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At least 973 records · Page 54Linked to original sources
PubMed1999

Assignment of disulfide linkages in chum salmon stanniocalcin.

Stanniocalcin (STC) is a hypocalcemic glycoprotein hormone secreted by the corpuscles of Stannius, endocrine glands unique to the bony fishes. Recently, two cDNAs encoding proteins, STC-1 and STC-2, homologous to fish STC have been identified in humans and rodents. The STC-1, but not STC-2, is identical to fish STC with respect to location of all Cys residues. The present study reports the localization of inter- and intra-molecular disulfide linkages in chum salmon STC. The chum salmon STC was deglycosylated and digested with several proteases in series. Six fragments, each of which consisted of two peptides connected by a single disulfide bond, were isolated by HPLC. Disulfide bonds were determined by sequence analysis after reduction and S-alkylation of each peptide fragment. Chum salmon STC is a homodimer connected by a single intermonomeric disulfide bond at Cys169. The monomer consists of 179 amino-acids, containing five intramonomeric disulfide bonds formed between Cys12-Cys26, Cys21-Cys41, Cys32-Cys81, Cys65-Cys95, and Cys102-Cys137.

Amino Acid Sequence↗
PubMed1995

Influence of protein conformation on disulfide bond formation in the oxidative folding of ribonuclease T1.

In oxidative protein folding the interdependence between the acquisition of an ordered native-like conformation and disulfide bond formation was investigated by using the C2S/C10N variant of ribonuclease T1 as a model. This protein of 104 residues has a single disulfide bond between Cys6 and Cys103. In the reduced form it is unfolded in the presence of urea, but native-like folded when > or = 1.5 M NaCl is present. The influence of a folded conformation on the individual thiol/disulfide exchange reactions between the protein and glutathione could thus be studied in oxidative folding by varying the urea and NaCl concentrations. When the reduced protein was folded native-like the initial formation of the mixed disulfide between the protein and glutathione was decelerated about fourfold. The attachment of a glutathionyl moiety in this step destabilizes the protein by about 5 kJ mol-1 and led to a local unfolding near the two Cys residues. The reacting thiol groups still remained in close proximity for the subsequent intramolecular thiol/disulfide exchange reaction, but an increase in the energy of the transition state (e.g. by a hydrophobic environment or by steric strain) could be avoided. As a consequence the formation of the protein disulfide in this reaction was 100-fold faster when the mixed-disulfide species was in this ordered conformation. These results illustrate the importance of a low stability and a high flexibility of folding intermediates.

Aspergillus oryzae↗
PubMed1994

Clearance of glutathione disulfide from rat mesenteric vasculature.

Organs of the digestive tract, including pancreas, small intestine, and colon, have mechanisms to modulate plasma thiol-disulfide balance. Because plasma glutathione disulfide (GSSG) concentration may be elevated from < 1 microM in control rats to over 25 microM during oxidative stress, we examined whether GSSG was cleared from rat mesenteric vasculature. When 100 microM GSSG was perfused through the gut via the superior mesenteric artery, an average of 45% was lost in a single pass. Results showed that gamma-glutamyltransferase (gamma-GT)-dependent and -independent mechanisms were involved in GSSG loss. Acivicin (AT125) treatment inhibited gamma-GT activity in the mesenteric vasculature by 94% and attenuated the loss of GSSG equivalents by 44%. These results supported a role for gamma-GT in GSSG loss from the mesenteric vasculature but indicated that still other mechanisms were involved in GSSG clearance. Elevations of portal levels of glutathione (GSH) and the mixed disulfide of cysteine and GSH (CySSG) also occurred with vascular GSSG perfusion and could account for about 40% of GSSG equivalents lost. Because portal elevations of GSH and CySSG were not inhibited by AT125, they were formed by a gamma-GT-independent mechanism(s). Given that cysteine was present in the mesenteric vasculature, the most likely mechanism to explain GSH and CySSG formation was via nonenzymatic thiol-disulfide exchange between GSSG and cysteine. Uptake of vascular GSSG by pancreas, small intestine (jejunum and ileum), or colon apparently did not occur as tissue contents of GSSG or GSH were not elevated, except for a small elevation of GSH in pancreas when mesenteric gamma-GT was inhibited with AT125. Additionally, GSSG was not transported from mesenteric vasculature into the small intestinal lumen because luminal levels of GSSG or GSH were not elevated. Further, total cysteine equivalents in lumen were unchanged indicating that GSSG was not transported to lumen and degraded to cystine by gamma-GT and dipeptidases localized to the intestinal brush-border. These results indicate that GSSG present in mesenteric vasculature is metabolized in the vascular compartment by gamma-GT-dependent and -independent reactions; together, these account for over 80% of lost GSSG equivalents. They also suggest that organs of the mesentery may play a quantitatively important role in plasma GSSG clearance and modulation of vascular thiol-disulfide balance.

Analysis of Variance↗
PubMed1977

Protein thiol-disulfide interchange and interfacing with biological systems.

Disulfide-containing proteins offer unique advantages for mechanistic studies of the formation of native three-dimensional structure from unordered, reduced precursors. The main advantage is that covalent intermediates are formed; by characterizing these intermediates, one obtains substantial information about the reaction pathway. Thiol-disulfide interchange is a major component of most oxidative mechanisms carrying thiol to disulfide; thus, it required some attention in its own right. Afinsen's descriptions of a "shuffle-ase" enzyme led us to examine the rates of the uncatalyzed exchange under physiologically plausible conditions. Somewhat surprisingly, we found that the rates for formation of several native proteins in uncatalyzed systems containing GSSG and GSH are as great as with the "shuffle-ase" enzyme, suggesting that a substantial portion of biological thiol oxidations proceed by uncatalyzed exchange. While thiol-disulfide exchange of course results in no net change in the oxidation level of a system, catalytic linkage of thiol or disulfide to other redox systems provides a mechanism for achieving net changes.

Chemical Phenomena↗
PubMed1997

Disulfide-mediated polymerization of whey proteins in whey protein isolate-stabilized emulsions.

The effects of protein polymerization in whey protein isolate-stabilized emulsions on emulsion properties were investigated. Polymerization, involving intermolecular disulfide bonds between whey proteins adsorbed at the oil-water interface, increased with increasing storage time following emulsion formation. Ageing resulted in increased aggregation of emulsion droplets, emulsion viscosity and susceptibility to creaming but these effects were lower when thiol-disulfide interchange reactions were inhibited by N-ethylmaleimide (NEM). Following heating to 75 degrees C, disulfide-mediated polymerization of whey proteins increased as did droplet aggregation, emulsion viscosity and creaming. While NEM lowered the extent of disulfide-mediated polymerization it did not affect the measured physical properties of the heated emulsions. Non-covalent interactions appeared to be the principal forces leading to aggregation of emulsion droplets but aggregates once formed were stabilized by disulfide bonds.

Animals↗
PubMed1987

Effect of exposure to carbon disulfide on tryptophan metabolism and the tissue vitamin B6 contents of rats.

Female Wistar rats were exposed to 100 ppm or 600 ppm carbon disulfide for 6 hours a day, 5 days a week for 12 weeks. During the exposure period, their urinary excretion of 4-pyridoxic acid was determined. The urinary excretions of xanthurenic acid and kynurenic acid after tryptophan loading were also determined. At the end of the exposure period, the rats were sacrificed and the levels of the five major forms of vitamin B6, pyridoxine, pyridoxal, pyridoxamine, pyridoxal phosphate and pyridoxamine phosphate, in the liver, kidneys and brain were determined by HPLC fluorometry. During the exposure the urinary excretions of xanthurenic acid and kynurenic acid after i.p. administration of tryptophan increased significantly in both experimental groups. However, urinary excretion of 4-pyridoxic acid decreased only slightly in the group exposed to carbon disulfide at 600 ppm and did not decrease in the group exposed to 100 ppm carbon disulfide. Furthermore, no significant changes were observed in the contents of vitamins B6 in any tissues examined. These results indicate that carbon disulfide does not cause vitamin B6 deficiency and thus that the disorders of tryptophan metabolism induced by carbon disulfide intoxication are not due to vitamin B6 deficiency.

Animals↗
PubMed1987

Experimental human exposure to carbon disulfide. II. Urinary excretion of 2-thiothiazolidine-4-carboxylic acid (TTCA) during and after exposure.

Six human volunteers were exposed to 10 and 20 ppm carbon disulfide at rest and to 3 and 10 ppm carbon disulfide under a 50 W level of physical exercise during four consecutive periods of 50 min. At the start of the experiments, at the end of the exposure periods and during the post-exposure period, urine was sampled and the concentration of 2-thiothiazolidine-4-carboxylic acid (TTCA) was determined. It was established that only a small percentage, ranging from 0.7 to 2.2% of the absorbed carbon sulfide was transformed into TTCA. The excretion rate of TTCA (mumol TTCA h-1) was found to be the best parameter in evaluating the respiratory uptake of carbon disulfide over a range of 37.9 to 163.3 mg CS2 compared to the urinary concentration of TTCA (mole TTCA ml-1) or the creatinine corrected concentration of TTCA (mmol TTCA mol-1 creatinine). The total amount of TTCA (mumol TTCA) excreted proved to be independent of the urinary flow (ml h-1), the estimates of the individual fatty tissue content and the urinary pH. No correlation was found between the respiratory uptake of carbon disulfide (mg CS2) and the excretion rate of TTCA within each exposure condition of 3, 10 or 20 ppm carbon disulfide, respectively.

Adult↗
PubMed1988

Local interactions favor the native 8-residue disulfide loop in the oxidation of a fragment corresponding to the sequence Ser-50-Met-79 derived from bovine pancreatic ribonuclease A.

A 30-residue peptide was obtained from ribonuclease A by chemical cleavage with cyanogen bromide, subsequent sulfitolysis with concomitant S-sulfonation, and finally enzymatic cleavage with Staphylococcus aureus protease. The peptide was converted to the free thiol form by reductive cleavage of the S-sulfo-protecting groups with D,L-dithiothreitol. This peptide consisted of residues 50-79 of the native sequence of ribonuclease A, with the exception that methionine-79 had been converted to homoserine. Included in this sequence are residues cysteine-65 and cysteine-72, which form a disulfide bond in the native enzyme, as well as cysteine-58. This molecule may form one of three possible intramolecular disulfide bonds upon thiol oxidation, viz. one loop of 15 and 2 of 8 residues each. These isomeric peptides were prepared by oxidation with cystamine, 2-aminoethanethiolation of residual thiols, and fractionation by reverse-phase high-performance liquid chromatography. Disulfide pairings were established by mapping the tryptic fragments and confirming their composition by amino acid analysis. After protracted incubation under oxidizing conditions at 25.0 degrees C and pH 8.0, the 26-member ring incorporating the native disulfide bond between residues 65 and 72 is the dominant product. Assuming that equilibrium is established, we infer that local interactions in the sequence of ribonuclease A significantly stabilize the native 8-residue disulfide loop with respect to the non-native 8-residue loop (delta G degree = -1.1 +/- 0.1 kcal mole-1). The implications of this observation for the oxidative folding of the intact protein are discussed.

Amino Acid Sequence↗
PubMed1990

Identification of disulfide-containing peptides in endocrine tissue extracts by HPLC-electrochemical detection and mass spectrometry.

A new procedure to selectively identify disulfide-containing peptides in extracts of biological tissues is described. Disulfide-containing peptides are detected by their UV absorbance and electrochemical (EC) activity after chromatographic separation, and subsequently identified by fast atom bombardment mass spectrometry (FABMS). This combination of fractionation by HPLC and selective detection is attractive because it is rapid, highly specific for disulfide-containing peptides, and applicable to all disulfide-containing peptides that may be present in complex biological mixtures. Useful procedures for applying the method are demonstrated with tissue extracts from bovine pituitary and catfish pancreas. In addition to finding the expected disulfide-containing peptides, evidence for two forms of catfish insulin are presented. The merits of this and other methods used to detect peptides in similar tissue extracts are discussed.

Amino Acid Sequence↗
PubMed1986

Rat heart gap junctions as disulfide-bonded connexon multimers: their depolymerization and solubilization in deoxycholate.

Unproteolyzed gap junctions isolated from rat heart and liver were analyzed for the presence of inter-subunit disulfide bonds by sodium dodecylsulfate polyacrylamide gel electrophoresis. Rat cardiac junctions contained multiple disulfide bonds connecting the Mr 47,000 subunits of the same connexon and of different connexons. Inter-subunit disulfide bonds were absent in liver junctions. Unproteolyzed rat heart gap junctions were resistant to deoxycholate in their oxidized state, but dissolved readily in the detergent when the disulfide bonds were cleaved with beta-mercaptoethanol. Disulfide bonding in proteolyzed cardiac junctions was limited to pairs of Mr 29,500 subunits. These junctions were not soluble in deoxycholate even in the presence of beta-mercaptoethanol. These results show that heart and liver junctions differ in their quarternary organization.

Animals↗
PubMed1994

Covalent structure of botulinum neurotoxin type A: location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains.

Botulinum neurotoxin Type A is synthesized by Clostridium botulinum as a approximately 150 kD single chain polypeptide. The posttranslational processing of the 1296 amino acid residue long gene product involves removal of the initiating methionine, formation of disulfide bridges, and limited proteolysis (nicking) by the bacterial protease(s). The mature dichain neurotoxin is made of a approximately 50-kD light chain and a approximately 100-kD heavy chain connected by a disulfide bridge. DNA derived amino acid sequence predicted a total of 9 Cys residues (Binz et al., 1990, J. Biol. Chem. 265, 9153-9158; Thompson et al., 1990, Eur. J. Biochem. 189, 73-81). Treatment of the dichain neurotoxin, dissolved in 6 M guanidine. HCl, with 4-vinylpyridine converted 5 Cys residues into S-pyridylethyl cysteine residues; but alkylation after mercaptolysis converted all 9 Cys residues in the S-pyridylethylated form. After confirming the predicted number of Cys residues by amino acid analysis, the positions of the 5 Cys residues carrying sulfhydryl groups and the 4 involved in disulfide bridges were determined by comparing the elution patterns in reversed-phase HPLC of the cyanogen bromide mixtures of the exclusively alkylated and the mercaptolyzed-alkylated neurotoxin. The chromatographically isolated components were identified by N-terminal amino acid sequence analysis. The HPLC patterns showed characteristic differences. The Cys residues predicted in positions 133, 164, 790, 966, and 1059 were found in the sulfhydryl form; Cys 429 and 453 were found disulfide-bridge connecting the light and heavy chains, and Cys 1234 and 1279 were found in an intrachain disulfide-bridge near the C-terminus in the heavy chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Relationship between biliary excretion of bilirubin and glutathione disulfide.

The effects of two glutathione-oxidizing agents, t-butyl hydroperoxide and diamide, on biliary excretion of bilirubin and glutathione disulfide were investigated in anesthetized male Sprague-Dawley rats. Bilirubin (unconjugated) was infused at a constant rate of 100 nmol/kg/min through the jugular vein. When biliary excretion of bilirubin was stabilized, either of the glutathione-oxidizing agents was administered via the mesenteric vein. Biliary excretion of glutathione disulfide increased temporarily after the administration and returned to its basal levels within 20 min. The biliary excretion of bilirubin decreased during the same period and returned to the former levels thereafter. Changes in bile flow rates remained within 20% of the basal levels. A linear correlation was found between the increments in the bile concentration of glutathione disulfide and the decrements in that of bilirubin. Furthermore, separate experiments revealed that reduction of hepatocellular glutathione per se had little effect on biliary excretion of bilirubin. The results thus indicate that the reduction of biliary excretion of bilirubin by glutathione-oxidizing agents was due to the increase in biliary excretion of glutathione disulfide, and suggest that a common biliary excretory mechanism is shared, at least partially, by bilirubin and glutathione disulfide in Sprague-Dawley rats.

Animals↗
PubMed1997

Acrylamide and carbon disulfide treatments increase the rate of rat brain tubulin polymerization.

Acrylamide and carbon disulfide produce central-peripheral distal axonopathy in experimental animals and humans. The main feature of this disease is the focal swellings containing neurofilaments in distal axons, followed by nerve degeneration beyond these swellings. We studied the possible role of tubulin assembly kinetics in this disease. The rats were either administered acrylamide (50 mg/kg, ip, saline) or exposed to carbon disulfide (700 ppm, 9 h) via inhalation for 12 and 15 d, respectively. Tubulin, purified from both acrylamide-(10.37 +/- 0.3 vs 11.3 +/- 0.15) and carbon disulfide-treated (9.72 +/- 0.5 vs 11.18 +/- 0.25) rat brains showed increase in Vmax (OD/min x 10(3)) of its polymerization. However, only acrylamide treatment showed a decrease in time to Vmax, when brain supernatant was used for tubulin polymerization. In vitro addition of acrylamide (0.1-1 mM) to bovine brain tubulin also showed a decrease in time to Vmax (16-21%) of its polymerization. Carbon disulfide treatment of rats, on the other hand, showed a decrease in MAP-2 and an increase in a 120-kDa peptide concentration. The latter showed immunoreactivity with anti-MAP-2. The increase in the rate of tubulin polymerization by acrylamide and carbon disulfide treatment may alter the rate of transport of axonal constituents, including neurofilament, and contribute toward their accumulation in the focal swellings observed in this neuropathy.

Acrylamides↗
PubMed2003

Identification of genes in thyrocytes regulated by unfolded protein response by using disulfide bond reducing agent of dithiothreitol.

Disulfide bonds are formed between the sulfhydryl groups in two cysteine residues of a protein. The formation of these bonds is necessary for the proper folding of a protein into its active three-dimensional form. In this study, the genes associated with disulfide bond formation of proteins from the rat thyroid cell line, FRTL-5 cell, were investigated using disulfide bond reducing agent of dithiothreitol (DTT), which prevented disulfide formation of newly synthesized proteins. The expression of six genes, they being the cAMP phosphodiesterase 7A1, neuronal cell death inducible putative kinase (NIPK), cytosolic LIM protein (Ajuba), Eker, early growth response 1 and the ferritin heavy chain, was specifically enhanced under both reductive conditions and various endoplasmic reticulum (ER) stresses inducing drugs such as Brefeldin A (BFA), calcium ionophore A23187 (A23187) and tunicamycin. These results suggest that a suitable redox environment is necessary for the correct disulfide bond conformation in thyrocytes in a complex system.

Animals↗
PubMed2006

Introduction of a disulfide bridge enhances the thermostability of a Streptomyces olivaceoviridis xylanase mutant.

Substitution of the N-terminus of Streptomyces olivaceoviridis xylanase XYNB to generate mutant TB has been previously shown to increase the thermostability of the enzyme. To further improve the stability of this mutant, we introduced a disulfide bridge (C109-C153) into the TB mutant, generating TS. To assess the effect of the disulfide bridge in the wild-type enzyme, the S109C-N153C mutation was also introduced into XYNB, resulting in XS. The mutants were expressed in Pichia pastoris, the recombinant enzymes were purified, and the effect of temperature and pH on enzymatic activity was characterized. Introduction of the disulfide bridge (C109-C153) into XYNB (XS variant) and TB (TS variant) increased the thermostability up to 2.8-fold and 12.4-fold, respectively, relative to XYNB, after incubation at 70 degrees C, pH 6.0, for 20 min. In addition, a synergistic effect of the disulfide bridge and the N-terminus replacement was observed, which extended the half-life of XYNB from 3 to 150 min. Moreover, XS and TS displayed better resistance to acidic conditions compared with the respective enzymes that did not contain a disulfide bridge.

Disulfides↗
PubMed2006

Carbon disulfide-induced changes in cytoskeleton protein content of rat cerebral cortex.

To investigate the mechanism of carbon disulfide-induced neuropathy, male wistar rats were administrated by gavage at dosage of 300 or 500 mg/kg carbon disulfide, five times per week for 12 weeks. By the end of the exposure, the animals produced a slight or moderate level of neurological deficits, respectively. Cerebrums of carbon disulfide-intoxicated rats and their age-matched controls were Triton-extracted and centrifuged at a high speed (100,000 x g) to yield a pellet fraction of NF polymer and a corresponding supernatant fraction, which presumably contained mobile monomer. Then, the contents of six cytoskeletal protein (NF-L, NF-M, NF-H, alpha-tubulin, beta-tubulin, and beta-actin) in both fractions were determined by immunoblotting. Results showed that the contents of the three neurofilament subunits in the pellet and the supernatant fraction decreased significantly regardless of dose levels (P<0.01). As for microtubule proteins, in the pellet fraction of cerebrum, the levels of alpha-tubulin and beta-tubulin demonstrated some inconsistent changes. However, in the supernatant fractions, the content of alpha-tubulin and beta-tubulin increased significantly in both two dose groups (P<0.01). In comparison to neurofilament and tubulin proteins, the content of beta-actin changed less markedly, only the supernatant fraction of the high dose group displayed significant increase (P<0.01), but the others remained unaffected. These findings suggested that the changes of cytoskeleton protein contents in rat cerebrum were associated with the intoxication of carbon disulfide, which might be involved in the development of carbon disulfide neurotoxicity.

Actins↗
PubMed2005

Structure and activity of the photosystem II manganese-stabilizing protein: role of the conserved disulfide bond.

The 33-kDa manganese-stabilizing protein (MSP) of Photosystem II (PS II) maintains the functional stability of the Mn cluster in the enzyme's active site. This protein has been shown to possess characteristics similar to those of the intrinsically disordered, or natively unfolded proteins. Alternately it was proposed that MSP should be classified as a molten globule, based in part on the hypothesis that its lone disulfide bridge is necessary for structural stability and function in solution. A site-directed mutant MSP (C28A,C51A) that eliminates the disulfide bond reconstitutes O(2) evolution activity and binds to MSP-free PS II preparations at wild-type levels. This mutant was further characterized by incubation at 90 degrees C to determine the effect of loss of the disulfide bridge on MSP thermostability and solution structure. After heating at 90 degrees C for 20 min, C28A,C51A MSP was still able to bind to PS II preparations at molar stoichiometries similar to those of WT MSP and reconstitute O(2) evolution activity. A fraction of the protein aggregates upon heating, but after resolubilization, it regains the ability to bind to PS II and reconstitute O(2) evolution activity. Characterization of the solution structure of C28A,C51A MSP, using CD spectroscopy, UV absorption spectroscopy, and gel filtration chromatography, revealed that the mutant has a more disordered solution structure than WT MSP. The disulfide bond is therefore unnecessary for MSP function and the intrinsically disordered characteristics of MSP are not dependent on its presence. However, the disulfide bond does play a role in the solution structure of MSP in vivo, as evidenced by the lability of a C20S MSP mutation in Synechocystis 6803.

Amino Acid Substitution↗
PubMed1987

Selective labeling of sulfhydryls and disulfides on blot transfers using avidin-biotin technology: studies on purified proteins and erythrocyte membranes.

Various strategies for the use of 3-(N-maleimido-propionyl) biocytin (MPB) as a general label for distinguishing between protein sulfhydryls and disulfides on blot transfers are presented. In the first approach, endogenous SH groups in proteins were labeled directly with MPB. For disulfide staining, endogenous sulfhydryls were blocked with N-ethylmaleimide, disulfides were then reduced with mercaptoethanol, and the newly formed SH groups were labeled with MPB. In this approach, all treatments were performed in vitro, and, between steps, excess reagent was removed by dialysis. The MPB-labeled proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (in the presence of mercaptoethanol), the labeled proteins were transferred to nitrocellulose, and the blotted proteins were detected by avidin-biotin technology. In the second approach, MPB treatment was performed directly on blot transfers. For SH labeling, proteins were subjected to SDS-PAGE in the absence of mercaptoethanol, thus retaining the status of endogenous sulfhydryl and disulfide groups. For S-S labeling, proteins were treated with N-ethylmaleimide in vitro and then subjected to SDS-PAGE in the presence of mercaptoethanol, such that endogenous sulfhydryls were blocked and endogenous disulfides were converted to SH groups. Subsequent treatments and washings were performed on blots. In the third approach, immobilized proteins (i.e., in artificial systems or in natural systems such as membrane preparations or intact cells) were treated essentially as described in the first approach, except that washings were carried out by centrifugation. In vitro treatments were performed before SDS-PAGE (carried out in the presence of mercaptoethanol) and subsequent blot transfer. The relative merits of the three strategies are discussed.

Avidin↗