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Two sulfhydryl reagents (N-ethyl maleimide and p-chloromercuribenzoate), used in a concentration of 0.1 mmol/l, increased both spontaneous and evoked acetylcholine secretion at the toad neuromuscular junction, the former in the absence of extracellular calcium ions.
2-Mercaptoethanol increases the optical density of assay solutions at wavelengths between 280 to 400 nm, and therefore interferes with the measurement of protein concentration by the microbiuret method. Protein concentration can be determined in the presence of 2-mercaptoethanol up to 6 mM by modification of the method as follows: after the precipitation of protein by trichloroacetic acid in the presence of deoxycholate, the precipitate is resolubilized with NaOH solution. Dithiothreitol interfered with the protein determinations could by made in the presence of 4 mM of dithiothreitol with the modified microbiuret method. This modified method is time-saving and more reliable than other methods for protein determination, such as Lowry's method, in the presence of 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.
The sulfhydryl group modifying reagent, p-hydroxymercuribenzoate, inhibited the cytochalasin binding activity of the actin nuclei in the spectrin-band 4.1-actin complex from the erythrocyte membrane and of muscle F-actin. Kinetic studies indicated that while the cytochalasin binding activity was immediately inhibited, the actin remained filamentous and depolymerized slowly over a period of 1 to 2 h. Scatchard analysis of the binding data revealed that initially only the KD was affected. However, prolonged incubation led to depolymerization of the F-actin and dissociation of the spectrin-band 4.1-actin complex, resulting in loss of binding sites. It thus appears that certain actin sulfhydryl group(s) are important for cytochalasin binding. However, the most reactive sulfhydryl group (cys-374) on actin does not appear to be involved.
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The effects of specific sulfhydryl reagents, N-ethylmaleimide (NEM), p-chloromercuribenzoic acid (PCMB) and 5-5'-dithiobis(2-nitrobenzoic acid) (DTNB), were tested on the vasoactive intestinal peptide (VIP) receptor binding capacity of the human superficial melanoma-derived IGR39 cells. On intact cell monolayers NEM and PCMB inhibit the specific [125I]VIP binding in a time and dose-dependent manner while DTNB has no effect at any concentration tested. Inhibitory effects of NEM and PCMB on high and low affinity VIP receptor are not identical. With NEM-treated cells, only low affinity sites remained accessible to the ligand. Their affinity constant is not modified. With PCMB-treated cells, the binding capacity of high affinity sites is reduced by 56% while the binding capacity of low affinity sites is not significantly affected. For both types of binding sites, the affinity constants remain in the same range of that of untreated cells. On cells made permeable by lysophosphatidylcholine, DTNB is able to inhibit the specific [125I]VIP binding in a time and dose-dependent manner. The three sulfhydryl reagents stabilize the preformed [125I]VIP receptor complex whose dissociation in the presence of native VIP is significantly reduced. Labeling of free SH groups with tritiated NEM after preincubation of cells with DTNB and VIP made possible the characterization of reacting SH groups which probably belong to the receptor. Taken together, these data allow us to define three classes of sulfhydryl groups. In addition, it is shown that high and low affinity sites have different sensibility to sulfhydryl reagents.
Effect of sulfhydryl reagents on the transport of tetraethylammonium, an organic cation, has been studied in brush border membrane vesicles isolated from rat renal cortex. H+ gradient-dependent uptake of tetraethylammonium by the vesicles was inhibited by various sulfhydryl reagents in a dose-dependent manner, and the potency of the reagents was followed in the order of HgCl2 greater than p-chloromercuribenzoate, p-chloromercuribenzene sulfonate (PCMBS) greater than N-ethylmaleimide. In the absence of H+ gradient, tetraethylammonium uptake and efflux also were inhibited by p-chloromercuribenzoate and PCMBS. The sulfhydryl reagents did not affect the dissipation rate of H+ gradient across the membranes. Pretreatment of brush border membranes with PCMBS resulted in an inhibition of tetraethylammonium uptake in the presence and absence of H+ gradient, and this inhibition was reversed by subsequent treatment of the vesicles with thiols such as dithiothreitol, glutathione and cysteine. The inhibitory effect by PCMBS pretreatment was protected in the preincubation with unlabeled tetraethylammonium. These results suggest that sulfhydryl reagents inhibit the transport of tetraethylammonium by their specific interaction with the active sites of the carrier, and that sulfhydryl groups are essential for organic cation transport system in renal brush border membranes.
Sulfhydryl reagents are good stimulators of the enzymatic conversion of T4 to T3. Previous studies have suggested that the underlying mechanism for this property is that these reagents may serve as a cofactor in the reaction, or they maintain the enzyme in its active reduced form. We examined another possibility, namely that sulfhydryl reagents affect T4 itself and hence increase T3 generation by the enzyme. The substrate T4 and sulfhydryl reagents dithiothreitol, mercaptoethanol, and reduced glutathione were incubated at 37 C for 1 h and then combined with rat liver homogenate and incubated for additional 15 min to permit the enzymatic formation of T3. For control, substrate T4 was not preincubated with reagents, and those reagents were added just before the incubation. Preincubation of T4 with these reagents markedly enhanced subsequent T3 generation by the homogenate. Without preincubation, however, reduced glutathione and mercaptoethanol did not enhance T3 generation by simultaneous incubation with the homogenate. Dithiothreitol increased T3 generation when introduced into the incubation medium simultaneously with the homogenate and incubated only 15 min. However, the magnitude of stimulation was far less than that obtained from T4 preincubated with this reagent. These procedures did not alter the nonenzymatic generation of T3 significantly. These results suggest that sulfhydryl reagents can affect T4 itself and make it more susceptible to enzymatic outer ring monodeiodination.
The effect of certain sulfhydryl reagents and metal ions were studied on the carnitine acetyltransferase (CAT) activity from the skeletal muscle of the Arabian camel (Camelus dromedarius). DTNB and iodoacetamide caused concentration and time dependent inhibition of CAT activity. The inhibition seen with these sulfhydryl reagents could be protected with prior incubation of the enzyme with acetyl-Co A, suggesting that these reagents might interact with the same site. Among the various metal ions tested, Cu2+, Zn2+ and Hg2+ caused total inhibition at very low concentrations, while, Mn2+, Mo6+ and Co2+ caused between 32-52% inhibition at 10 mM concentrations. Alkali earth divalent metals Mg2+ and Ca2+ caused less than 15% inhibition at this concentration. These metal ions are probably interacting at certain nucleophilic groups in the enzyme thus disrupting its tertiary structure.
The addition of 1 X 10(-3) M or 1 X 10(-2) M 2-mercaptoethanol (2-MEt), a sulfhydryl reagent, produced a leftward displacement (potentiation) of the dose-response curves of mesenteric arterial strips for histamine, norepinephrine, serotonin, angiotensin II, prostaglandin F2 alpha and KCl. N-ethylmaleimide abolished the 2-MEt-induced potentiation of the arterial responses. Dithiothreitol (DTT) and cysteine also potentiated the contraction by these agonists in mesenteric arterial strips, suggesting that the sulfhydryl group plays a role in the arterial responses to various contractile agonists. In contrast to the mesenteric arterial strips, 2-MEt abolished the contraction by angiotensin II without greatly affecting contraction by norepinephrine in the thoracic aorta, femoral, renal and carotid arterial strips. These data suggest that there are regional differences in the responsiveness of rabbit arteries to sulfhydryl reagents. In mesenteric arterial strips treated with 1 X 10(-6) M histamine, 6 X 10(-8) M norepinephrine or 1 X 10(-9) M angiotensin II after exposure to Ca2+-free Krebs' bicarbonate solutions containing 0.1 mM EGTA, the addition of 2.5 mM CaCl2 caused a contraction (agonist-induced Ca2+-contraction). Sulfhydryl reagents potentiated each agonist-induced Ca2+-contraction in this artery. Moreover, in thoracic aortic strips, sulfhydryl reagents enhanced only histamine-induced Ca2+-contraction and attenuated norepinephrine- and angiotensin II-induced Ca2+-contractions. It is concluded that the reduction of a disulfide bond of the arterial strips to a sulfhydryl group affects the pharmacological receptor activities of the strips and that the changes in the receptor activities may be related to the changes in the transmembrane influx of calcium.
Secretory phospholipase A(2)s (sPLA(2)s) have been implicated in physiological and pathological events, but the regulatory mechanism(s) of their activities in cells remains to be solved. Previously, we reported that phenylarsine oxide (PAO), a sulfhydryl reagent, stimulated arachidonic acid (AA) release in rat pheochromocytoma PC12 cells. In this study, we examined the effects of thimerosal, another sulfhydryl reagent, to clarify the sulfhydryl modification and activation of sPLA(2) molecules in cells. Like PAO, thimerosal-stimulated AA release in an irreversible manner and the responses were not additive. Dithiol compounds such as dithiothreitol inhibited AA release from both the thimerosal- and the PAO-treated cells, and monothiol compounds (l-Cys and glutathione) decreased the thimerosal response. Both sulfhydryl reagents stimulated AA release from the HEK293T cells expressing human sPLA(2)X, and stimulated the sPLA(2) activities of bee venom sPLA(2) and the soluble fraction of sPLA(2)X-expressing cells. Our results suggest that the sPLA(2)s in cells are inactive and modification of disulfide bonds in the molecules can be a trigger of sPLA(2) activation in cells. Sulfhydryl reagents are useful tools for studying the regulatory mechanism(s) of sPLA(2) activity in cells.
Purified D-alanine carboxypeptidase IA of Escherichia coli is inhibited by penicillin G and binds penicillin G reversibly. The binding of penicillin to the enzyme is relatively insensitive to sulfhydryl reagents, while release of penicillin from the enzyme is severely inhibited by these reagents. The inhibition of release parallels the inhibition of carboxypeptidase activity by the sulfhydryl reagents. In the presence of the sulfhydryl reagent p-chloromercuribenzoate, an acyl-enzyme intermediate, produced by the reaction of carboxypeptidase IA with diacetyl-L-lysyl-D-alanyl-D-alanine, accumulates and can be isolated. These results indicate that binding of penicillin to carboxypeptidase IA occurs by an acylation step of the carboxypeptidase reaction, while penicillin release occurs by a deacylation step of the reaction. Only the latter is inhibited by sulfhydryl reagents.
The present study was designed to address the reactivity and accessibility of the particular class of sulfhydryl groups involved in the regulatory process of renin secretion. Both mercurial (such as P-chloromercuriphenyl sulfonate [PCMPS] and non-mercurial sulfhydryl reagents (for example, 6,6-dithiodinicotinic acid [DTDN]), which very slowly penetrate the cell membrane of intact cells, stimulated renin secretion. The membrane permeant sulfhydryl reagent N-ethylmaleimide had no effect on renin secretion but its membrane impermeant derivative, stilbene maleimide, strongly stimulated secretion. Furthermore, disulfide reducing agents such as dithiothreitol (DTT) had no effect on renin secretion at low concentrations, but strongly inhibited it at high concentrations. Several reagents which are known to primarily deplete cellular reduced glutathione were without effect on renin secretion. The stimulation of renin secretion by PCMPS was rapid in onset, and prevented and reversed by DTT and L-cysteine. Furthermore, the maximal stimulatory effect of PCMPS was not additive to that by diuretics with sulfhydryl reactivity (such as, ethacrynic acid and mersalyl). The stimulatory effect of PCMPS was not affected by diuretics which lack sulfhydryl reactivity (such as, bumetanide and furosemide). These results suggest that sulfhydryl reagents of both with and without diuretic activity stimulate renin secretion by reacting with specific class of sulfhydryl groups which are readily accessible from the extracellular compartment. In addition, these results provide further support the possibility that a sulfhydryl-disulfide interchange in the membrane may play a regulatory role in the renin secretory process.
The effect of sulfhydryl reagents on the Na+ permeability mechanisms of toad urinary bladder vesicles was examined. The reagents 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), iodosobenzoate, and ethylenimine were able to decrease amiloride-inhibited sodium uptake into vesicles when used at low concentrations. When used at higher concentrations these reagents were able to induce large increases in vesicle Na+ permeability that were not sensitive to amiloride. The reagent p-chloro-mercuribenzene sulfonate was able to induce such leaks even at low concentrations. The reagent N-ethylmaleimide was incapable of substantially affecting vesicle Na+ transport in any way. All of the effects observed could be reversed by removing the reagents from the solution surrounding the vesicles. Our results help explain the varied actions of sulfhydryl reagents on intact epithelial tissue.
Exposure to peroxides is known to increase the sensitivity of platelets towards activation by agonists. Similar platelet-activating effects are induced by sulfhydryl reagents that evoke Ca2+-induced Ca2+ release (CICR) by stimulating the Ca2+-releasing property of the inositol-1,4,5-trisphosphate receptor. We questioned whether these compounds may act by mobilising intracellular calcium in platelets by altering the intracellular glutathione redox state. Using FURA2-loaded, aspirin-treated platelets, Ca2+ signals were studied following exposure to the membrane-permeable sulfhydryl reagents, thimerosal and disulfiram, the glutathione peroxidase substrate, tert-butyl hydroperoxide, and the inhibitor of glutathione reductase, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In single platelets monitored by fluorescence imaging techniques, thimerosal and disulfiram elicited repetitive spiking in [Ca2+]i after variable lag times, indicating that these compounds stimulated CICR. BCNU caused [Ca2+]i spiking of only low amplitude, whereas tert-butyl hydroperoxide was inactive. In platelets in suspension devoid of extracellular CaCl2, the sulfhydryl reagents, at concentrations which decreased glutathione by 25%, strongly increased the Ca2+ responses of agonists that stimulated phospholipase C (thrombin) or acted independently of phospholipase C stimulation (thapsigargin). However, Ca2+ release was only slightly promoted by concentrations of BCNU that resulted in substantial depletion of the glutathione level. Tert-butyl hydroperoxide was without effect on glutathione, but partially inhibited Ca2+ mobilisation with these agonists. It is concluded that, in platelets, the potent CICR-promoting effects of sulfhydryl reagents are not solely due to their reaction with intracellular glutathione, but that extensive reduction in glutathione content is associated with Ca2+ mobilisation and CICR.
Organomercurial diuretics stimulate renin secretion although the underlying cellular mechanisms remain undefined. Since organomercurials are also known to react with sulfhydryl groups, the present studies determined the effects of sulfhydryl reagents on renin secretion. The effects of the non-diuretic mercurial agent, parachloromercuriphenyl-sulfonate (PCMPS), as well as that of other sulfhydryl reagents, N-ethylmaleimide (NEM), N-phenylmaleimide (NPM) and monobromotrimethylammoniobimane (qBBR), on renin secretion were determined in rabbit renal cortical slices. All four reagents stimulated renin secretion. NEM, which has a high membrane permeability, stimulated secretion to a relatively small extent and its effects were not apparent for at least one hour. Conversely, PCMPS, which is much less permanent than NEM, produced the largest stimulation and these effects were apparent within one hour. The stimulation of secretion by sulfhydryl reagents was independent of the concentration of Ca2+, Na+, and K+ in the incubation media, suggesting that the stimulation is not secondary to alterations of intracellular ion concentrations. These results raise the possibility of direct involvement of sulfhydryl groups of particular membrane protein(s) of the juxtaglomerular (JG) cells in some steps leading to renin secretion, and raise the possibility that sulfhydryl reactivity might in part account for the stimulatory effects of organomercurial and other diuretics.
The vacuolar class of (H+)-ATPases are highly sensitive to sulfhydryl reagents, such as N-ethylmaleimide. The cysteine residue which is responsible for inhibition of the coated vesicle (H+)-ATPase upon modification by N-ethylmalemide is located in subunit A and is able to form a disulfide bond with the cysteine moiety of cystine through an exchange reaction. This unique property distinguishes this cysteine residue from the remaining cysteine residues of the (H+)-ATPase. Using this reaction, we selectively labeled the cystine-reactive cysteine residue of subunit A with fluorescein-maleimide. After complete digestion of the labeled subunit A by V8 protease, a single labeled fragment of molecular mass 3.9 kDa was isolated and the amino-terminal sequence was determined. This fragment contains 2 cysteine residues, Cys240 and Cys254. Since Cys254 is conserved among all vacuolar (H+)-ATPases whereas Cys240 is not, it is likely that Cys254 is the residue which is responsible for the sensitivity of the vacuolar (H+)-ATPase to sulfhydryl reagents.
The effect of sulfhydryl reagents on phagocytosis and concomitant enzyme release and on ionophore A 23187 + Ca2+-induced exocytosis in rabbit polymorphonuclear leukocytes (PMN's) was studied. Membrane-penetrating sulfhydryl reagents such as cytochalasin A and N-naphthylmaleimide in micromolar concentrations inhibit both phagocytosis and exocytosis. Poorly penetrating reagents such as p-chloromercuribenzene sulfonate (pCMBS) and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), inhibit only in high concentrations (pCMBS), or they are ineffective as inhibitors (DTNB). Inhibition by pCMBS is not reversed by glutathione or dithiothreitol; this suggests that some pCMBS probably enters the cell. Specific intracellular sulfhydryl compounds appear to be essential in the cellular apparatus involved in phagocytosis and exocytosis; various possibilities are considered. A concentration of N-naphthylmaleimide which completely inhibits phagocytosis and exocytosis leaves cellular ATPase activity intact.