Oxidative stress of human erythrocytes by iodate and periodate. Reversible formation of aqueous membrane pores due to SH-group oxidation

Human erythrocytes were exposed to oxidative stress by iodate and periodate. Oxidation causes a time- and concentration-dependent increase in membrane permeability for hydrophilic molecules and ions. The induced leak discriminates nonelectrolytes on the basis of molecular size and exhibits a very low activation energy (Ea = 1-4 kcal.mol-1). These results are reconcilable with the formation of aqueous pores. The pore size was approximated to be between 0.45 and 0.6 nm. This increase in permeability is reversible upon treatment with dithioerythritol. Blocking of membrane thiol groups with N-ethylmaleimide protects the membranes against leak formation. The oxidation causes dithioerythritol-reversible modification of membrane proteins as indicated by the gel electrophoretic behavior. These modifications can also be suppressed by blocking the membrane thiol groups with N-ethylmaleimide. About half of the membrane methionine is oxidized to acid hydrolysis-stable derivatives. A fast saturating increase in diene conjugation was observed in whole cells but not in isolated membranes, with only minor degradation of fatty acid chains. The oxidation of cell membrane lipids as well as oxidation of cell surface carbohydrates are not involved in leak formation. Taken together with earlier data (Deuticke, B., Poser, B., Lütkemeier, P. and Haest, C.W.M. (1983) Biochim. Biophys. Acta 731, 196-210), these findings indicate that formation of disulfide bonds by different oxidative mechanisms results in leaks with similar properties.     

Permeability of a Cell Membrane Junction : Dependence on energy metabolism

The ion permeability of the membrane junctions between Chironomus salivary gland cells is strongly depressed by treatments that are generally known to inhibit energy metabolism. These treatments include prolonged cooling at 6°–8°C, and exposure to dinitrophenol, cyanide, oligomycin, and N-ethylmaleimide. Intracellular injection of ATP appears to prevent depression of junctional permeability by dinitrophenol or to reverse it. Ouabain, azide, p-chloromercuriphenylsulfonic acid, reserpine, and acetazolamide fail to depress junctional permeability. Thus the ion permeability of the junctional membranes appears to depend on energy provided by oxidative phosphorylation. Possible energy-linked processes for maintaining junctional permeability are discussed, including processes involving transport of permeability-modifying species such as Ca⁺⁺.     

Vanadate inhibition of ATP-dependent H+ transport in membrane vesicles from turtle bladder epithelial cells

The ATP-dependent proton transport into vesicles of a mixed membrane fraction obtained from turtle bladder epithelial cells consists of at least two kinetically defined moieties: one, which is maximally inhibited by 25% with nanomolar levels of vanadate, but not inhibited at all with equimolar levels of N-ethylmaleimide, and another, which is maximally inhibited by 70% with micromolar levels of N-ethylmaleimide and by 25% with equimolar levels of vanadate. In contrast to the transport function, the associated enzymatic function (the ouabain-resistant ATPase activity) in these membranes, not inhibited by nanomolar levels of vanadate or N-ethylmaleimide, is maximally inhibited by 40% with micromolar levels of vanadate and by 13% with equimolar levels of N-ethylmaleimide. Independent of these kinetic differences between the enzyme and the transport functions, membranes containing the N-ethylmaleimide-sensitive proton transport function are electrophoretically separable from those containing the vanadate-sensitive transport function. For example, the kinetically defined, vanadate-sensitive proton transport function is recovered exclusively and kinetically identified in one of four electrophoretic membrane fractions, EF-II; while the N-ethylmaleimide-sensitive function is recovered in EF-III as well as in EF-II. Membranes of EF-IV, maximally enriched in ouabain-resistant ATPase activity, possess no proton transport function at all, even in the absence of N-ethylmaleimide or vanadate. Additional data under in vivo as well as under in vitro conditions are required to prove that the vanadate-sensitive proton transport in these vesicles is an in vitro manifestation of the mechanism responsible for generating the vanadate-sensitive luminal acidification process under in vivo conditions in the intact turtle bladder.     

The functional role of cysteine residues for c-Abl kinase activity

S-glutathionylation, the formation of mixed disulfides of glutathione with cysteine residues of proteins, is a broadly observed physiological modification that occurs in response to oxidative stress. Since cysteine residues are particularly susceptible to oxidative modification by reactive oxygen species, S-glutathionylation can protect proteins from irreversible oxidation. In this study, we show that the kinase activity of the non-receptor tyrosine kinase c-Abl is inhibited by in vitro thiol modification; specifically, the cysteine residues of c-Abl are modified by S-glutathionylation and by thiol alkylating agents such as 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid and N-ethylmaleimide. Modification of cysteine residues of c-Abl tyrosine kinase using glutathione disulfide and thiol alkylating agents corresponds to a concomitant loss of kinase activity. We also demonstrate that S-glutathionylation of c-Abl can be reversed using a physiological system involving glutaredoxin and this reversal restores c-Abl kinase activity. To our knowledge, these are the first data to show S-glutathionylation of c-Abl, and this modification may represent a mechanism of regulation of c-Abl kinase activity in cells under oxidative stress.     

Some differences in the conjugation of o-aminophenol and p-nitrophenol by the uridine diphosphate transglucuronylase of mouse-liver homogenates

1. A study of the catalysis of the formation of the glucuronides of o-aminophenol and p-nitrophenol by the uridine diphosphate transglucuronylase of homogenates of female mouse liver has been made, with reference to the effect of reagents reacting with thiol groups. 2. The synthesis of both glucuronides was completely inhibited by organic mercurials and N-ethylmaleimide. The inhibition was only partial with arsenite and the arsenoxides, iodoacetamide and o-iodosobenzoate. 3. The o-aminophenol system was much more sensitive than that for p-nitrophenol to all the thiol reagents, except N-ethylmaleimide, which was equally active in both systems. 4. At very low concentrations of the organic mercurials, the o-aminophenol system was activated. 5. With o-aminophenyl glucuronide formation, complete protection was given by glutathione and cysteine against the organic mercurials, N-ethylmaleimide and iodoacetamide, and partial protection against the arsenicals. Reversal was complete against the mercurials, and very limited against the arsenicals and iodoacetamide. The effects of N-ethylmaleimide and o-iodosobenzoate were irreversible. Results with p-nitrophenol were very similar. 6. Uridine diphosphate transglucuronylase was partially protected against p-chloromercuribenzoate and lewisite oxide by uridine diphosphate glucuronate, but not by o-aminophenol. 7. Glutathione did not prevent the decline in the rate of conjugation of o-aminophenol when homogenates were aged by incubation at 30°. Cysteine was unable to prevent or reverse inactivation by ultrasonic radiation.     

The inhibition of the uridine diphosphate-transglucuronylase activity of mouse-liver homogenates by thiol reagents

1. A study of the catalysis of the formation of the glucuronides of o-aminophenol and p-nitrophenol by the uridine diphosphate transglucuronylase of homogenates of female mouse liver has been made, with reference to the effect of reagents reacting with thiol groups. 2. The synthesis of both glucuronides was completely inhibited by organic mercurials and N-ethylmaleimide. The inhibition was only partial with arsenite and the arsenoxides, iodoacetamide and o-iodosobenzoate. 3. The o-aminophenol system was much more sensitive than that for p-nitrophenol to all the thiol reagents, except N-ethylmaleimide, which was equally active in both systems. 4. At very low concentrations of the organic mercurials, the o-aminophenol system was activated. 5. With o-aminophenyl glucuronide formation, complete protection was given by glutathione and cysteine against the organic mercurials, N-ethylmaleimide and iodoacetamide, and partial protection against the arsenicals. Reversal was complete against the mercurials, and very limited against the arsenicals and iodoacetamide. The effects of N-ethylmaleimide and o-iodosobenzoate were irreversible. Results with p-nitrophenol were very similar. 6. Uridine diphosphate transglucuronylase was partially protected against p-chloromercuribenzoate and lewisite oxide by uridine diphosphate glucuronate, but not by o-aminophenol. 7. Glutathione did not prevent the decline in the rate of conjugation of o-aminophenol when homogenates were aged by incubation at 30°. Cysteine was unable to prevent or reverse inactivation by ultrasonic radiation.     

Biochemical aspects of the visual process XXVIII. Classification of sulfhydryl groups in rhodopsin and other photoreceptor membrane proteins

Reaction of isolated bovine rod outer segment membrane with radioactiveN-ethylmaleimide, both in the presence and absence of 1% dodecyl sulfate followed by dodecyl sulfate-polyacrylamide gel electrophoresis, shows that six sulfhydryl groups (96% of total sulfhydryl in this membrane) are located on the rhodopsin molecule.On the basis of their reactivity towardsp-chloromercuribenzoate andp-chloromercuribenzene sulfonate in suspensions of outer segment membranes, the sulfhydryl groups of rhodopsin can be divided into three pairs. One pair is rapidly modified, both in light and darkness. This modification does not impair the recombination capacity of opsin with 11-cis retinaldehyde under regeneration of rhodopsin. A second pair is modified upon prolonged interaction with thep-chloromercuriderivatives in darkness. Modification of this pair leaves the typical rhodopsin absorbance at 500 nm intact, but a proportional loss of recombination capacity does occur. The third pair is only modified after illumination and is probably located in the vicinity of the chromophoric center.The difference between these results and those obtained by modification with dithiobis-(2-nitrobenzoic acid) orN-ethylmaleimide in suspension, where even upon prolonged exposure to light as well as in darkness only two sulfhydryl groups of rhodopsin are modified, is explained by the detergent-like character of thep-chloromercuri-derivatives.     

On the Opening of an Insensitive Cyclosporin A Non-specific Pore by Phenylarsine Plus Mersalyl

The purpose of this work was addressed to provide new information on the effect of thiol reagents on mitochondrial non-specific pore opening, and its response to cyclosporin A (CSA). To meet this proposal phenylarsine oxide (PHA) and mersalyl were employed as tools to induce permeability transition and CSA to inhibit it. PHA-induced mitochondrial dysfunction, characterized by Ca²⁺ efflux, swelling, and membrane de-energization, was inhibited by N-ethylmaleimide and CSA. Conversely, mersalyl failed to inhibit the inducing effect of phenylarsine oxide, it rather strengthened it. In addition, the effect of mersalyl was associated with cross-linking of membrane proteins. The content of membrane thiol groups accessible to react with PHA, mersalyl, and PHA plus mersalyl was determined. In all situations, permeability transition was accompanied by a significant decrease in the whole free membrane thiol content. Interestingly, it is also shown that mersalyl hinders the protective effect of cyclosporin A on PHA-induced matrix Ca²⁺ efflux.     

The reaction of myosin with N-ethylmaleimide in the presence of ADP

Myosin reacted with N-ethylmaleimide in the presence of ADP lost its ability to be activated by actin. Subfragment 1 behaved similarly. About 2 moles of N-ethylmaleimide per mole of Subfragment 1 were required to eliminate actin activation of the Mg²⁺-ATPase activity. At the point at which actin activation was lost the K⁺-EDTA-ATPase activity was also lost, but the Ca²⁺-activated ATPase activity was increased. Kinetic measurements indicated that the labelling with N-ethylmaleimide in the presence of ADP reduced V (the ATPase activity at infinite actin concentration) but did not effect K_app (which is related to the dissociation constant of the actin-Subfragment 1 complex). The Mg²⁺-activated activity of the reacted myosin alone remained unaltered and the ability to bind actin was retained. We propose that the N-ethylmaleimide labelling blocked the actin activation by preventing the accelerated release of hydrolysis products from the myosin.     

THE STRUCTURE OF THE COLLODION MEMBRANE AND ITS ELECTRICAL BEHAVIOR : XI. THE PREPARATION AND PROPERTIES OF "MEGAPERMSELECTIVE" COLLODION MEMBRANES COMBINING EXTREME IONIC SELECTIVITY WITH HIGH PERMEABILITY

1. The electronegative membranes described in the literature which show a high degree of ionic selectivity (permitting cations to pass and restricting the anions) have serious shortcomings: their absolute permeability is extremely low, much too small for convenient experimentation; their ionic selectivity in most cases is not as perfect as would be desirable, and is moreover adversely affected by prolonged contact with electrolyte solutions. 2. A method has been worked out to prepare membranes substantially free from these defects. Porous collodion membranes were cast on the outside of rotating tubes and then oxidized with 1 M NaOH. By allowing the oxidized porous membranes to dry in air on the tubes membranes of desirable properties are obtained. These membranes are smooth, have a well defined shape, and allow considerable handling without breaking. 3. This new type membrane when tested for ionic selectivity by the measurement of the "characteristic concentration potential," consistently gives potentials of 54 to 55 mv., the maximum thermodynamically possible value (at 25°C.) being 55.1 mv. This high degree of ionic selectivity is not lost on prolonged contact with water, and is only very slowly affected by electrolyte solutions. 4. The absolute permeability of the new type membranes can be varied over a very wide range by changing the time of oxidation. Under optimum conditions membranes can be obtained with a resistance in 0.1 N KCl solution of only 0.5 ohms per 50 cm.² membrane area. The absolute rate of cation exchange through these membranes between solutions of different uni-univalent electrolytes is very high, in one case, e.g. 0.9 m.eq. cations per 4 hours, the anion leak being 0.02 m.eq. Thus, the absolute permeability of the new type membranes is two to four orders of magnitude greater than the permeability of the dried collodion membranes and the oxidized ("activated") dried collodion membranes used heretofore. Because of the characteristic properties of the new type membranes the term "megapermselective" (or "permselective") collodion membranes is proposed for them.     

Redox proteomics of thiol proteins in mouse heart during ischemia/reperfusion using ICAT reagents and mass spectrometry

There is strong evidence for the involvement of reactive oxygen species in ischemia/reperfusion injury. Although oxidation of individual thiol proteins has been reported, more extensive redox proteomics of hearts subjected to ischemia/reperfusion has not been performed. We have carried out an exploratory study using mass spectrometry with isotope-coded affinity tags (ICAT) aimed at identifying reversible oxidative changes to protein thiols in Langendorff perfused isolated mouse hearts subjected to 20 min ischemia with or without aerobic reperfusion for 5 or 30 min. Reduced thiols were blocked by adding N-ethylmaleimide during protein extraction, then reversibly oxidized thiols in extracts of control perfused and treated hearts were reduced and labeled with the light and heavy ICAT reagents, respectively. Protein extracts were mixed in equal amounts and relative proportions of the isotope-labeled peaks were used to quantify oxidative changes between the control and the treated groups. Approximately 300 peptides with ICAT signatures were reliably identified in each sample, with 181 peptides from 118 proteins common to all treatments. A proportion showed elevated ICAT ratios, consistent with reversible thiol oxidation. This was most evident after early reperfusion, with apparent reversal after longer reperfusion. In comparison, there was gradual accumulation of protein carbonyls and loss of GSH with longer reperfusion. Many of the thiol changes were in mitochondrial proteins, including components of electron transport complexes and enzymes involved in lipid metabolism. The results are consistent with mitochondria being a major site of oxidant generation during early cardiac reperfusion and mitochondrial thiol proteins being targets for oxidation.     

Does Oxidation of Mitochondrial Cardiolipin Trigger a Chain of Antiapoptotic Reactions?

Oxidative stress causes selective oxidation of cardiolipin (CL), a fourtail lipid specific for the inner mitochondrial membrane. Interaction with oxidized CL transforms cytochrome c into peroxidase capable of oxidizing even more CL molecules. Ultimately, this chain of events leads to the pore formation in the outer mitochondrial membrane and release of mitochondrial proteins, including cytochrome c, into the cytoplasm. In the cytoplasm, cytochrome c promotes apoptosome assembly that triggers apoptosis (programmed cell death). Because of this amplification cascade, even an occasional oxidation of a single CL molecule by endogenously formed reactive oxygen species (ROS) might cause cell death, unless the same CL oxidation triggers a separate chain of antiapoptotic reactions that would prevent the CL-mediated apoptotic cascade. Here, we argue that the key function of CL in mitochondria and other coupling membranes is to prevent proton leak along the interface of interacting membrane proteins. Therefore, CL oxidation should increase proton permeability through the CL-rich clusters of membrane proteins (CL islands) and cause a drop in the mitochondrial membrane potential (MMP). On one hand, the MMP drop should hinder ROS generation and further CL oxidation in the entire mitochondrion. On the other hand, it is known to cause rapid fission of the mitochondrial network and formation of many small mitochondria, only some of which would contain oxidized CL islands. The fission of mitochondrial network would hinder apoptosome formation by preventing cytochrome c release from healthy mitochondria, so that slowly working protein quality control mechanisms would have enough time to eliminate mitochondria with the oxidized CL. Because of these two oppositely directed regulatory pathways, both triggered by CL oxidation, the fate of the cell appears to be determined by the balance between the CL-mediated proapoptotic and antiapoptotic reactions. Since this balance depends on the extent of CL oxidation, mito-chondria-targeted antioxidants might be able to ensure cell survival in many pathologies by preventing CL oxidation.     

Redox Regulation of Large Conductance Ca2+-activated K+ Channels in Smooth Muscle Cells

The effects of sulfhydryl reduction/oxidation on the gating of large-conductance, Ca²⁺-activated K⁺ (maxi-K) channels were examined in excised patches from tracheal myocytes. Channel activity was modified by sulfhydryl redox agents applied to the cytosolic surface, but not the extracellular surface, of membrane patches. Sulfhydryl reducing agents dithiothreitol, β-mercaptoethanol, and GSH augmented, whereas sulfhydryl oxidizing agents diamide, thimerosal, and 2,2′-dithiodipyridine inhibited, channel activity in a concentration-dependent manner. Channel stimulation by reduction and inhibition by oxidation persisted following washout of the compounds, but the effects of reduction were reversed by subsequent oxidation, and vice versa. The thiol-specific reagents N-ethylmaleimide and (2-aminoethyl)methanethiosulfonate inhibited channel activity and prevented the effect of subsequent sulfhydryl oxidation. Measurements of macroscopic currents in inside-out patches indicate that reduction only shifted the voltage/nP_o relationship without an effect on the maximum conductance of the patch, suggesting that the increase in nP_o following reduction did not result from recruitment of more functional channels but rather from changes of channel gating. We conclude that redox modulation of cysteine thiol groups, which probably involves thiol/disulfide exchange, alters maxi-K channel gating, and that this modulation likely affects channel activity under physiological conditions.     

Author index for volume 178, number 2

Ribonucleoside triphosphate reductase from Lactobacillus leichmannii, after reduction by exposure to dithiothreitol, has been alkylated with N-ethylmaleimide. Under conditions where the unreduced enzyme does not incorporate N-ethylmaleimide residues, the reduced enzyme is rapidly alkylated to the extent of one N-ethylmaleimide per molecule of enzyme. Loss of enzyme activity parallels the incorporation of N-ethylmaleimide. The value of the second-order rate constant for the alkylation at 0 °C of the reduced enzyme is influenced by the presence of some of the effectors of the enzyme, e.g., dATP at 200 μm reduces this parameter from 0.61 to 0.33 mm^?1 min^?1. The addition of coenzyme B₁₂ did not significantly affect the rate of alkylation of the reduced enzyme nor did it change the rate of alkylation of the dATP-reduced enzyme complex. Reduced enzyme, freed of dithiol, was shown to be unable to convert CTP stoichiometrically to dCTP when all of the usual enzyme assay components, except the dithiol, were present, nor did addition of CTP to the otherwise complete mixture decrease the level of N-ethylmaleimide-reactive thiol. However, the subsequent addition of dithiol was found to result in essentially complete reduction of CTP to dCTP. Hence, although reduction of the enzyme is probably required to generate an active form of the enzyme, the reduced enzyme does not appear to be capable of transferring its reducing equivalents stoichiometrically to the substrate to form dCTP from CTP. These results are discussed in terms of the mechanism of action of this enzyme.     

Biochemical aspects of the visual process. XXIII. Sulfhydryl groups and rhodopsin photolysis

1. The number of exposed sulfhydryl groups in cattle rod photoreceptor membranes has been determined in suspension and after solubilization in various detergents both before and after illumination.2. In suspensions, two sulfhydryl groups are modified per mole of rhodopsin, both by Ellman's reagent 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide, while no extra SH groups are uncovered upon illumination. Neither reagent affects the spectral integrity of rhodopsin at 500 nm and the recombination capacity is retained upon modification of both rhodopsin and opsin.3. However, in detergents (digitonin, Triton X-100 and cetyltrimethylammonium bromide (CTAB)) 2–3 additional sulfhydryl groups appear upon illumination, in agreement with earlier reports.4. A total number of six sulfhydryl groups and two disulfide bridges are found in rod photoreceptor membranes, expressed per mole of rhodopsin.5. DTNB reacts somewhat faster with membrane suspensions after than before illumination. The less reactive sulfhydryl modifying agents O-methylisourea and methyl-p-nitrobenzene sulfonate show a similar behavior.6. It is concluded that illumination of rhodopsin in vivo will not uncover additional SH groups, although the reactivity of one exposed SH group may increase somewhat. These findings also exclude a role of SH groups in the covalent binding of the chromophore.     

Heat and chilling induced disruption of redox homeostasis and its regulation by hydrogen peroxide in germinating rice seeds (Oryza sativa L., Cultivar Ratna)

Extremes of temperature (both heat and chilling) during early inbibitional phase of germination caused disruption of redox-homeostasis by increasing accumulation of reactive oxygen species (superoxide and hydrogen peroxide) and significant reduction of antioxidative defense (assessed in terms of total thiol content and activities of superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) in germinating tissues of rice (Oryza sativa L., cultivar Ratna). Imbibitional heat and chilling stress also induced oxidative damage to newly assembled membrane system by aggravating membrane lipid peroxidation and protein oxidation [measured in terms of thiobarbituric acid reactive substances (TBARS), free carbonyl content (C = O groups) and membrane protein thiol level (MPTL)]. Treatment with standardized low titer hydrogen peroxide during early imbibitional phase of germination caused significant reversal in oxidative damages to the newly assembled membrane system imposed by heat and chilling stress [evident from the data of TBARS, C = O, MPTL, ROS accumulation, membrane permeability status, membrane injury index and oxidative stress index] in seedlings of experimental rice cultivar. Imbibitional H₂O₂ pretreatment also caused up-regulation of antioxidative defense (activities of superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and total thiol content) in the heat and chilling stress-raised rice seedlings. When the parameters of early growth performances were assessed (in terms of relative growth index, biomass accumulation, relative germination performance, mean daily germination, T₅₀ value), it clearly exhibited significant improvement of early growth performances of the experimental rice cultivar. The result proposes that an ‘inductive pulse’ of H₂O₂ is required to switch on some stress acclimatory metabolism through which plant restores redox homeostasis and prevents or repairs oxidative damages to newly assembled membrane system caused by unfavorable environmental cues during early germination to the rice cultivar Ratna. The importance of mitigating oxidative damages to membrane lipid and protein necessary for post-germinative growth under extremes of temperature is also suggested.     

Evaluation of a dithiocarbamate derivative as a model of thiol oxidative stress in H9c2 rat cardiomyocytes

Thiol redox state (TRS) refers to the balance between reduced thiols and their corresponding disulfides and is mainly reflected by the ratio of reduced and oxidized glutathione (GSH/GSSG). A decrease in GSH/GSSG, which reflects a state of thiol oxidative stress, as well as thiol modifications such as S-glutathionylation, has been shown to have important implications in a variety of cardiovascular diseases. Therefore, research models for inducing thiol oxidative stress are important tools for studying the pathophysiology of these disease states as well as examining the impact of pharmacological interventions on thiol pathways. The purpose of this study was to evaluate the use of a dithiocarbamate derivative, 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA), as a pharmacological model of thiol oxidative stress by examining the extent of thiol modifications induced in H9c2 rat cardiomyocytes and its impact on cellular functions. The extent of thiol oxidative stress produced by 2-AAPA was also compared to other models of oxidative stress including hydrogen peroxide (H₂O₂), diamide, buthionine sulfoximine, and N,N׳-bis(2-chloroethyl)-N-nitroso-urea. Results indicated that 2-AAPA effectively inhibited glutathione reductase and thioredoxin reductase activities and decreased the GSH/GSSG ratio by causing a significant accumulation of GSSG. 2-AAPA also increased the formation of protein disulfides as well as S-glutathionylation. The alteration in TRS led to a loss of mitochondrial membrane potential, release of cytochrome c, and increase in reactive oxygen species production. Compared to other models, 2-AAPA is more potent at creating a state of thiol oxidative stress with lower cytotoxicity, higher specificity, and more pharmacological relevance, and could be utilized as a research tool to study TRS-related normal and abnormal biochemical processes in cardiovascular diseases.     

Protein synthesis catalyzed by thiol blocked ribosome preparations

Ribosome preparations respond heterogeneously to thiol blocking agents such as N-ethylmaleimide. About 55% of the particles rapidly lose the ability to catalyze peptide bond formation. The remaining 45% are immune. The only function related to peptide bond formation that appears abnormal in inactivated ribosomes is messenger RNA-directed transfer RNA binding. The change, however, is small relative to the alteration in activity observed. Thus it appears that thiol blocking, which selectively damages 30 S subunits, renders about half the 30 S subunits unable to co-ordinate properly with 50 S subunits in peptide bond formation. The implications of these results with regard to ribosome heterogeneity are discussed.     

Erythrocyte Lii-Nao countertransport system Inhibition by N-ethylmaleimide probes for a conformational change of the transport system

Human erythrocytes were treated by a series of SH-reagents, including maleimides, iodo compounds, mercurials and oxidizing agents. Rates of Li efflux into Na-rich medium, Li leak and Li_i-Na_o countertransport were then determined. Of the 13 different reagents studied, only N-ethylmaleimide, iodoacetamide and iodoacetate inhibited selectively the countertransport activity. The effect of the various reagents indicates that the sensitive SH-groups of the countertransport system are not externally exposed. N-Ethylmaleimide was used to probe for changes elicited by substrate cations in Li_i-Na_o countertransport. In Na- and Li-free medium, inhibition of Li_i-Na_o countertransport by N-ethylmaleimide of 35% was reached within 2 s. In Na or Li medium, maximal inhibition was twice as great, but was attained much more slowly, within 10 min. Kinetic data and Hill plot analysis indicate the involvement of two classes of SH-groups: one expressed in the various media with and without substrate cations, and an additional one, which becomes specifically available to N-ethylmaleimide in the presence of external Na or Li. The affinity of Na to the site promoting inhibition by N-ethylmaleimide (apparent K_m  12 mM) is higher than the affinity of Na to its external countertransport site (apparent K_m  25 mM), as reported by Sarakadi, B., Alifimoff, J.K., Gunn, R.B. and Tosteson, D.C. (1978) J. Gen. Physiol. 72, 249–265). Reactivity of $\text{N-}\text{ethyl}\text{[}{}^{14}\text{C}\text{]}\text{maleimide}$ was not modified by the media tested. It is concluded that external Na and Li cause a conformational change in the protein(s) of the countertransport system in human erythrocytes.