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PubMed1993

Assignment of the three disulfide bridges of huwentoxin-I, a neurotoxin from the spider selenocosmia huwena.

The positions of the disulfide bonds of huwentoxin-I, a neurotoxin from the spider Selenocosmia huwena, have been determined. The existence of three disulfide bonds in the native toxin was demonstrated by mass spectroscopy and the lack of reactivity with a thiol reagent. The assignment procedure involved a combination of tryptic digestion of the native toxin and sequence analysis of both intact and in situ S-carboxymethylated toxin. In situ carboxymethylation is shown to be a useful procedure in sequencing of cysteine- and cystine-containing peptides. Sequence analysis of the intact, cross-linked toxin indicated that no amino acid phenylthiohydantoin (PTH) derivative is seen for the first half-cystine in a cross-linked pair, but that the PTH of dehydroalanine, which can be detected at 313 nm, is seen at the position of the second half-cystine. By sequencing disulfide cross-linked tryptic fragments, the three disulfide linkages in huwentoxin-I could be assigned as Cys2-Cys17, Cys9-Cys22, and Cys16-Cys29.

Amino Acid Sequence↗

PubMed1977

A unique property of fetal bovine serum: high levels of protein-glutathione mixed disulfides.

Fetal bovine serum has been reported to delay or inhibit "spontaneous" neoplastic transformation in vitro as compared with all other sera tested. The present results indicate that fetal bovine serum is also unique in containing high levels of protein-glutathione mixed disulfides (3 to 7 microng glutathione as mixed disulfide per ml serum). The level of mixed disulfide appears to vary in accordance with the period of gestation of the fetal calves used to prepare the serum, decreasing below detectable levels (less than 0.2 microng per ml) with near-term fetal calves. Calf, adult bovine, fetal horse, and swine sera did not contain detectable levels of this type of mixed disulfide.

Animals↗

PubMed1995

Age-dependent effects of t-BuOOH on glutathione disulfide reductase, glutathione peroxidase, and malondialdehyde in the brain.

Intracerebroventricular t-butyl hydroperoxide has been reported to induce damage to many types of brain cells. t-Butyl hydroperoxide administration increases glutathione disulfide levels and decreases levels of glutathione. Young adult mice may be more protected from t-butyl hydroperoxide than mature mice due to their higher glutathione levels, even after the administration of t-butyl hydroperoxide. This leads to our current study, investigating glutathione peroxidase and glutathione disulfide reductase in 2-mo-old and 8-mo-old mice. Furthermore, malondialdehyde levels were measured with the thiobarbituric acid assay and compared between the two age groups. Mature mice detoxify glutathione disulfide less readily than young adult mice. Glutathione disulfide reductase activity increases in young adult mice after t-butyl hydroperoxide administration, but not in mature mice. Glutathione peroxidase activity is significantly lower in 8-mo-old than 2-mo-old mouse striatum after t-butyl hydroperoxide administration. Furthermore, malondialdehyde levels in the 8-mo-old striatum increase significantly 20 min after t-butyl hydroperoxide administration. This suggests that age plays a factor in protective mechanisms that are involved in oxidative stress in the brain.

Aging↗

PubMed2002

Role of protein disulfide isomerase in molecular fate of thyroglobulin and its regulation by endogenous oxidants and reductants.

The molecular fate of thyroglobulin (Tg) is controlled by oligomerization, a means of storing Tg at high concentrations, and deoligomerization. The oligomerization of bovine Tg are intermolecular reactions that occur through oxidative processes, such as disulfide and dityrosine formation, as well as isopeptide formation; disulfide formation is primarily responsible for Tg oligomerization. Here, the protein disulfide isomerase (PDI) and/or peroxidase-induced oligomerization of unfolded thyroglobulins, which were prepared by treating bovine Tg with heat, urea or thiol/urea, was investigated using SDS-PAGE analyses. In addition, the enzymatic oligomerization was compared with non-enzymatic oligomerization. The thermally-induced oilgomerization of Tg, dependent on glutathione redox state, was affected by the ionic strength or the presence of a surfactant. Meanwhile, PDI-catalyzed oligomerization, time and pH-dependent, was the most remarkable with unfolded/reduced Tg, which was prepared from a treatment with urea/DTT, while the thermally-unfolded Tg was less sensitive. Similarly, the oligomerization of unfolded/reduced Tg was also mediated by peroxidase. However, PDI showed no remarkable effect on the peroxidase-mediated oligomerization of either the unfolded or unfolded/reduced Tg. Additionally, the reductive deoligomerization of oligomeric Tg was exerted by PDI in an excessively reducing state. Based on these results, it is proposed that PDI catalyzes the oligomerization of Tg through the disulfide linkage and its deoligomerization in the molecular fate, and this process may require a specific molecular form of Tg, optimally unfolded/reduced, in a proper redox state.

Animals↗

PubMed1997

Analysis of intermolecular disulfide bonds and free sulfhydryl groups in hepatitis B surface antigen particles.

The envelope proteins of hepadnaviruses are highly cross-linked by disulfide bonds in complete virions and 20 nm subviral envelope particles. We have previously shown which of the cysteines in the envelope proteins of the human hepatitis B virus (HBV) are essential for assembly and secretion of 20 nm particles and for the structure of the major antigenic determinants (HBsAg). Now we have analyzed the intermolecular disulfide bonds between S proteins. We have constructed mutants lacking cysteines and have analyzed their capacity for oligomerization in COS-7 cells. We demonstrate that C121 and C147 located in the second hydrophilic region carrying the major antigenic determinants of the HBV S protein participate in intermolecular disulfide bonding. A disulfide bond involving C124 blocks the accessibility of arginine/lysine at position 122, as shown by trypsin digestion of cysteine mutants. Alkylation studies using N-ethyl-maleimide indicate that C76, C90, and/or C221 carry the only free sulfhydryl group(s) present in 20 nm particles secreted from cell lines.

Animals↗

PubMed2006

Direct evidence that two cysteines in the dopamine transporter form a disulfide bond.

We have generated a fully functional dopamine transporter (DAT) mutant (dmDATx7) with all cysteines removed except the two cysteines in extracellular loop 2 (EL2). Random mutagenesis at either or both EL2 cysteines did not produce any functional transporter mutants, suggesting that the two cysteines cannot be replaced by any other amino acids. The cysteine-specific reagent MTSEA-biotin labeled dmDATx7 only after a DTT treatment which reduces disulfide bond. Since there are no other cysteines in dmDATx7, the MTSEA-biotin labeling must be on the EL2 cysteines made available by the DTT treatment. This result provides the first direct evidence that the EL2 cysteines form a disulfide bond. Interestingly, the DTT treatment had little effect on transport activity suggesting that the disulfide bond is not necessary for the uptake function of DAT. Our results and previous results are consistent with the notion that the disulfide bond between EL2 cysteines is required for DAT biosynthesis and/or its delivery to the cell surface.

Amino Acid Sequence↗

PubMed2005

Human papillomavirus 16 minor capsid protein L2 helps capsomeres assemble independently of intercapsomeric disulfide bonding.

The human papillomavirus (HPV) capsomeres (pentamers of major capsid protein L1), which constitute along with L2 the virion capsid, can assemble themselves alone into the L1-capsid particles in vivo and in vitro, depending on intercapsomeric disulfide bonds. To study a possible role of L2 in capsid assembly, we examined the interaction between HPV16 L2 and capsomeres under the conditions that inhibit the formation of disulfide bonds in vitro and in vivo. The purified L2 bound to free capsomeres prepared by disassembling L1-capsids but not to the L1-capsids in vitro. And the L2 was found to help capsomeres assemble into smaller capsid-like particles independently of intercapsomeric disulfide bonding. Similar particles were obtained from the Sf9 cells co-infected with baculoviruses expressing L2 and an L1 mutant that lacks a C-terminal cysteine (C428S) and can form capsomeres but no capsids when expressed alone. These findings suggest that L2, which is known to bind both viral DNA and L1, may contribute to the formation of the virion by linking viral DNA and capsomeres and by helping capsomeres assemble before the virion capsid structure is completed by dintercapsomeric disulfide bonding.

Animals↗

PubMed1988

Identification of disulfide-containing peptides by performic acid oxidation and mass spectrometry.

In addition to reducing the analysis time, the direct examination of proteolytic digests by fast atom bombardment mass spectrometry (FABMS) greatly extends the information that is available from peptide mapping experiments. Mass spectral data are particularly useful for identifying post-translationally modified peptides. For example, the molecular weight of a disulfide-containing peptide may be used to locate the disulfide bond in the protein from which the peptide was derived. This paper describes a new procedure, which is useful for identifying disulfide-bonded peptides. Peptides are treated with performic acid to modify certain residues and thereby cause a characteristic change in the peptide molecular weight. This change in molecular weight is determined by FABMS and used to help identify peptides. Results for a series of small peptides demonstrate that Cys, Met, and Trp are the only residues that undergo a change in molecular weight under the conditions used here. Furthermore, these changes in molecular weight are diagnostic for each of the residues. Cysteinyl-containing peptides are of particular interest, because their identification is essential for locating disulfide bonds. The molecular weight of a peptide increases by 48 mu for each cysteinyl residue present. This approach is used to identify peptides that contain both cysteinyl and cystinyl residues in the peptic digest of bovine insulin. The method is extended to the analysis of a tryptic digest of cyanogen bromide-treated ribonuclease A. A computer-assisted analysis procedure is used to demonstrate the specificity with which peptide molecular weight is related to specific segments of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, High Pressure Liquid↗

PubMed1988

Measurement of biological disulfides by postcolumn sulfitolysis following separation by HPLC.

We developed a method to measure disulfides which is applicable to biological fluids. It consisted of two parts. First, certain thiols and disulfides were separated by HPLC. Second, the eluted materials were submitted to postcolumn reaction with 2-nitro-5-thiosulfobenzoate in the presence of sulfite. The resultant yellow product, 2-nitro-5-thiobenzoate, was measured by its absorbance at 412 nm. We determined the elution characteristics of the thiols and disulfides derived from cysteine, glutathione, alpha-mercaptopropionylglycine (Thiola), and cysteamine. Penicillamine and its disulfide did not react. Cystine in the urine of 22 cystinuric patients, measured by this method, was compared with results obtained by automatic amino acid analysis.

Chromatography, High Pressure Liquid↗

PubMed1987

Importance of disulfide linkage for constructing the biologically active human interleukin-2.

Recombinant human interleukin-2 (rIL-2) produced in Escherichia coli possesses a free thiol group at Cys-125 and a disulfide linkage between Cys-58 and Cys-105, as in the case for natural human interleukin-2. Treatment of rIL-2 with 200 mM dithiothreitol resulted in the cleavage of the Cys-58-Cys-105 disulfide bond. The reduced form of rIL-2 thus obtained retained only 10% of the in vitro biological activity of the native form, as measured by the ability to stimulate the growth of an IL-2-dependent mouse natural killer cell line, NKC3. Far-uv circular dichroism studies indicated that the cleavage of the disulfide bond results in a decrease of alpha-helix content. Near-uv circular dichroism studies suggested that the native molecule is folded into a rigid tertiary structure, while the reduced form showed a spectrum similar to that of rIL-2 denatured in the presence of 6 M guanidine.HCl. The once-reduced molecule was readily reoxidized in the presence of 10 microM Cu2+ to form the native molecule with full biological activity. These results strongly demonstrate that the Cys-58-Cys-105 disulfide linkage in the IL-2 molecule is essential for constructing a rigid and biologically active form of IL-2.

Amino Acids↗

PubMed1980

Sulfhydryl groups in photosynthetic energy conservation. VI. Subunit distribution of sulfhydryl groups and disulfide bonds in chloroplast coupling factor and ATPase activity.

The subunit distribution of sulfhydryl groups and disulfide bonds of spinach chloroplasts coupling factor I has been determined. Native coupling factor I with a latent ATPase activity has eight sulfhydryl groups distributed 4 : 2 : 0 : 0 : 2 in the alpha, beta, gamma, delta and epsilon subunits, respectively. Heat treatment of coupling factor I, in addition to the activation of its ATPase activity, induces a dithiol-disulfide interchange between the gamma and the alpha subunit, changing the sulfhydryl groups' distribution to 2 : 2 : 2 : 0 : 2. Reduction of disulfide bonds of coupling factor I by dithioerythritol during heat treatment gives a subunit distribution of 4 : 4 : 4 : 0 : 2, suggesting that native coupling factor I has three disulfide bonds, two in the gamma subunit and one in one of the beta subunits. The results suggest an asymmetric redox state of some of the subunits of coupling factor I and an asymmetric positioning of some of them in the molecular structure of coupling factor I.

Adenosine Triphosphatases↗

PubMed1989

Formation of an intramolecular disulfide bond of glycolipid transfer protein.

Functional and structural differences between the two forms of glycolipid transfer protein (GLTP) with (the faster component) and without (the slower component) an intramolecular disulfide bond were studied. GLTP treated with N-ethylmaleimide (NEM) and Na2S4O6 had a transfer activity of about 70% and 55%, respectively, of the control GLTP. No significant decrease was found in the binding affinity of NEM-treated GLTP to pyrene-labeled galactosylceramide (PyrGalCer). A small decrease in the binding affinity was found in the Na2S4O6-treated GLTP. Oxidation of NEM-treated and Na2S4O6-treated GLTP catalyzed by CuSO4 resulted in a stoichiometric conversion of the slower component to the faster component. The faster component thus formed was quantitatively reduced back to the slower component by treatment with 2-mercaptoethanol in the presence of 1% SDS. These results provided strong evidence for the conversion of the slower component to the faster component as a result of the formation of an intramolecular disulfide bond. The transfer activity of the NEM-treated and oxidized GLTP (the faster component) was 1.7-fold higher than that of the original GLTP and 2.4-fold higher than that of the NEM-treated GLTP. The transfer activity of the Na2S4O6-treated and oxidized GLTP was 2-fold higher than that of the original GLTP and 3.6-fold higher than that of the Na2S4O6-treated GLTP. The binding affinity of the faster components, produced from both the NEM-treated GLTP and the Na2S4O6-treated GLTP, to PyrGalCer was found to be twice that of the respective modified GLTPs before oxidation. By circular dichroism measurements, it was found that a small decrease in the magnitude of mean residue ellipticity but no significant change in ellipticity spectrum was brought about either upon the modification of GLTP by Na2S4O6 or upon the formation of an intramolecular disulfide bond in GLTP. The results suggest that the formation of an intramolecular disulfide bond results in only a small change in the secondary and tertiary structure of GLTP. The results presented in this paper suggest that the transfer activity of GLTP may be regulated by the redox state of sulfhydryl groups present in GLTP.

Animals↗

PubMed1987

Interchange reaction of disulfides and denaturation of oxytocin by copper(II)/ascorbic acid/O2 system.

The interchange reaction of disulfides was caused by the copper(II)/ascorbic acid/O2 system. The incubation of two symmetric disulfides, L-cystinyl-bis-L-phenylalanine (PP) and L-cystinyl-bis-L-tyrosine (TT), with L-ascorbic acid and CuSO4 in potassium phosphate buffer (pH 7.2, 50 mM) resulted in the formation of an asymmetric disulfide, L-cystinyl-L-phenylalanine-L-tyrosine (PT), and the final ratio of PP:PT:TT was 1:2:1. As the reaction was inhibited by catalase and DMSO only at the initial time, hydroxyl radical generated by the copper(II)/ascorbic acid/O2 system seemed to be responsible for the initiation of the reaction. Oxytocin and insulin were denatured by this system, and catalase and DMSO similarly inhibited these denaturations. As the composition of amino acids was unchanged after the reaction, hydroxyl radical was thought to cause the cleavage and/or interchange reaction of disulfides to denature the peptides.

Amino Acids↗

PubMed1989

Calcium regulates folding and disulfide-bond formation in alpha-lactalbumin.

Refolding and disulfide bond formation in reduced denatured bovine alpha-lactalbumin is shown to be Ca2+-dependent. Whereas in the absence of Ca2+ only about 2% of the native active protein is regenerated, in the presence of Ca2+, almost quantitative renaturation is obtained. A close coupling between Ca2+-binding and native disulfide bond formation is also indicated by spontaneous disulfide scrambling in the apoprotein in the presence of low concentrations of thiols. This phenomenon is not found in other disulfide-containing proteins including the homologous chicken lysozyme. It is proposed that the alpha-lactalbumin Ca2+-binding site has the in vivo function of imposing Ca2+ regulation on the folding of nascent alpha-lactalbumin and thereby on lactose synthesis.

Animals↗

PubMed1989

Modification of protein stability by introduction of disulfide bridges and prolines: geometric criteria for mutation sites.

We define geometrical parameters to characterize disulfide bridges using x-ray crystal structure data on small molecules and use them to suggest replacements of amino acids by cysteines in order to introduce disulfide bridges to increase thermal stability in proteins. We also define geometric parameters to identify target amino acids for replacements by prolines in order to conserve desired structural attributes in the vicinity of disulfide mutations leading to further structural and thermal stability of proteins. The geometric criteria are applied to the serine protease, subtilisin, to model stereochemically favorable disulfide mutants without altering the active site geometry, implying conservation of native biological activity.

Binding Sites↗

PubMed1986

Effect of disulfide-bond reducing agents on the specific binding of growth hormone to microsomal membrane preparations from rabbit liver.

The effect of the disulfide-bond reducing agents, mercaptoethanol (ME) and dithiothreitol (DTT), on the specific binding of 125I-labeled human growth hormone (hGH) to microsomal membrane preparations from rabbit liver was investigated. The presence of ME or DTT caused a time- and dose-related inhibition of [125I]hGH binding to both particulate and solubilized somatotrophic receptors of rabbit liver membranes. Maximum inhibition was 20-30%. Disulfide bond reduction also caused a marked increase in the extent of reversibility of [125I]hGH binding. These effects were not due to effects on the GH, itself, but appeared to be directed at the receptor. Scatchard analysis showed that ME and DTT caused a change in the nature of the binding interaction, with at least partial conversion of receptors into sites with reduced affinity. These data together suggest that the partial effect of reducing agents on somatotrophic receptors of rabbit liver may reflect two types of receptors within the microsomal membrane preparations--one (approximately 30% of total receptors) involving disulfide bonding and the remaining type (70%) being independent of disulfide bonds--or, alternatively, that the state of reduction affects a single class of receptors in a rather more complex manner. These studies provide further intriguing insights into the overall mechanism(s) involved in the interaction of GH with its target cell receptors.

Animals↗

PubMed1991

Inhibition of muscarinic cholinergic receptors by disulfide reducing agents and arsenicals. Differential effect on locust and rat.

Muscarinic receptors are altered by sulfhydryl reagents. Arsenic compounds, which have been used as insecticides, exert their toxic effects by combining with sulfhydryl groups. We compared the action of arsenicals and other sulfhydryl reagents on the muscarinic receptor from invertebrate and vertebrate species (locust and rat). Disulfide-reducing reagents dithiothreitol (DTT) and British Anti-Lewisite (BAL), but not arsenicals, inhibited [3H]quinuclidinyl benzilate ([3H]QNB) binding. However, after disulfide reduction, arsenicals caused a further inhibition of muscarinic binding. The effect of DTT + arsenicals was largely irreversible. The locust receptors were more sensitive to the action of both disulfide reagents either in the absence or presence of arsenicals than the rat receptors. The sulfhydryl reagent p-chloromercuric benzoate (PCMB) was more effective at inhibiting the locust receptors than the rat receptors, but addition of arsenicals did not cause further inhibition in either the locust or rat receptors. In locust, DTT + cacodylate and DTT + arsenite caused a reduction in the number of sites without modifying the affinity of [3H]QNB binding. In rat, DTT + arsenite caused a decrease in the affinity, while DTT + cacodylate caused a decrease in the affinity of [3H]QNB binding and its number of sites. Competition experiments after DTT + cacodylate showed that the IC50 and the Hill coefficient (nH) remained unchanged in the locust. In the rat, the IC50 for atropine was increased without alteration in the nH, and both parameters were increased for carbachol. These results are explained assuming that the binding site of the locust receptor has a disulfide group similar to that of the mammalian receptor, but that the hydrophobic interactions within the binding site are weaker in the locust receptor. The higher sensitivity of the insect receptor to sulfhydryl reagents could be of interest for developing methods of pest control.

Animals↗

PubMed1991

Association of topoisomerase II with the hepatoma cell nuclear matrix: the role of intermolecular disulfide bond formation.

Previous studies have resulted in conflicting data regarding the recovery of the nuclear enzymes topoisomerase (topo) II and topo I in the nuclear matrix fraction. In the present study we have assessed the effect of systematically altering a single extraction procedure on the distribution of these enzymes during the subfractionation of nuclei from HTC hepatoma tissue culture cells. When nuclear monolayers (prepared by treating attached cells in situ with the neutral detergent Nonidet-P40 at 4 degrees C) were isolated in the presence of the irreversible sulfhydryl blocking reagent iodoacetamide, subsequent treatment with DNase I and RNase A followed by 1.6 M NaCl resulted in structures which were extensively depleted of intranuclear components as assessed by phase contrast microscopy and conventional transmission electron microscopy. These structures contained 12 +/- 4% of the total protein present in the original nuclear monolayers. The lamins and polypeptides with molecular weights comparable to those of actin and vimentin were the predominant polypeptides present on SDS-polyacrylamide gels. Western blotting revealed that less than 5% of the total nuclear topo II molecules were present in these structures. In contrast, when the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) was substituted for iodoacetamide, the same extraction procedure yielded structures containing components of the nucleolus and an extensive intranuclear network. These structures contained a wide variety of nonlamin, nonhistone nuclear polypeptides including 23 +/- 4% of the total nuclear topo II. SDS-polyacrylamide gel electrophoresis performed under nonreducing conditions revealed that topo II in these nuclear matrices was present as part of a large disulfide cross-linked complex. Treatment of these structures with reducing agents in 1.6 M NaCl released the topo II. In contrast, topo I did not form disulfide cross-linked oligomers and was not detectable in any of these nuclease- and salt-resistant structures prepared at 4 degrees C. To assess the effect of in vitro heat treatment on the distribution of the topoisomerases, nuclear monolayers (isolated in the absence of iodoacetamide and NaTT) were heated to 37 degrees C for 1 h prior to treatment with nucleases and 1.6 M NaCl. The resulting structures (which retained 26 +/- 5% of the total nuclear protein) were morphologically similar to the NaTT-stabilized nuclear matrices and contained 15 +/- 4% of the total nuclear topo II. High-molecular-weight disulfide cross-linked oligomers of topo II were again demonstrated. Attempts to demonstrate these disulfide cross-linked oligomers in intact cells were unsuccessful.

Carcinoma, Hepatocellular↗