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PubMed1997

Structural change and receptor binding in a chemokine mutant with a rearranged disulfide: X-ray structure of E38C/C50AIL-8 at 2 A resolution.

The characteristic CXC chemokine disulfide core of interleukin-8 (IL-8) has been rearranged in a variant replacing the 9-50 disulfide with a 9-38 disulfide. The new variant has been characterized by its binding affinity to IL-8 receptors A and B and the erythrocyte receptor DARC. This variant binds the three receptors with affinities between 500- and 2,500-fold lower than wild-type IL-8. Binding affinity results are also reported for the variant with alanine substituted for both cysteines 9 and 50. The Glu38-->Cys/Cys50-->Ala IL-8 crystallizes in space group P2(1)2(1)2(1) with cell parameters a = 46.4, b = 49.2, and c = 69.5 A, and has been refined to an R-value of 19.4% for data from 10 to 2 A resolution. Analysis of the structure confirms the new disulfide arrangement and suggests that changes at Ile10 may be the principal cause of the lowered affinities.

Antigens, CD↗
PubMed1998

[The dynamic indices of the thiol-disulfide system and of the electrocardiogram under conditions of surgical stress].

Changes of the thiol-disulfide balance and electrocardiographic indices were studied in ischemic heart disease patients undergoing operation stress. It was shown that the operative procedure resulted in a shift of the redox state to oxidation in the thiol-disulfide system, especially in its protein part. The revealed correlation between the value of the thiol-disulfide ratio and ECG changes attest to the significance of the state of the blood thiol-disulfide system for the assessment of the degree of ischemic alterations in the myocardium.

Aged↗
PubMed1997

Human carbonic anhydrase IV: in vitro activation and purification of disulfide-bonded enzyme following expression in Escherichia coli.

Human carbonic anhydrase IV (CA IV) expressed in Escherichia coli was refolded and activated in cell extracts with the help of endogenous periplasmic protein disulfide isomerase, DsbA, in the presence of oxidized glutathione. The refolding and activation were inhibited by bacitracin but not affected by known cofactors or activators of other chaperones. Although the yield of the purified CA IV recovered from cell extracts was maximal when activated at 4 degrees C in the presence of 2 mM oxidized glutathione, the rate of refolding and activation was much more rapid at 25 and 37 degrees C. The enzyme purified from the E. coli cell extracts following activation in vitro showed similar structural stability and functional properties as CA IV purified from secretion medium from a stably transfected CHO cell line. These studies suggest that the soluble truncated form of human CA IV expressed in E. coli, which is disulfide-bonded zinc metalloenzyme, can provide a useful model enzyme for studies of protein folding and enzyme activation in vitro. Furthermore, the procedure described for recovery of CA IV following expression in E. coli may be useful for in vitro activation and subsequent purification of other disulfide-containing proteins.

Bacitracin↗
PubMed2005

Insights on a new PDI-like family: structural and functional analysis of a protein disulfide oxidoreductase from the bacterium Aquifex aeolicus.

A potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been attributed to a new family of protein disulfide isomerase (PDI)-like proteins. Members of this family are characterized by a molecular mass of about 26kDa and by two Trx folds, each comprising a CXXC active site motif. We report on the functional and structural characterization of a new member of this family, which was isolated from the thermophilic bacterium Aquifex aeolicus (AaPDO). Functional studies have revealed the high catalytic efficiency of this enzyme in reducing, oxidizing and isomerizing disulfide bridges. Site-directed mutagenesis experiments have suggested that its two active sites have similar functional properties, i.e. that each of them imparts partial activity to the enzyme. This similarity was confirmed by the analysis of the enzyme crystal structure, which points to similar geometrical parameters and solvent accessibilities for the two active sites. The results demonstrated that AaPDO is the most PDI-like of all prokaryotic proteins so far known. Thus, further experimental studies on this enzyme are likely to provide important information on the eukaryotic homologue.

Amino Acid Sequence↗
PubMed1993

Determination of the reduction-oxidation potential of the thioredoxin-like domains of protein disulfide-isomerase from the equilibrium with glutathione and thioredoxin.

Protein disulfide-isomerase (PDI) contains two thioredoxin-like domains with the active-site sequence: Cys-Gly-His-Cys. Reduction of the two active-site disulfides in PDI by NADPH and bovine thioredoxin reductase was not reversible by addition of excess NADP+, consistent with a redox potential (E0') above -200 mV. Redox states of PDI and a mutated Escherichia coli thioredoxin, P34H Trx, were determined by quantitative analysis of cysteine residues by alkylation in equilibrium mixtures of oxidized and reduced forms of the two proteins. From the known E0' of P34H Trx (-235 mV), an E0' value of -190 +/- 10 mV was calculated for PDI. Similarly, with defined redox buffers of glutathione, the redox-active dithiols in PDI were shown to have an equilibrium constant of 3 mM (E0' = -175 +/- 15 mV). The results showed that PDI has a high redox potential and therefore is a good oxidant of nascent protein thiols. Direct transfer of reducing equivalents from PDI to NADP+ via thioredoxin reductase during protein disulfide formation seems unlikely due to the unfavorable equilibrium. The thioredoxin domains in PDI have a widely different redox potential compared with that of thioredoxin. A Pro to His exchange in the active site contributes to half of the change; the other half remains to be identified in the structure of PDI.

Amino Acid Sequence↗
PubMed1992

Early disulfide bond formation prevents heterotypic aggregation of membrane proteins in a cell-free translation system.

We previously demonstrated that a heterotypic complex of the two rat asialoglycoprotein receptor subunits was assembled during cell-free translation (Sawyer, J. T., and D. Doyle. 1990. Proc. Natl. Acad. Sci. USA. 87:4854-4858). We have characterized this system further by analyzing polypeptide interactions under both reducing and oxidizing translation conditions. This report shows that the complex represents a heterogeneous interaction between reduced membrane proteins rather than a specific oligomeric structure. In the reduced state membrane proteins interact in this system to form aggregates of diverse size and composition. The aggregated nascent polypeptides interact with the immunoglobulin heavy chain binding protein but this protein is not an integral component of the aggregate. Aggregation occurs via the exoplasmic domain, rather than the transmembrane domain, and the folding of this domain by the formation of intramolecular disulfides, prevents the interaction from occurring. Additionally, the folded molecules containing intramolecular disulfides lack high affinity binding activity and thus appear to resemble the earliest folding intermediates seen in vivo (Olson, J. T., and M. D. Lane. 198. FASEB (Fed. Am. Soc. Exp. Biol.) J. 3:1618-1624). These results lead us to suggest that the formation of intramolecular disulfides during early biogenesis serves to prevent nonspecific associations between nascent polypeptides.

Animals↗
PubMedDate not supplied

[Enzymes of thiol-disulfide metabolism of proteins].

Occurrence, properties and physiological role of protein disulfide reductases (EC 1.6.4.4 and 1.8.4.2), protein disulfide isomerase (EC 5.3.4.1), and thiol oxidase (EC 1.8.3.2) catalyzing thiol-disulfide interchange reactions in proteins are reviewed with a particular emphasis on seed storage proteins. An important role of the enzymes in the formation and degradation of seed storage protein complexes is discussed.

Animals↗
PubMed1984

Quantitative determination of captopril in blood and captopril and its disulfide metabolites in plasma by gas chromatography.

A sensitive, quantitative gas chromatographic-electron capture (GC-EC) method for the determination of captopril in blood and captopril and its disulfide metabolites (collectively) in plasma was developed. After addition of an internal standard and N-ethylmaleimide to the biological samples, excess N-ethylmaleimide and naturally occurring interfering substances were removed by extraction with benzene followed by acidification and extraction with hexane. The N-ethylmaleimide adducts of captopril and of the internal standard were then extracted with benzene and converted to their hexafluoroisopropyl esters. For the assay of captopril and its disulfide metabolites, tributylphosphine was used to reduce the disulfide metabolites to captopril prior to derivatization. The hexafluoroisopropyl esters of the N-ethylmaleimide adducts of captopril and of the internal standard, the 4-ethoxyproline analogue of captopril, were separated by GC on a column packed with 3% OV-101 on Chromosorb W-HP. The lower limits of sensitivity were 20 ng/mL for captopril in blood and 50 ng/mL for captopril and its disulfide metabolites in plasma. Linearity, precision, and accuracy were excellent. The method was validated by comparison of results obtained for total captopril in dog plasma by the GC-EC assay with results obtained by a published GC-MS method. The assay was applied to dog and human samples to explore its general utility.

Administration, Oral↗
PubMed1998

An allosteric drug, o,o'-bismyristoyl thiamine disulfide, suppresses HIV-1 replication through prevention of nuclear translocation of both HIV-1 Tat and NF-kappa B.

The efficacy of o,o'-bismyristoyl thiamine disulfide (BMT) was examined in detail against HIV-1 laboratory isolates (HTLV-IIIB, JRFL, and MN), primary isolates (KMT and KMO), and simian immunodeficiency virus (SIVmac251) in vitro. BMT inhibited the replication of HIV-1 in both laboratory and primary isolates in vitro. In addition, BMT exhibited antiviral activity against SIVmac251. Minimizing energy studies of BMT structure reveal that a trans-disulfide of thiamine (holo drug) disulfide (TDS, protodrug) is allosterically transited to the reactive twisted disulfide of BMT (allo drug) by o,o'-bismyristoyl esterification of TDS. BMT inhibits nuclear translocation of both HIV-1 transactivator (TAT) and the cellular transcriptional nuclear factor-KB (NF-kappa B), resulting in the suppression of HIV-1 replication.

Allosteric Site↗
PubMed1987

The pharmacokinetics of captopril and captopril disulfide conjugates in uraemic patients on maintenance dialysis: comparison with patients with normal renal function.

We have measured the plasma concentrations of captopril and total disulfide conjugates of captopril after a 50 mg oral dose in 6 uraemic patients on maintenance dialysis and in 8 hypertensive subjects with normal renal function. The mean peak plasma concentration of captopril was 2.5 times higher (0.447 micrograms X ml-1 vs 0.181 micrograms X ml-1) and the concentrations of the disulfides 4 times higher (3.62 micrograms X ml-1 vs 0.924 micrograms X ml-1) in the uraemic patients. Moreover captopril disulfide conjugates in the uraemic subjects reached peak concentrations at 8 h after the dose and subsequently felt. The apparent plasma half-time was 46 +/- 19 h. Only 15% of these conjugates were removed by dialysis. This marked accumulation of captopril conjugates was associated with a sustained fall in both systolic and diastolic blood pressures. In uraemic patients the mean maximum reduction in systolic and diastolic blood pressures were 37 +/- 7 mmHg and 24 +/- 9 mmHg respectively, occurring 6 h after the dose, compared with 8 +/- 7 and 8 +/- 1 mmHg respectively at 30 min in normal renal function patients. These results are consistent with the results of animal experiments, which show that captopril disulfides can be converted back to free captopril and can contribute to the antihypertensive effect of the drug. They provide a reationale for reducing the dose and frequency of administration of captopril in patients with significant renal impairment.

Adult↗
PubMed1991

Simultaneous detection of thiol- and disulfide-containing peptides by electrochemical high-performance liquid chromatography with identification by mass spectrometry.

A new HPLC electrochemical detector that can be used to detect selectively and simultaneously both thiol- and disulfide-containing peptides is described. One electrode responds only to thiol-containing peptides, while a second electrode located downstream from a third electrode responds to thiol- as well as disulfide-containing peptides. All three electrodes are located in a single block and sample the HPLC effluent simultaneously. Since the electrochemical detector responds only to peptides that contain thiol or disulfide functionalities, it is ideal for detecting these types of peptides when they are present in complex mixtures. Peptides can be identified by their retention times if reference peptides are available, or by their molecular weights if the appropriate chromatographic fractions are collected and analyzed by mass spectrometry. The utility of the three-electrode electrochemical detector for detecting thiol- and disulfide-containing peptides in a single chromatographic analysis is illustrated with proteolytic digests of bovine alpha A-crystallin and partially reduced bovine insulin.

Amino Acid Sequence↗
PubMed1986

Analysis of the internal nuclear matrix. Oligomers of a 38 kD nucleolar polypeptide stabilized by disulfide bonds.

When rat liver nuclei are treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) prior to nuclease treatment and extraction with 1.6 M NaCl, residual nucleoli and an extensive non-chromatin intranuclear network remain associated with the nuclear envelope. Subsequent treatment of this structure with 1 M NaCl containing 20 mM dithiothreitol (DTT) solubilizes the intranuclear material, while the nuclear envelope remains structurally intact. We have isolated and partially characterized a major polypeptide of the disulfide-stabilized internal nuclear matrix. The polypeptide, which has an apparent molecular mass 38 kD and isoelectric point 5.3, has been localized to the nucleolus of rat liver nuclei by indirect immunofluorescence using a specific polyclonal chicken antiserum. Based on its molecular mass, isoelectric point, intracellular localization and amino acid composition, the 38 kD polypeptide appears to be analogous to the nucleolar phosphoprotein B23 described by Prestayko et al. (Biochemistry 13 (1974) 1945) [20]. Immunologically related polypeptides have likewise been localized to the nucleoli of both hamster and human tissue culture cell lines as well as the cellular slime mold Physarum polycephalum. By immunoblotting, a single 38 kD polypeptide is recognized by the antiserum in rat, mouse, hamster and human cell lines. The antiserum has been utilized to investigate the oligomeric structure of the 38 kD polypeptide and the nature of its association with the rat liver nuclear matrix. By introducing varying numbers of disulfide bonds, we have found that the 38 kD polypeptide becomes incorporated into the internal nuclear matrix in a two-step process. Soluble disulfide-bonded homodimers of the polypeptide are first formed and then are rendered salt-insoluble by more extensive disulfide cross-linking.

Animals↗
PubMed2006

The effects by neuroleptics, antimycotics and antibiotics on disulfide reducing enzymes from the human pathogens Acanthamoeba polyphaga and Naegleria fowleri.

This paper discusses the effects of two neuroleptic agents, chlorpromazine and trifluoperazine; three antimycotics, amphotericin B, ketoconazole and miconazole and four antibiotics, pentamidine, rifampicin, mepacrine and metronidazole on the NADPH-dependent disulfide reducing enzymes cystine reductase (CysR), glutathione reductase (GR) trypanothione reductase (TR) and a putative disulfide reductase for compound X in Acanthamoeba polyphaga from the human pathogens A. polyphaga and Naegleria fowleri. Against A. polyphaga, all nine drugs studied had the capacity to inhibit the putative disulfide reductase from the trophozoites at a concentration of 32microg/ml during a 24h incubation and they were: the neuroleptics trifluoperazine (100%) and chlorpromazine (96%), the antimycotics miconazole (89%) ketoconazole (81%) and amphotericin B, (53%) and the antibiotics pentamidine (89%), rifampicin (64%), mepacrine (57%) and metronidazole (14%). Only six of the nine drugs simultaneously inhibited CysR, GR and the putative disulfide reductase. In N. fowleri, the most potent inhibitors of trypanothione reductase were amphotericin B and miconazole which inhibited 100% at a concentration of 32microg/ml during the 24h incubation followed by the neuroleptics trifluoperazine (92%) and chlorpromazine (80%) and the antibiotic mepacrine (70%). All these also inhibited CysR and GR from the trophozoites other than mepacrine which inhibited only CysR and TR. Ketoconazole, rifampicin (which did not affect CysR), pentamidine and metronidazole had opposite effects since they did not inhibit but increased the amount of the three thiols.

Acanthamoeba↗
PubMed1997

Maurotoxin, a four disulfide bridge toxin from Scorpio maurus venom: purification, structure and action on potassium channels.

A new toxin acting on K+ channels, maurotoxin (MTX), has been purified to homogeneity from the venom of the chactoid scorpion Scorpio maurus. MTX is a basic single chain 34 amino acid residue polypeptide, amidated at its C terminal, and crosslinked by four disulfide bridges. It shows 29-68% sequence identity with other K+ channel toxins, and presents an original disulfide pattern, the last two half-cystine residues (31-34) being connected. Although the first three disulfide bonds have not been defined experimentally, modelling based on the structure of charybdotoxin favored two combinations out of six, one of which has two bridges (3-24 and 9-29) in common with the general motif of scorpion toxins. The last bridge would connect residues 13 and 19. MTX inhibits the binding to rat brain synaptosomal membranes of both [125I]apamin, a SK(Ca) channel blocker (IC50 5 nM), and [125I]kaliotoxin, a Kv channel blocker (IC50 30 pM). MTX blocks the Kv1.1, Kv1.2 and Kv1.3 currents expressed in Xenopus oocytes with IC50 of 45, 0.8 and 180 nM, respectively. MTX represents a member of a new class of short toxins with 4 disulfide bridges, active on voltage-dependent K+ channel and also competing with apamin for binding to its receptor.

Amino Acid Sequence↗
PubMed1998

Sulfitolysis and thioredoxin-dependent reduction reveal the presence of a structural disulfide bridge in spinach chloroplast fructose-1,6-bisphosphatase.

A significant difference between cytosolic and chloroplastic fructose-1,6-bisphosphatase (FbPase) is an extra peptide in the middle of chloroplast FbPase which contains three additional cysteine residues. Sit-directed mutagenesis experiments have shown that at least two of these cysteine residues are involved in forming the regulatory disulfide bridge [Jacquot, J.-P. et al., FEBS Lett. 401 (1997) 143-147] which is the presupposition for the thioredoxin-dependent control of chloroplast FbPase activity. Here we report that each subunit of the FbPase contains an additional structural disulfide bridge which has been observed by combined application of thioredoxins and sulfitolysis. Observation of the structural disulfide bridges by sulfitolysis was only possible when the FbPase was already specifically reduced by the homologous thioredoxin species TRm. and TRf from spinach chloroplasts. Interestingly, the accessibility of the structural disulfide bridge for sulfite ions depends on the thioredoxin species engaged in the thioredoxin/FbPase complex.

Chloroplasts↗
PubMed1998

The functional properties of DsbG, a thiol-disulfide oxidoreductase from the periplasm of Escherichia coli.

Genetic studies have recently identified DsbG, a new member of the dsb group of redox proteins, which catalyze protein disulfide bond formation in the periplasm of Escherichia coli. We now demonstrate that DsbG functions primarily as an oxidant during protein disulfide bond formation, which is consistent with the low stability of its active site disulfide bond. There are indications, however, that the substrate range of DsbG may be narrower than the other periplasmic oxidative enzymes, DsbA and DsbC. Our observations further elaborate the pathway of disulfide bond formation in E. coli.

Aprotinin↗
PubMed2003

Direct electrochemical oxidation of disulfides at anodically pretreated boron-doped diamond electrodes.

Anodically oxidized diamond electrodes have been used to oxidize disulfides, thiols, and methionine in aqueous acidic media and tested for amperometric detection of these compounds after chromatographic separation. Cyclic voltammetric signals for 1 mM glutathione disulfide (GSSG) were observed at 1.39 and 1.84 V vs SCE, the values being less positive than those of its as-deposited counterpart as well as glassy carbon electrode. The voltammetric and chronocoulometric results have indicated the high stability of the electrode with negligible adsorption. A positive shift in the peak potential with increasing pH indicated the attractive electrostatic interaction between the anodically oxidized diamond surface and the positively charged GSSG in acidic media that promoted its analytical performance. The results of the electrolysis experiments of disulfides and thiols showed that the oxidation reaction mechanism of glutathione (GSH) and GSSG involves oxygen transfer. Following separation by liquid chromatography (LC), the determination of both GSH and GSSG in rat whole blood was achieved at a constant potential (1.50 V vs Ag/AgCl), and the limits of detection for GSH and GSSG were found to be 1.4 nM (0.028 pmol) and 1.9 nM (0.037 pmol) with a linear calibration range up to 0.25 mM. These detection limits were much lower than those reported for the amperometry using Bi-PbO2 electrodes and LC-mass spectrometry, and the LC method using diamond electrodes were comparable with enzymatic assay in real sample analysis. The high response stability and reproducibility together with the possibility of regeneration of the electrode surface by on-line anodic treatment at 3 V for 30 min further support the applicability of anodically pretreated diamond for amperometric detection of disulfides.

Journal Article↗
PubMed1986

An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of lambda repressor.

Site-directed mutagenesis has been used to replace Tyr-88 at the dimer interface of the N-terminal domain of lambda repressor with cysteine. Computer model building had suggested that this substitution would allow formation of an intersubunit disulfide without disruption of the dimer structure [Pabo, C. O., & Suchanek, E. G. (1986) Biochemistry (preceding paper in this issue)]. We find that the Cys-88 protein forms a disulfide-bonded dimer that is very stable to reduction by dithiothreitol and has increased operator DNA binding activity. The covalent Cys88-Cys88' dimer is also considerably more stable than the wild-type protein to thermal denaturation or urea denaturation. As a control, Tyr-85 was replaced with cysteine. A Cys85-Cys85' disulfide cannot form without disrupting the wild-type structure, and we find that this disulfide bond reduces the DNA binding activity and stability of the N-terminal domain.

Amino Acid Sequence↗