Disulfide Cleavage Research Articles

Inhibition of a reductive function of the plasma membrane by bacitracin and antibodies against protein disulfide-isomerase.

Evidence had been provided that a disulfide-linked [125I]iodotyramine/poly(D-lysine) conjugate was reductively cleaved when bound nonspecifically to the surface of Chinese hamster ovary (CHO) cells and that this cleavage was abolished by membrane-impermeant sulfhydryl blockers. The same blockers were subsequently found to inhibit the cytotoxicity of diphtheria toxin, a disulfide-linked heterodimer that binds to a specific surface receptor and must undergo chain separation to exert its cytotoxicity. This suggested that the disulfides of both macromolecules might be cleaved by a thiol-disulfide interchange reaction, possibly mediated by protein disulfide-isomerase (PDI, EC 5.3.4.1). We tested whether inhibitors of PDI--in particular, bacitracin and anti-PDI antibodies--might mimic the two effects of sulfhydryl blockers. Both bacitracin and anti-PDI antibodies were effective in inhibiting both reductive processes. This strongly suggests that the disulfide cleavage in the two membrane-bound macromolecules is mediated by PDI and that this enzyme, besides its known retention in the endoplasmic reticulum, must also be exposed at the plasma membrane. This paper points to other potentially important disulfide reductions that might be catalyzed by surface-associated PDI. It thereby broadens the known functions of an enzyme already known for its multifunctional properties.

Alteration of fibrinogen secondary structure by cis-diamminedichloroplatinum(II) and calcium protection.

The structural alteration of human fibrinogen (Fbg) by cis-diamminedichloroplatinum(II) (cis-DDP) was investigated using intrinsic fluorescence and circular dichroism (CD) spectra. In a preceding report, incubation of Fbg with cis-DDP under physiological conditions resulted in the cleavage of disulfide (S-S) bonds (N. Ohta, T. Yotsuyanagi and K. Ikeda, J. Pharmacobio-Dyn., 15, 611 (1992)). The intensity of fluorescence for cis-DDP-treated Fbg, in which 6.7 S-S bonds per mol of protein were cleaved, was reduced to 42% relative to native Fbg. CD studies suggested that the alpha-helix content decreased from 42% in its natural state to 29% in the cis-DDP-treated form. The extent of the fluorescence quenching and the decrease in helix correlated with the number of cleaved S-S bonds. The results indicate that cis-DDP leads to a secondary conformational alteration of Fbg involving a structural perturbation of nearby tryptophan residue(s) through S-S bond cleavage. In a buffer containing calcium, however, the smaller fluorescence quenching and the smaller helix decline were observed in cis-DDP-treated-Fbg, even though the same level of S-S cleavage occurred. Calcium binding would protect Fbg in terms of structural alteration as well as S-S cleavage by both dithiothreitol (DTT) and cis-DDP.

Disulfide bonds are essential for the stability of the sindbis virus envelope

Sindbis virus is a membrane-containing virus which has two glycoproteins organized in an icosahedral lattice. Protein-protein associations have been identified which participate in the formation of thtxte icosahedron and these associations are stabilized by intramolecular disulfide bridges (Anthony, R. P., and Brown, D. T., 1990, J. Virol. 65, 1187–1194). The present study further examines the role of disulfides in the structure and function of Sindbis virus by following the effect of dithiothreitol on the protease sensitivity of envelope proteins as well as the electron microscopic appearance and infectivity of Sindbis virus. Treatment of isolated virus with 5 m M dithiothreitol for 6 hr causes a marked increase in trypsin sensitivity of both E1 and E2, profound morphological alterations in the viral envelope, increased susceptibility of the nucleocapsid to RNase, and 95% loss of infectivity. These effects are greatly enhanced and accelerated when treatment with DTT is preceded by a brief exposure of the virus to pH 5.3, suggesting that acid-induced conformational changes render structurally critical disulfides more accessible to reductive cleavage by DTT. When compared to other manipulations known to change the conformation of the viral envelope, such as heating to 51 or 60° or exposure to acid pH, only the exposure to DTT with or without prior acid treatment caused marked structural changes correlated with a loss of infectivity. These data provide electron microscopic and functional evidence that intact disulfide bonds are critical for the stability of the virus envelope and suggest that the cleavage of critical disulfide(s) may play a role in the process of virus infection.

Internalization and intracellular fate of anti-CD5 monoclonal antibody and anti-CD5 ricin A-chain immunotoxin in human leukemic T cells

We have investigated the entry and subsequent intracellular fate of T101 monoclonal antibody (MoAb) and T101-ricin A-chain (RTA) immunotoxin (IT) directed against the CD5 antigen (Ag) expressed on human leukemic CEM cells. We provide direct evidence for the internalization of T101 MoAb and the corresponding IT. Both the MoAb and IT were internalized at a relatively low rate. This slow internalization process could be related to the partial recycling of the MoAb/Ag or IT/Ag complexes. Analysis of the internalized molecules showed that their molecular weight was only partially altered after internalization and that no free A-chain could be found inside the cells, indicating that lysosomal degradation and cleavage of disulfide- linked conjugates is a quantitatively minor phenomenon compared with the localization of internalized anti-CD5 ITs in an endosomo-Golgi compartment, followed by their recycling to the cell surface. We believe that this is the major factor explaining the low efficacy of anti-CD5 IT when assayed in the absence of potentiating substances. Finally, we showed that the presence of ammonium chloride and monensin, which both dramatically enhance the kinetics of IT activity, did not affect the rate of internalization or the intracellular localization of the conjugate, suggesting that these activators could act at the postendocytotic level on a limited number of IT molecules.

Characterization of disulfide bond position in proteins and sequence analysis of cystine-bridged peptides by tandem mass spectrometry

Tandem mass spectrometry employing high-energy, collisionally activated dissociation (CAD) is shown to be a useful method for sequencing through the cystine bridge of intermolecularly disulfide-bonded peptides. A characteristic triplet of intense fragment ions is observed corresponding to cleavage through and to either side of the disulfide bridge. These fragments define the masses of the linked peptides. Fragments due to peptide chain cleavage are also observed at lower abundance in the product-ion spectra and can be sufficient to sequence both of the disulfide-linked peptides without any prior knowledge of the peptide or protein sequence. Even in cases where the peptide sequence-related product-ion yields are poor, the intensities of the disulfide cleavage ions are usually sufficient to determine the molecular weights of the component cystine-bridged peptides. In this paper we demonstrate that the high-energy CAD tandem MS approach may be used to characterize disulfide-bonded peptides directly in complex enzymatic or chemical digests of native proteins. This obviates the need for individual purification of intermolecularly disulfide-linked peptides prior to analysis. The techniques are illustrated here for synthetic, inter- and intramolecularly disulfide-linked peptides and for human transforming growth factor-α (des-Val-Val-TGF-α), a compact protein containing 48 residues and three disulfides.

Synthesis of a bis(aminoethanethiol) ligand with an activated ester group for protein conjugation and 99mTc labeling

AbstractThe N‐hydroxysuccinimide (NHS) ester of the bis(aminoethanethiol) (BAT) ligand, 6‐(4″‐(4″‐carboxyphenoxy) butyl)‐2, 10‐dimercap‐to‐2, 10‐dimethyl‐4, 8‐diazaundecane, was synthesized by reacting 7‐ (4′‐bromobutyl) ‐3,3, 11, 11‐tetramethyl‐1, 2‐dithia‐5, 9‐diazacycloundecane with the sodium salt of 4‐hydroxybenzoic acid ethyl ester, followed by ester hydrolysis, reductive disulfide cleavage with dithiothreitol (DTT) and activation of the carboxyl group with NHS and dicyclohexylcarbodiimide (DCC). The resulting NHS‐BAT ester was conjugated to monoclonal antibodies at pH 8.5 with about 40% yield. 2–4 BAT ligands conjugated to the antibody did not change the binding characteristics significantly. Proof of the amount of covalent bound BAT ligands was attained by UV‐laser desorption/ionization (UV‐LD/I) mass spectrometry. Complexation with 99mTc was accomplished by using the tin‐reduction method. The labeling efficiency was >90%.

Removal of dodecyl sulfate ions bound to bovine serum albumin and chymotrypsinogen from the proteins. Effects of reduction of disulfide bridges and cleavage of peptide bonds on the removal extent

The removal of dodecyl sulfate (DS) ions which had bound to proteins, bovine serum albumin (BSA) and chymotrypsinogen, was attempted by precipitating the surfactant ions by the addition of a dipotassium hydrogen and potassium dihydrogen phosphate mixture. The effects of reduction of disulfide bridges and cleavage at peptide bonds on the removal of DS ion were examined. The amount of DS ion remaining on the proteins after the removal treatment sharply depended on the potassium ion concentration up to 150 m M in each case. At high concentrations of potassium ion, the amount of the remaining DS ion was approximately 25 and 10 mol/mol for the intact BSA and the disulfide bridges-reduced BSA, respectively. The amount was approximately 35 and 10 mol/mol for the intact chymotrypsinogen and the disulfide bridges-reduced chymotrypsinogen, respectively. No appreciable amount of DS ion remained on doubly-cleaved BSA and doubly-cleaved chymotrypsinogen, that is, α-chymotrypsin (in both cases the resulting three polypeptide chains produced on cleavage are joined with disulfide bridges). On the other hand, each protein was recovered with no appreciable loss at any potassium ion concentration. The recovery of protein conformation was examined on the intact BSA by circular dichroism measurements. The secondary structure of the protein was recovered to the original one in spite of the remaining DS ion of about 25 mol/mol.

A factor that activates oscillatory chloride currents in Xenopus oocytes copurifies with a subfraction of serum albumin.

Vertebrate blood sera contain a factor that elicits oscillatory chloride currents in Xenopus oocytes through activation of the phosphatidylinositol second messenger system. This factor was purified from rabbit and human sera by a sequence of Blue-Agarose chromatography, concanavalin A affinity chromatography, and hydroxyapatite fractionation, yielding a single active protein band (67 kDa). This protein is a subfraction of serum albumin, as revealed by its molecular mass, isoelectric properties, peptide maps, amino acid composition, and NH2-terminal sequence. Moreover, the factor could be purified with a monoclonal antibody to serum albumin and its ability to elicit oscillatory currents was inhibited by several polyclonal-monospecific antibodies to serum albumin. Various commercial high purity albumin preparations elicited oscillatory currents in oocytes. The activity of albumin was partially reduced by charcoal absorption and was greatly diminished when crystalline albumin was extracted with dry methanol. However, the activity was resistant to extraction with chloroform/ether, disulfide cleavage, and denaturation with 8 M urea, 6 M guanidinium chloride, and 1% sodium dodecyl sulfate. Trypsin or lipase treatment substantially reduced the potency of the active albumin, but neither treatment alone abolished the factor even after prolonged digestion. In contrast to serum or serum albumin, freshly collected blood plasma or purified plasma albumin did not evoke oscillatory currents. This indicates that some of the plasma albumin changes during blood coagulation and acquires a "factor" that makes it capable of activating the phosphatidylinositol-Ca2+ system in Xenopus oocytes. The serum factor also activates the phosphatidylinositol system in a variety of mammalian cells, suggesting that the modified albumin may play a role in cellular events related to tissue repair following injury.

Aqueous high-temperature chemistry of carbo- and heterocycles. Part 16. Model sulfur compounds: a study of hydrogen sulfide generation

The aquathermolysis reactivity of nine organic sulfur compounds was investigated to determine their potential to generate hydrogen sulfide during the cyclic steam stimulation process to recover Cold Lake bitumen. Dioctyl sulfide, 1-decanethiol, didecyl disulfide, 1-naphthalenethiol, 1,1'-dinaphthyl sulfide, 1,1'-dinaphthyl disulfide, 1-naphthyl 1-octyl sulfide, tetrahydrothiophene, and thiophene were selected as model compounds and subjected to neutral, basic, and acidic aquath-ermolysis conditions at 250 and 300 o C. Thiols, and thiols formed by cleavage of sulfides and disulfides were found to be the source of H 2 S evolution. Aliphatic sulfides, in addition to their usual C-S bond cleavage, showed α,β C-C bond cleavage

Cell surface sulfhydryls are required for the cytotoxicity of diphtheria toxin but not of ricin in Chinese hamster ovary cells.

A previous study on cleavage of disulfide bonds in endocytosed model compounds had shown that an initial phase of cleavage was totally inhibited by membrane-impermeant sulfhydryl inhibitors and thus was mediated by cell surface sulfhydryls (Feener, E. P., Shen, W.-C., and Ryser, H. J.-P. (1990) J. Biol. Chem. 265, 18780-18785). This paper uses the same inhibitors (5,5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuriphenylsulfonic acid) to examine the role of surface sulfhydryls in the cytotoxicity of diphtheria toxin (DT). Since the interchain disulfide of endocytosed DT must be cleaved prior to translocation of chain A from endosomes to cytoplasm, it was postulated that surface sulfhydryls might mediate the cleavage of that disulfide bond as well. Both sulfhydryl blockers did indeed markedly inhibit DT cytotoxicity. This effect was not due to inactivation of unbound DT, inhibition of receptor-mediated endocytosis, or impairment of acidification of endosomes. We conclude that cell surface sulfhydryls susceptible to blockage by 5,5'-dithiobis(2-nitro-benzoic acid) and p-chloromercuriphenylsulfonic acid are required for the cytotoxicity of DT and, most likely, for the reductive cleavage of DT's interchain disulfides. Ricin cytotoxicity was not decreased; this is consistent with the view that ricin reaches the cytoplasm from a late endocytic structure and with the finding that endocytosed disulfides are also cleaved in a cell fraction containing elements of the Golgi apparatus (Feener, E. P., Shen, W.-C., and Ryser, H. J.-P. (1990) J. Biol. Chem. 265, 18780-18785).

Isolation of variants of chinese hamster ovary cells with abnormally low levels of GSH: Decreased ability to cleave endocytosed disulfide bonds

Mutants of Chinese hamster ovary cells were selected for resistance to a 3 hour exposure to 4 microgram/ml N-methyl-N'-nitro-N-nitrosoguanidine and tested for glutathione (GSH) levels. Six of eight clones that survived the initial treatment had reduced GSH levels ranging from 26 to 61% of wild-type values. These eight cell lines were tested for their susceptibility to a drug conjugate in which methotrexate (MTX) is disulfide-linked to poly(D-lysine) (MTX-SS-PDL) to test their capacity to cleave the endocytosed disulfide bond and release free MTX from this otherwise undegradable drug conjugate. We had shown that wild-type cells were killed by approximately 1 x 10(-7) M MTX given as free drug, as MTX-poly(L-lysine) or as MTX-SS-PDL, but were not affected by MTX-poly(D-lysine). All six lines with abnormally low levels of GSH were resistant to MTX-SS-PDL. The variants with the lowest levels of GSH (MNR-5 and MNR-10) were tested further and showed near-normal sensitivity to MTX and MTX poly(L-lysine). As expected, both lines were hypersensitive to melphalan. They were, however, normally sensitive to diphtheria toxin and ricin, indicating that some cleavage of the interchain disulfides in these protein toxins occurs even when cellular GSH is abnormally low. The lesser GSH requirement for toxin activation may be due to their extraordinary potency.

A New Type of Reductive Cleavage of Disulfide in Quadruply ortho-Substituted Diphenyl Disulfide by Neighboring Thiolate Anion

Abstract The reaction of bis[2,6-bis(bromomethyl)phenyl] disulfide with thiourea or sodium sulfide showed an unusual reductive. cleavage of disulfide linkage with elimination of bromine by neighboring-group participation of the generated thiolate anion. Its reaction gave the structurally intriguing thiols which indicated the facile anodic oxidation.

Specific disulfide cleavage is required for ubiquitin conjugation and degradation of lysozyme.

Both ubiquitin conjugation and ubiquitin-dependent degradation of chicken egg white lysozyme in a reticulocyte lysate depend on the presence of a reducing agent. We present evidence that the reduction of a specific disulfide bond, namely that at Cys6-Cys127, facilitates ubiquitination and is a prerequisite to the formation of a multiubiquitin chain on one of at least four chain initiation sites on lysozyme. The Cys6-Cys127 disulfide bond in lysozyme can be specifically reduced, and the modified protein can be isolated after carboxymethylation of the 2 resulting cysteines. This modified lysozyme no longer requires the presence of a reducing agent for ubiquitin conjugation and degradation. Inhibition of ubiquitination by the dipeptide Lys-Ala revealed that this modified lysozyme, like the unmodified protein, is recognized via the binding of the ubiquitin protein ligase, E3, to the substrate's N-terminal lysyl residue. Both the rate and the extent of ubiquitin-lysozyme conjugation, however, are significantly higher with this modified substrate. Likewise, ubiquitin-dependent degradation of 6,127-reduced/carboxymethylated lysozyme was 2-4-fold faster than degradation of the unmodified counterpart. These results are consistent with an interpretation that the modified lysozyme mimics an intermediate formed at the rate-limiting step of the degradation of lysozyme in the reticulocyte lysate. Reduction of the Cys6-Cys127 disulfide bond is expected to unhinge the N-terminal region of lysozyme, and we propose that the recognition of this otherwise stable protein by the ubiquitin pathway is due to facilitated binding of E3 that results from such a conformational transition.

Effect of Salts on the Thiol-dependent Gelation of Bovine Serum Albumin.

A previous report [M. Hirose, Y. Nishizawa and J. Y. Lee, J. Food Sci., 55, 915 (1990)] has shown that the thio-dependent gelation of bovine serum albumin was strongly inhibited by high concentrations of salts. The inhibition mechanism was investigated in relation to the conformation and disuliide cleavage of the protein. The effects on the thiol-dependent disulfide cleavage of the protein varied with the salt species, especially with the anion species. The circular dichroism spectra and difference UV-spectra showed that anions induced some conformational changes in the tertiary structure, but not in the secondary structure. With regard to a chaotropic anion, perchlorate, this conformational change was closely correlated with the inhibition of gelation and disulfide reduction. In contrast, a lyotropic anion such as sulfate inhibited the gelation at a higher concentration than the concentrations that induced the conformational change. In addition, this anion showed no inhibition effect on disulfide reducti...

Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes.

Whereas there is biological evidence that the reductive cleavage of disulfide bonds is critical for the activation of endocytosed macromolecules such as toxins, immunotoxins, and other drug carriers, virtually nothing is known about the specifics of this cleavage. To study this process, a model compound was synthesized in which a radioiodinated tyramine was linked through a disulfide bond to an undegradable carrier, poly(D-lysine), known to be efficiently endocytosed. Cultured Chinese hamster ovary cells were pulse-labeled with this probe, and the disulfide cleavage was measured as released acid-soluble radioactivity at different times of chase. Pulse-labeled cells were also subjected to subcellular fractionation to identify intracellular structures associated with disulfide cleavage. Cleavage began without lag, amounted to about approximately 7% of the initial cell-bound radioactivity in the first hour and continued for more than 6 h. It was abolished in the presence of N-ethylmaleimide. When sulfhydryl groups present at the cell surface were blocked with cell-impermeant sulfhydryl reagent, the initial phase of disulfide cleavage was inhibited, indicating that cleavage began at the cell surface. A long-lasting intracellular phase of disulfide cleavage began after about approximately 30 min of chase. Subcellular fractionation and kinetic analysis indicated that neither lysosomes nor endosomes were participating in that phase, leaving the Golgi apparatus as the most probable site of endocytic disulfide cleavage.

Sodium Telluride-Mediated Sulfenylation of α-Halo Carbonyl Compounds with Diphenyl Disulfide

Abstract Under mild aprotic conditions α-halo carbonyl compounds react with diphenyl disulfide in the presence of sodium telluride to give the corresponding α-phenylthio derivatives in good to moderate yields. The reaction appears to proceed involving the sulfur–sulfur bond cleavage of diphenyl disulfide by sodium telluride.

ChemInform Abstract: A Mild and Efficient Cleavage of Symmetrical Disulfides Using Semicarbazide and Thiosemicarbazide.

ChemInformVolume 20, Issue 1 Preparative Organic Chemistry ChemInform Abstract: A Mild and Efficient Cleavage of Symmetrical Disulfides Using Semicarbazide and Thiosemicarbazide. S. N. MAITI, S. N. MAITI SynPhar Lab. Inc., Edmonton, Alberta T6E 5V2, Can.Search for more papers by this authorM. P. SINGH, M. P. SINGH SynPhar Lab. Inc., Edmonton, Alberta T6E 5V2, Can.Search for more papers by this authorP. SPEVAK, P. SPEVAK SynPhar Lab. Inc., Edmonton, Alberta T6E 5V2, Can.Search for more papers by this authorR. G. MICETICH, R. G. MICETICH SynPhar Lab. Inc., Edmonton, Alberta T6E 5V2, Can.Search for more papers by this authorA. V. N. REDDY, A. V. N. REDDY SynPhar Lab. Inc., Edmonton, Alberta T6E 5V2, Can.Search for more papers by this author S. N. MAITI, S. N. MAITI SynPhar Lab. Inc., Edmonton, Alberta T6E 5V2, Can.Search for more papers by this authorM. P. SINGH, M. P. SINGH SynPhar Lab. Inc., Edmonton, Alberta T6E 5V2, Can.Search for more papers by this authorP. SPEVAK, P. SPEVAK SynPhar Lab. Inc., Edmonton, Alberta T6E 5V2, Can.Search for more papers by this authorR. G. MICETICH, R. G. MICETICH SynPhar Lab. Inc., Edmonton, Alberta T6E 5V2, Can.Search for more papers by this authorA. V. N. REDDY, A. V. N. REDDY SynPhar Lab. Inc., Edmonton, Alberta T6E 5V2, Can.Search for more papers by this author First published: January 3, 1989 https://doi.org/10.1002/chin.198901099Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume20, Issue1January 3, 1989 RelatedInformation

ChemInform Abstract: Reductive Cleavage of Symmetrical Disulfides with Hydrazines.

AbstractThe symmetrical dihetaryl disulfides (I) are reductively cleaved with hydrazine (IIa) or the methylated hydrazines (IIb) and (IIc) to produce the thiols (III).

Reductive Cleavage of Symmetrical Disulfides with Hydrazines

Abstract An efficient method for the reductive cleavage of symmetrical disulfides with hydrazines is described.

ChemInform Abstract: Selenium, Carbon Monoxide, and Water as a New Reduction System: Reductive Cleavage of Disulfides and Diselenides to Thiols and Selenols.

AbstractThe disulfides (I) are reduced with selenium, carbon monoxide and water, yielding the mercaptans (II) which are coupled with methyl vinyl ketone (III) to form the sulfenylethyl methyl ketones (IV).