nonprotein associated DMPS disulfides (36.6%) and unaltered DMPS (0.9%). Protein-bound DMPS consisted of a DMPS-albumin complex (84%) and a higher molecular weight protein complex (16%), perhaps albumin aggregates. DMPS was released from the isolated DMPS-albumin complex after treatment with dithiothreitol, indicating that it was bound via a disulfide linkage. The half-life of unaltered DMPS was 1.8 hr, whereas that of altered DMPS was 20 hr, suggesting that the DMPS-albumin disulfide complex is stable and that DMPS was released from it slowly. In addition, the biotransformation of OMPS to disulfide forms was extensive. By 9 hr after administration, 10% of the total urinary DMPS was unchanged drug and 90% was altered DMPS. The latter was converted to DMPS by dithiothreitol, indicating that the altered DMPS consisted of disulfides. In 2- to 4-hr urine, DMPS disulfides included cyclic polymeric DMPS disulfides (97%), DMPS-cysteine (1:2) mixed disulfide (2.5%) and acyclic DMPS disulfide (0.5%). The cyclic polymeric DMPS disulfides were present in a major (91.5%) and minor (5.5%) form. DMPS-albumin mixed disulfide and nonprotein DMPS disulfides may prolong the heavy metal mobilizing activity of DMPS and thus may represent reservoirs of DMPS which can be released by disulfide reduction in vivo.

Adult↗
PubMed1990

Kinetics of disulfide bond reduction in alpha-lactalbumin by dithiothreitol and molecular basis of superreactivity of the Cys6-Cys120 disulfide bond.

Kinetics of disulfide reduction in alpha-lactalbumin by dithiothreitol are investigated by measuring time-dependent changes in absorption at 310 nm and in CD ellipticity at 270 nm (pH 8.5 or 7.0, and 25 degrees C). When the disulfide-intact protein is folded, the kinetics are biphasic. The disulfide bond between the half-cystines-6 and -120 is reduced in the fast phase, and the other three disulfide bonds are reduced in the slow phase. The apparent rate constants of the two phases are both proportional to the concentration of dithiothreitol, indicating that both phases are expressed by bimolecular reactions. However, detailed molecular mechanisms that determine the reaction rates are markedly different between the two phases. The slow phase shows a sigmoidal increase in the reaction rate with increasing concentration of a denaturant, urea, and is also accelerated by destabilization of the native state on removal of the bound Ca2+ ion in the protein. The disulfide bonds are apparently protected against the reducing agent in the native structure. The fast phase reaction rate is, however, decreased with an increase in the concentration of urea, and the disulfide bond shows extraordinary superreactivity in native conditions. It is 140 times more reactive than normal disulfides in the fully accessible state, and three-disulfide alpha-lactalbumin produced by the fast phase assumes nativelike structure under a strongly native condition. As ionic strength does not affect the superreactivity of this disulfide bond, electrostatic contributions to the reactivity must be negligible. Inspection of the disulfide bond geometry based on the refined X-ray coordinates of baboon alpha-lactalbumin [Acharya et al. (1989) J. Mol. Biol. 208, 99-127] and comparison of the geometry with those in five other proteins clearly demonstrate that the superreactivity arises from the geometric strain imposed on this disulfide bond by the native structure folding. Relationships of the disulfide strain energy to the protein stability and the disulfide reactivity are discussed.

Chemical Phenomena↗
PubMed1987

Reversibility of disulfide formation. Comparison of chemical and enzyme-mediated reduction of penicillamine and captopril disulfides.

The reduction of penicillamine disulfide by reductants in aqueous solutions has been studied and compared with that for captopril disulfide. Whereas near quantitative reduction for captopril disulfide was achieved with tributyl phosphine (200 mM), no detectable penicillamine was formed from penicillamine disulfide. Thiol reductants (25 mM) were, however, partially able to reduce penicillamine disulfide with the most effective agent being glutathione (15% reduction) following by dithioerythritol (8%) and cysteine (5.1%). The reduction of penicillamine-cysteine disulfide by glutathione was 6-fold higher than for penicillamine disulfide. Kinetic analysis showed that the initial rate of reduction and equilibrium constant for the reduction of penicillamine disulfides by glutathione were 267- and 875-fold less than for captopril disulfide at pH 7.4. Biotransformation studies in the cytosol fraction of rat blood cells demonstrated that whereas 48% of the reduction of captopril disulfide was enzyme-mediated only 19% of the penicillamine formed was enzyme-mediated for penicillamine disulfide. Accumulation of disulfides of penicillamine in patients taking penicillamine may therefore be a problem during chronic therapy.

Animals↗