Role Of Sulfhydryls Research Articles

The role of cell surface sulfhydryl and amine binding sites in the removal of Cr(VI) from solution by Bacillus subtilis bacterial cells

A range of bacterial species are known to be capable of reducing soluble Cr(VI) to Cr(III), causing the precipitation of Cr(III) phases from solution, but the mechanism responsible for the initial interaction between anionic aqueous Cr(VI) and the negatively charged cell surface has not been identified. Our study examines Cr(VI) removal from solution by the Gram-positive soil bacterial species Bacillus subtilis. We measured the kinetics of Cr(VI) removal as a function of pH in B. subtilis suspensions with different Cr(VI):cell ratios, and we measured the effect of pH on the extent of Cr(VI) removal at a fixed time in experiments with B. subtilis biomass (cells plus exudates) and in experiments involving B. subtilis exudates alone. The roles of sulfhydryl and amine binding sites in Cr(VI) removal were constrained using site–specific blocking molecules, and the impact of the site blocking on Cr(VI) removal was studied as a function of pH. Our results indicate that Cr(VI) removal by B. subtilis cells under the experimental conditions is at least partially non-reversible and is dependent on binding site concentration, pH, and metal loading. Our results are consistent with a two step removal process: Cr(VI) first adsorbs reversibly onto a cell wall binding site, followed by Cr(VI) reduction to Cr(III) likely via electron transfer from cell wall electron transport chain molecules. B. subtilis exudates are capable of removing a relatively small fraction of Cr(VI) from solution, and hence our results indicate that the dominant mechanism of Cr(VI) removal requires interaction with bacterial biomass. The presence of either the sulfhydryl-specific blocking molecule or the amine-specific blocking molecule or both in the experimental systems dramatically reduces the extent of Cr(VI) reduction, especially under circumneutral pH conditions, strongly suggesting that both sulfhydryl and amine binding site types participate in the initial attachment of Cr(VI) onto the cell surface. The experiments with either sulfhydryl or amine sites blocked both exhibited a similar reduction in Cr(VI) removal to the experiments with both types of sites blocked, strongly suggesting that both types of sites are involved simultaneously in binding Cr(VI) species to the cell surface. For example, these sites could both be involved in forming a bidentate bond with Cr(VI) species, or the positive charge of an amine site in close proximity to a sulfhydryl site could be necessary in order to enable the Cr(VI) species to approach the cell wall and to bind to a sulfhydryl site. The results of this study are the first to propose a viable mechanism that can explain the binding of anionic Cr(VI) onto an overall negatively charged cell surface as a first step in the reduction to Cr(III) and subsequent removal of Cr from solution.

Analysis using fluorescence labeling and mass spectrometry of disulfide-mediated interactions of soy protein when heated.

It is well-known that disulfide-mediated interactions are important when soy protein is heated, in which soy proteins are dissociated and rearranged to some new forms. In this study, the disulfide bond (SS) linked polymer, which was composed of the acidic (A) polypeptides of glycinin, basic (B) polypeptides of glycinin, and a small amount of α' and α of β-conglycinin, was formed as the major product, accompanied by the formation of SS-linked dimer of B and monomer of A as minor products. The role of sulfhydryl (SH) of different subunits/polypeptides for forming intermolecular SS was investigated. The SH of B in glycinin (Cys298 of G1, Cys289 of G2, Cys440 of G4) was transformed into SS in polymer identified by mass spectrometry analysis. The SH content of polymer was lower than that of glycinin and β-conglycinin subunits when heated. The SH content of B in polymer was lower than that in glycinin subunit, and both of them were decreased by heating. The SH content of A in polymer was increased and higher than that of B in polymer and A in glycinin subunit when heated. These results indicated that the SH of B in glycinin subunit was subjected to not only SH oxidation but also SH-SS exchange (with SS of A) for forming intermolecular SS of polymer. The SH oxidation and SH-SS exchange were proven by the change of SH content for the first time. The SH of B was suggested to be reactive for forming intermolecular SS of polymer.

Acid-base formalism in dispersion-stabilized S-H···Y (Y═O, S) hydrogen-bonding interactions.

The role of sulfhydryl (S-H) group as hydrogen bond donor is not as well studied as that of hydroxyl (O-H). In this work we report on the hydrogen-bonding properties of S-H donor in 1:1 complexes of H2S with diethyl ether (Et2O), dibutyl ether (Bu2O), and 1,4-dioxane (DO). The complexes were prepared in supersonic jet and investigated using infrared predissociation spectroscopy based on VUV photoionization detection. The IR spectra of all the complexes showed the presence of a broad, intensity-enhanced, and red-shifted hydrogen-bonded S-H stretching transition. The S-H stretching frequency was red-shifted by 46, 63, and 49 cm(-1) in H2S-Et2O, H2S-Bu2O, and H2S-DO complexes, respectively, suggesting that all the complexes are S-H···O bound. Computationally, two different S-H···O bound structures, namely, "coplanar" and "perpendicular", were obtained as the minimum energy structures for these complexes at the MP2/6-311++G** level, with the former being the global minimum. However, with Dunning-type basis sets (aug-cc-pVDZ and aug-cc-pVTZ) only the perpendicular structures were found to be stable at the MP2 level. The large widths of the bound S-H stretch observed in the experimental spectra (fwhm of 35 to 80 cm(-1)) were attributed to inhomogeneous broadening due to multiple conformations of the alkyl chains in the coplanar and perpendicular structures populated in the jet. The frequency shifts in the hydrogen-bonded S-H stretching mode as well as the bond dissociation energies of all S-H···Y (Y═O,S) complexes of H2S, which includes the H2S dimer and H2S-methanol (H2S-MeOH) complexes reported in our previous work (ChemPhysChem 2013, 14, 905-914), were found to scale linearly with the proton affinity of the acceptor molecule. In this regard the S-H group, like O-H, is found to conform to the widely accepted acid-base nature of hydrogen-bonding interactions.

Protective effect of silymarin against ethanol-induced gastritis in rats: Role of sulfhydryls, nitric oxide and gastric sensory afferents

Silymarin has been known to exert antioxidant, anti-carcinogenic and anti-inflammatory effects. In this study, we examined the effect of silymarin on gastritis in rats. Oral administration of silymarin dose-dependently decreased gastric lesions in ethanol-induced gastritis model. Silymarin also significantly suppressed the development of gastric lesions in aspirin- or water immersion-restraint stress-induced gastritis models. Further study demonstrated that the gastroprotective effect of silymarin was blocked by nitric oxide (NO) synthase inhibitor l-NAME, SH blocker N-ethylmaleimide or TRPV1 antagonist capsazepine in ethanol-induced gastritis model. In addition, ex vivo analysis revealed that ethanol-induced decrease in gastric mucus and non-protein sulfhydryl (NPSH) groups was significantly reversed by silymarin treatment and lipid peroxidation was also suppressed by silymarin in ethanol-induced gastritis model. Taken together, these results suggest that silymarin exerts gastroprotective effects and the gastroprotective effects of silymarin might be related to the protection of gastric mucosal NO and NP-SH and the modulation of capsaicin-sensitive gastric sensory afferents.

Thermal instability of rat muscle sarcoplasmic reticulum Ca(2+)-ATPase function.

To examine the thermal instability and the role of sulfhydryl (SH) oxidation on sarcoplasmic reticulum (SR) Ca(2+)-ATPase function, crude homogenates were prepared from the white portion of the gastrocnemius (WG) adult rat muscles (n = 9) and incubated in vitro for < or =60 min either at a normal resting body temperature (37 degrees C) or at a temperature indicative of exercise-induced hyperthermia (41 degrees C) with DTT and without DTT (CON). In general, treatment with DTT resulted in higher Ca(2+)-ATPase and Ca(2+) uptake values (nmol. mg protein(-1). min(-1), P < 0.05), an effect that was not specific to time of incubation. Incubations at 41 degrees C resulted in lower (P < 0.05) Ca(2+) uptake rates (156 +/- 18 and 35.9 +/- 3.3) compared with 37 degrees C (570 +/- 54 and 364 +/- 26) at 30 and 60 min, respectively. At 37 degrees C, ryanodine (300 microM), which was used to block Ca(2+) release from the calcium release channel, prevented the time-dependent decrease in Ca(2+) uptake. A general inactivation (P < 0.05) of maximal Ca(2+)-ATPase activity (V(max)) in CON was observed with incubation time (0 > 30 > 60 min), with the effect being more pronounced (P < 0.05) at 41 degrees C compared with 37 degrees C. The Hill slope, a measure of co-operativity, and the pCa(50), the cytosolic Ca(2+) concentration required for half-maximal activation of Ca(2+)-ATPase activity, decreased (P < 0.05) at 41 degrees C only. Treatment with DTT attenuated the alterations in enzyme kinetics. The increase in V(max) with the Ca(2+) ionophore A-23187 was less pronounced at 41 degrees C compared with 37 degrees C. It is concluded that exposure of homogenates to a temperature typically experienced in exercise results in a reduction in the coupling ratio, which is mediated primarily by lower Ca(2+) uptake and occurs as a result of increases in membrane permeability to Ca(2+). Moreover, the decreases in Ca(2+)-ATPase kinetics in WG with sustained heat stress result from SH oxidation.

Hans Selye and the development of the stress concept. Special reference to gastroduodenal ulcerogenesis.

Hans Selye has a historic role in the development of the stress concept. Before his short article in Nature in 1936, the neuroendocrine response to nonspecific injury was thought to be restricted to the release of catecholamines, as recognized by Cannon. Selye was the first to appreciate the crucial role of the adrenal cortex/hypophysis axis in the stress response. He also insisted on the nonspecificity of this neuroendocrine response, and he named the stress-causing agent "stressors". His last major contribution was the distinction between negative, that is, distress, and positive, that is, eustress reactions. The "triad of stress" (adrenal hypertrophy, gastrointestinal ulcers, thymolymphatic atrophy) was also first described by Selye, who was fascinated by the fact that in stressed rodents only gastric and not duodenal ulcers would develop. It was not until the recognition of duodenal ulcerogenic properties of propionitrile and cysteamine as well as the subsequent quantitative structure-activity studies predicting the duodenal ulcerogenic action of complex molecules that pathogenetic investigations allowed a molecular and mechanistic approach for studying the etiology and pathogenesis of duodenal ulceration. Recent studies on the role of sulfhydryls, TRH, ET, and growth factors provide new insights into central and peripheral pre-ulcer pathways. We were surprised to learn that an organ-specific ET-1 release may play a role both in ulcer induction and healing, which seems to start with the expression of immediate early genes such as egr-1 and stimulation of the local synthesis of growth factors such as bFGF and PDGF. Thus, a historic review originating with Hans Selye and extending through the next 60 years allows a cellular and molecular approach to the better understanding of stress-related disorders such as gastroduodenal ulceration.

The Role of SH and S-S Groups in Bacillus cereus β-Amylase

The properties of sulfhydryl (SH) and disulfide (S-S) groups in Bacillus cereus BQ10-S1 Spo III beta-amylase have been investigated to clarify their roles in the enzyme action. Two out of three cysteine residues in B. cereus beta-amylase were found to form an S-S bond, which was found to be located between Cys91 and Cys99 by the analysis of an S-S containing peptide. The replacement of the soybean beta-amylase model around L3 loop 1 revealed that the S-S bond is located at the root of this flexible loop that moves between open and closed forms during catalysis. The analysis of fluorescence labeled peptides revealed that the remaining free SH group was Cys331. Modification of Cys331 with N-ethylmaleimide or p-chloromercuribenzoic acid (PCMB) caused inactivation of the enzyme. The rate constants for the reactions were consistent with those of Cys343 in soybean enzyme. The binding affinity of the PCMB-modified enzyme to maltose was also decreased. These results indicate that the modification of Cys331, which exists as a free SH group in B. cereus beta-amylase caused inactivation by a similar mechanism to that in the case of Cys343 in soybean beta-amylase as assumed from the sequence homology. This cysteine residue has a common role in beta-amylases irrespective their origin.

Sulfhydryl-depleting agents, but not deferoxamine, modulate EDRF action in cultured pulmonary arterial cells.

The potential role of intracellular sulfhydryls and iron on the biological activity of endothelium-derived relaxing factor (EDRF) released basally from bovine pulmonary arterial endothelial (BPAE) cells was investigated in a cultured cell bioassay system, by measuring N omega-nitro-L-arginine-sensitive guanosine 3',5'-cyclic monophosphate (cGMP) accumulation in rabbit pulmonary arterial smooth muscle (SM) cells. The role of sulfhydryls in the biosynthesis of EDRF was studied by selectively exposing the endothelial cells to thiol-depleting agents. Both N-ethylmaleimide (NEM) and maleic acid diethyl ester (DEM) inhibited EDRF-induced cGMP accumulation in a dose-dependent manner. To study the requirement of SM thiols in the metabolism of EDRF to a stimulator of cGMP formation, SM were selectively exposed to NEM and DEM before bioassay with control, untreated BPAE. DEM and NEM inhibited cGMP formation in response to EDRF by 30 and 68%, respectively. The requirement of SM sulfhydryls was further investigated in the stimulation of SM cGMP accumulation elicited by nitrosothiols [S-nitroso-L-cysteine, S-nitroso-mercaptoproprionic acid, and sodium nitroprusside (SNP)]. NEM pretreatment of SM cells abolished cGMP responses to all vasodilators; DEM did not affect the nitrosothiol responses but reduced by 30% the cGMP accumulation to SNP. The role of iron in the endothelial synthesis of EDRF was assessed by chelating endothelial low-molecular-weight iron compounds. Exposure of BPAE to deferoxamine mesylate had no effect on cGMP accumulation in SM, suggesting that deferoxamine-available iron is not necessary for the endothelial stimulation of SM cGMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of carboxyl and sulfhydryl residues on rabbit small intestinal brush-border membrane Na(+)-glucose cotransporter.

The role of sulfhydryl (SH) and carboxylic acid residues in Na(+)-dependent glucose uptake, Na(+)-dependent phlorizin binding, and substrate exchange by the rabbit small intestinal brush-border membrane (BBM) Na(+)-glucose cotransporter was examined in sodium dodecyl sulfate-BBM vesicles. The sulfhydryl reagent p-chloromercuribenzoate (PCMB) inhibited all three measures of cotransporter function in a dithiothreitol-sensitive manner with similar K0.5 values (concn of PCMB resulting in 50% inhibition). PCMB sulfonate had no effect on Na(+)-glucose cotransporter function < 250 microM. The carboxylic acid reagent 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide no effect on Na(+)-glucose cotransporter function. N,N'-dicyclohexylcarbodiimide (DCCD) inhibited all three measures of cotransporter function with similar K0.5 values for inhibition. Inhibition by DCCD did not require addition of a nucleophile. In contrast, PCMB-pretreated cotransporter was insensitive to DCCD in the absence of added nucleophile with respect to substrate transport (Na(+)-dependent glucose uptake) but not Na(+)-dependent phlorizin binding. These results indicate an intravesicular or lipophilic environment for both the PCMB-reactive SH residue and the DCCD-reactive carboxylic acid residues, suggesting that a SH residue may act as an endogenous nucleophile for interaction of DCCD with the Na(+)-glucose cotransporter and suggesting that different carboxylic acid residues may be involved in Na(+)-dependent glucose uptake and Na(+)-dependent phlorizin binding.

Structural properties of cyanase. Denaturation, renaturation, and role of sulfhydryls and oligomeric structure in catalytic activity.

Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to give ammonia and bicarbonate. The enzyme is composed of 8-10 identical subunits (Mr = 17,008). The objective of this study was to clarify some of the structural properties of cyanase for the purpose of understanding the relationship between oligomeric structure and catalytic activity. Circular dichroism studies showed that cyanase has a significant amount of alpha-helix and beta-sheet structure. The one sulfhydryl group per subunit does not react with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) unless cyanase is denatured. Denaturation is apparently complete in 10 M urea or 6 M guanidine hydrochloride, but is significantly reduced in 10 M urea by the presence of azide (analog of cyanate) and is incomplete in 8 M urea. Denatured cyanase could be renatured and reactivated (greater than 85%) by removal of denaturants. Reactivation was greatly facilitated by the presence of certain anions, particularly bicarbonate, and by high ionic strength and protein concentration. The catalytic activity of renatured cyanase was associated only with oligomer. Cyanase that had been denatured in the presence of DTNB to give a cyanase-DTNB derivative could also be renatured at 26 degrees C to give active cyanase-DTNB oligomer. The active oligomeric form of the cyanase-DTNB derivative could be converted reversibly to inactive dimer by lowering the temperature to 4 degrees C or by reduction of the ionic strength and removal of monoanions. These results provide evidence that free sulfhydryl groups are not required for catalytic activity and that catalytic activity may be dependent upon oligomeric structure.

Possible role of sulfhydryls in mucosal protection induced by aluminum hydroxide.

The involvement of sulfhydryl-containing compounds was investigated in the cytoprotective effect of certain antacids against ethanol-induced gastric mucosal damage in the rat. Not only the protective effect of a sulfhydryl containing compound, N-acetylcysteine, was abolished after pretreatment with N-ethylmaleimide, but also the adaptive cytoprotection induced by 20% ethanol. Pretreatment of the animals with the cyclooxygenase inhibitor indomethacin or with the thiol reagent N-ethylmaleimide significantly diminished the cytoprotection evoked by aluminum hydroxide. However, the protective effect of aluminium hydroxide could only be totally abolished by pretreatment with the combination of indomethacin and N-ethylmaleimide. The present results suggest the additional role of sulfhydryl-containing compounds localized in the gastric mucosa in the cytoprotection induced by a mild irritant or by aluminium hydroxide and aluminium hydroxide-containing antacids as well.

Role of Sulfhydryls in the Hepatotoxicity of Organic and Metallic Compounds

Role of Sulfhydryls in the Hepatotoxicity of Organic and Metallic Compounds. KLAASSEN, C. D., BRACKEN, W. M., DUDLEY, R. E., GOERING, P. L., HAZELTON, G. A., AND HJELLE, J. J. (1985). Fundam. Appl. Toxicol.. 5,806-815. Endogenous sulfhydryl compounds serve a critical role in maintaining the function and viability of living systems. Glutathione (GSH) is the most abundant of these nonprotein thiols. During the past decade it has been demonstrated that sulfhydryls such as GSH also serve an important role in protecting vital nucleophilic sites in the liver from electrophilic attack by numerous classes of reactive chemicals. Organocompounds such as bromobenzene and acetaminophen which undergo microsomal metabolism yield reactive intermediates that are specifically inactivated by conjugation with sulfhydryls in the form of GSH. Thus, for organocompounds GSH is extremely important in protecting against toxic insults. More recently, other sulfhydryl compounds also have been found to serve a specific but as yet less defined role in protecting biological systems against chemically induced injury. Metals such as cadmium have a high affinity for sulfhydryls and the metal binding protein metallothionein binds cadmium with high affinity. The highly specific association of the metal with this sulfhydryl-enriched protein serves to effectively sequester the reactive cadmium ion. The central role of sulfhydryl equivalents in the detoxication of organc- and metallocompounds is similar, however, the mechanism by which this is achieved is fundamentally different.

Potentiation in various agonists-induced contractions of rabbit mesenteric artery by sulfhydryl reagents.

The role of sulfhydryl and disulfide groups as determinants of rabbit mesenteric arterial responses to various contractile agonists were determined. The addition of 1 X 10(-3) M or of 1 X 10(-2) M 2-mercaptoethanol (2-MEt), a sulfhydryl reagent, produced a leftward displacement (potentiation) of the concentration-response curves of mesenteric arterial strips for KCl. Dithiothreitol (DTT), a reagent that reduces disulfide bonds to sulfhydryl groups, also potentiated the contractile response to KCl in this strip. In mesenteric arterial strips treated with 14 mM KCl after exposure to Ca2+-free Krebs' bicarbonate solutions containing 0.1 mM EGTA, the addition of CaCl2 in a concentration of 2.5 mM caused a contraction (14 mM KCl-induced Ca2+-contraction). The presence of 2-MEt or DTT expectedly potentiated this 14 mM KCl-induced Ca2+-contraction. Verapamil, a calcium antagonist, inhibited the 14 mM KCl-induced Ca2+-contraction both in the presence and the absence of these sulfhydryl reagents. 2-MEt also potentiated the contractile responses of mesenteric arterial strips to histamine, norepinephrine, angiotensin II and prostaglandin F2 alpha suggesting that the potentiation by the sulfhydryl reagent is a nonspecific effect. This sulfhydryl reagent potentiated the each agonist-induced Ca2+-contraction. It is concluded that reduction of a disulfide bridge to a sulfhydryl group at Ca2+-channels increases transmembrane influx of Ca2+ in strips of rabbit mesenteric artery and the increased Ca2+ influx in turn accompanied the contractile responses to various agonists.

POTENTIATION IN VARIOUS AGONISTS-INDUCED CONTRACTIONS OF RABBIT MESENTERIC ARTERY BY SULFHYDRYL REAGENTS

The role of sulfhydryl and disulfide groups as determinants of rabbit mesenteric arterial responses to various contractile agonists were determined. The addition of 1 x10−3 M or of 1 x10−2 M 2-mercaptoethanol (2-MEt), a sulfhydryl reagent, produced a leftward displacement (potentiation) of the concentration-response curves of mesenteric arterial strips for KCl. Dithiothreitol (DTT), a reagent that reduces disulfide bonds to sulfhydryl groups, also potentiated the contractile response to KCl in this strip. In mesenteric arterial strips treated with 14 mM KCl after exposure to Ca2+-free Krebs’ bicarbonate solutions containing 0.1 mM EGTA, the addition of CaCI2 in a concentration of 2.5 mM caused a contraction (14 mM KCI-induced Ca2+-contraction). The presence of 2-MEt or DTT expectedly potentiated this 14 mM KCI-induced Ca2+-contraction. Verapamil, a calcium antagonist, inhibited the 14 mM KCI-induced Ca2+-contraction both in the presence and the absence of these sulfhydryl reagents. 2-MEt also potentiated the contractile responses of mesenteric arterial strips to histamine, norepinephrine, angiotensin II and prostaglandin F2α suggesting that the potentiation by the sulfhydryl reagent is a nonspecific effect. This sulfhydryl reagent potentiated the each agonist-induced Ca2+-contraction. It is concluded that reduction of a disulfide bridge to a sulfhydryl group at Ca2+-channels increases transmembrane influx of Ca2+ in strips of rabbit mesenteric artery and the increased Ca2+ influx in turn accompanied the contractile responses to various agonists.

Role of sulfhydryl groups in aortic responses using N-ethylmaleimide.

This study examines the effects of N-ethylmaleimide (NEM) 10(-7) M on agonist-induced contraction, and the relaxation following drug washout of vascular smooth muscle (VSM) segments. 45Ca flux, cyclic 3':5'-adenosine-monophosphate (cAMP), and cyclic 3':5'-guanosine-monophosphate (cGMP) were analyzed in an attempt to understand the observed physiological modifications with NEM. Cyclic nucleotides were measured by 125I-radioimmunoassay. Contractile responses to norepinephrine (NE) 5.9 x 10(-7) M, angiotensin II (AT) 9.8 x 10(-8) M, and potassium chloride (KCl) 2.2 x 10(-2) M were significantly depressed when VSM segments were exposed to NEM prior to agonist challenge. An inhibition of relaxation occurred when NEM was added after the development of a maximal contractile response to NE, AT, or KCl. VSM relaxation was significantly inhibited when compared to controls. Altered 45Ca efflux, increased cGMP, and decreased cAMP levels were all associated with an inhibition of relaxation of VSM. These results suggest a regulatory role of sulfhydryl (SH) groups in contraction and relaxation of VSM involving both the control of Ca2+ flux and cAMP and cGMP metabolism.

Role of sulfhydryls in in vitro growth of Mycobacterium lepraemurium.

In an attempt to evaluate various factors that influence the growth of Mycobacterium lepraemurium in NC-5 medium, the effects of sulfur and --SH compounds were investigated. Cysteine could be replaced by equimolar concentrations of other --SH compounds containing carboxyl group, and at lower concentrations by nonpolar sulfhydryl compounds. The oxidized form of sulfhydryls, as well as certain organic and inorganic reducing agents, did not support growth. The results suggest that the function of sulfhydryl compounds is to maintain low reducing potential in the medium and also to participate in metabolic or biosynthetic pathways or both. A combination of dithiothreitol and thioglycolate gave better results than when these compounds were incorporated individually in the medium. This suggests the protective action of dithiothreitol in preventing oxidation of monothiols.

Studies on the role of sulfhydryls in the myosin ATPase. Characterization of the site of modification by the bifunctional sulfhydryl reagent p-N,N'-phenylenedimaleimide.

Myosin has been modified with near stoichiometric amounts of the bifunctional reagent [14C]p-N,N'-phenylenedimaleimide (pPDM) in the presence of MgADP under conditions which abolish its ATPase activity. Subsequent carboxymethylation and CNBr cleavage results in the 14C label being associated with a single polypeptide of Mr approximately 10,000. Amino acid composition and partial sequence analysis of this peptide showed that it corresponded to the peptide containing -SH1 and -SH2 sequenced by Elzinga and Collins (Elzinga, M., and Collins, J.H. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4281-4284) and to the peptide labeled at -SH1 or -SH2 by N-ethylmaleimide by Kunz et al. (Kunz, P.A., Walser, J.T., Watterson, J.G., and Schaub, M.C. (1977) FEBS Lett. 83, 137-140). These data indicating that pPDM does label the -SH1- and -SH2-containing region in myosin by covalently bridging them and shows that in the presence of MgADP these thiols can approach to within 12 to 14 A.

The effect of oxidant stress on diamide-treated human granulocytes

The role of sulfhydryls in the protection of human polymorphonuclear neutrophils against extracellular oxidant attack was investigated by simultaneously exposing polymorphonuclear neutrophils to the thiol-oxidizing agent diamide and the oxidant-generating system xanthine-xanthine oxidase. Neither diamide nor the oxidants generated by the xanthine-xanthine oxidase system alone impaired the burst in chemiluminescence, hexose monophosphate shunt activity or formate oxidation normally seen during polymorphonuclear neutrophil phagocytosis. Incubation of the polymorphonuclear neutrophils simultaneously with diamide and xanthine-xanthine oxidase markedly impaired polymorphonuclear neutrophil phagocytosis, hexose monophosphate shunt activity, chemiluminescence and formate oxidation. Although the polymorphonuclear neutrophils exposed to diamide and xanthine-xanthine oxidase did not respond to a variety of phagocytizable stimuli, trypan blue exclusion was normal and hexose monophosphate shunt activity could be stimulated by diamide. The damaging effect of the diamide xanthine-xanthine oxidase system could be blocked by the addition of superoxide dismutase or catalase, but not by hydroxyl radical or singlet oxygen scavengers. We hypothesize that an unidentified population of thiols may play a role in protecting the polymorphonuclear neutrophil from endogenously derived oxidants.

Differential Effects of p-Chloromercuriphenyl Sulfonate on Arterial Responses to Epinephrine and Serotonin

SummaryThe purpose of this study was to gain insight into the role of sulfhydryl (SH) groups in arterial receptors for vasoactive simulants. We studied the effects of p-chloromercuriphenyl sulfonate (PCMBS, 10-6 to 10-4 M), a potent and specific SH binding agent, on contractile responses produced with 10-5M epinephrine and 10-5M serotonin in helical strips of arterial smooth muscle prepared from tail arteries of the rat. Responses to epinephrine were rapidly, markedly, and reversibly inhibited with low concentrations of PCMBS. In contrast, responses to serotonin were slowly inhibited when high concentrations of PCMBS were tested. The inhibitory effect of PCMBS (10-5M) on epinephrine-induced contractions was prevented by 10-3 M cysteine. Alpha-adrenergic blockade with phentol-amine (10-4 or 5 × 10-6 M) virtually abolished responses to epinephrine and significantly decreased responses to serotonin. These findings are consistent with the view that arterial receptors for contractile responses to epinephrine a...

Temperature sensitivity of myosin and actomyosin.

The thermal denaturation of myosin and actomyosin was studied by active site analysis (enzymatic activity) and measurements were related to overall conformational changes (viscosity) over the temperature range 19–65 °C. The role of sulfhydryl (SH) groups on the temperature-induced denaturation of actomyosin was investigated. The temperature of maximum change in the overall conformation (the melting temperature, TM) was unaffected by the binding of F-actin to myosin. For both myosin and actomyosin the TM was 43 ± 2 °C. However, the range of temperature over which large conformational changes were observed was affected by the binding of F-actin to myosin. For myosin and dissociated actomyosin, changes were observed between 37 and 50 °C, while for actomyosin large changes were observed between 20 and 50 °C. With actomyosin there was an irreversible increase in titratable sulfhydryl groups from 19 to 60 °C. Temperature effects on the calcium-activated ATPase were studied. The temperature of maximum enzymatic activity for actomyosin was 45 ± 2 °C, which corresponds to the TM and the temperature at which increases in SH content were apparent. However, for myosin the temperature of maximum enzymatic activity was 33 °C, considerably below the TM. Overall conformational changes were reversible below the TM, while changes in enzymatic activity were reversible below the temperature of maximum enzymatic activity. Actin offers considerable protection to the temperature inactivation of the active site of myosin even though the F-actin–myosin complex is very highly dissociated in the presence of ATP. However, there is no significant stabilization of myosin by F-actin in terms of the temperature sensitivity of the overall conformation.