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Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl(-)/H(+) antiport current (I(ClC-3)) in HEK293 cells. The outward rectification and time dependence of I(ClC-3) closely resemble an endogenous HEK293 cell acid-activated Cl(-) current (ICl(acid)) seen at extracellular pH
Mitochondria are the major source of potentially damaging reactive oxygen species in most cells. Since ascorbic acid, or vitamin C, can protect against cellular oxidant stress, we studied the ability of mitochondria prepared from guinea pig skeletal muscle to recycle the vitamin from its oxidized forms. Although ascorbate concentrations in freshly prepared mitochondria were only about 0.2 mM, when provided with 6 mM succinate and 1 mM dehydroascorbate (the two-electron-oxidized form of the vitamin), mitochondria were able to generate and maintain concentrations as high as 4 mM, while releasing most of the ascorbate into the incubation medium. Mitochondrial reduction of dehydroascorbate was strongly inhibited by 1,3-bis(chloroethyl)-1-nitrosourea and by phenylarsine oxide. Despite existing evidence that mitochondrial ascorbate protects the organelle from oxidant damage, ascorbate failed to preserve mitochondrial alpha-tocopherol during prolonged incubation in oxygenated buffer. Nonetheless, the capacity for mitochondria to recycle ascorbate from its oxidized forms, measured as ascorbate-dependent ferricyanide reduction, was several-fold greater than total steady-state ascorbate concentrations. This, and the finding that more than half of the ascorbate recycled from dehydroascorbate escaped the mitochondrion, suggests that mitochondrial recycling of ascorbate might be an important mechanism for regenerating intracellular ascorbate.
Reduction of extracellular ferricyanide by intact cells reflects the activity of an as yet unidentified trans-plasma membrane oxidoreductase. In human erythrocytes, this activity was found to be limited by the ability of the cells to recycle intracellular ascorbic acid, its primary trans-membrane electron donor. Ascorbate-dependent ferricyanide reduction by erythrocytes was partially inhibited by reaction of one or more cell-surface sulfhydryls with p-chloromercuribenzene sulfonic acid, an effect that persisted in resealed ghosts prepared from such treated cells. However, treatment of intact cells with the sulfhydryl reagent had no effect on NADH-dependent ferricyanide or ferricytochrome c reductase activities of open ghosts prepared from treated cells. When cytosol-free ghosts were resealed to contain trypsin or pronase, ascorbate-dependent reduction of extravesicular ferricyanide was doubled, whereas NADH-dependent ferricyanide and ferricytochrome c reduction were decreased by proteolytic digestion. The trans-membrane ascorbate-dependent activity was also found to be inhibited by reaction of sulfhydryls on its cytoplasmic face. These results show that the trans-membrane ferricyanide oxidoreductase is limited by the ability of erythrocytes to recycle intracellular ascorbate, that it does not involve the endofacial NADH-dependent cytochrome b(5) reductase system, and that it is a trans-membrane protein that contains sensitive sulfhydryl groups on both membrane faces.
Interleukin-1beta (IL-1beta) increases the production of complement component C3 in enterocytes. Heat shock regulates the response to cytokines and other inflammatory mediators in various cell types. We tested the hypothesis that the heat-shock response regulates IL-1beta-induced C3 production in the enterocyte. Cultured Caco-2 cells, a human intestinal epithelial cell line, were treated with sodium arsenite (10-500 microM) for 1 h or subjected to hyperthermia (43 degrees C) for 1-4 h, and allowed to recover for 1 h. The cells were then treated with IL-1beta (0.5 ng/ml) for up to 24 h, whereafter C3 levels were measured by ELISA and C3 mRNA by Northern blot analysis. Heat-shock protein of 72 kDa (hsp72) was determined by Western blot analysis. Treatment of the cells with sodium arsenite or subjecting them to hyperthermia induced the expression of hsp72. The IL-1beta-induced expression of C3 mRNA and C3 production were down-regulated by hyperthermia and sodium arsenite in a dose-dependent fashion. The results suggest that the stress response induced by hyperthermia or sodium arsenite decreases IL-1beta-induced C3 production in human enterocytes.
Treatment with the sulfhydryl oxidant diamide denatures and aggregates cellular proteins, which prior studies have implicated as an oxidative damage that activates the heat shock transcription factor and induces thermotolerance. This study was initiated to further characterize cellular response to diamide-denatured proteins, including their involvement in diamide cytotoxicity. Cytotoxic diamide exposures at 37.0 degrees C denatured and aggregated cellular proteins in a manner that was proportional to cell killing, but this correlation was different than that established for heated cells. Diamide exposures at 24.0 degrees C were orders of magnitude less cytotoxic, with little additional killing occurring after diamide was removed and cells were returned to 37.0 degrees C. Thus, protein denaturation that occurred at 37.0 degrees C, after proteins were chemically destabilized by diamide at 24.0 degrees C [Freeman et al., J. Cell. Physiol., 164:356-366 (1995); Senisterra et al., Biochemistry 36: 11002-11011 (1997)], had little effect on cell killing. Thermotolerance protected cells against diamide cytotoxicity but did not reduce the amount of denatured and aggregated protein observed immediately following diamide exposure. However, denatured/aggregated proteins in thermotolerant cells were disaggregated within 17 h following diamide exposure, while no disaggregation was observed in nontolerant cells. This more rapid disaggregation of proteins may be one mechanism by which thermotolerance protects cells against diamide toxicity, as it has been postulated to do against heat killing. As with heat shock, nontoxic diamide exposures induced maximal tolerance against heat killing; however, there was no detectable, increased synthesis of heat shock proteins. Thus, diamide treatment proved to be a reproducible procedure for inducing a phase of thermotolerance that does not require new heat shock protein (HSP) synthesis, without having to use transcription or translation inhibitors to suppress HSP gene expression. These results complement those from studies with other stresses to establish the importance of protein denaturation/aggregation as a cytotoxic consequence of stress and a trigger for thermotolerance induction. The data also illustrate that differences in how proteins are denatured and aggregated can affect their cytotoxicity and the manner in which thermotolerance is expressed.
Previous studies have demonstrated that mutation in prostaglandin endoperoxide synthase-1 of Cys313 or Cys540 to Ser residues reduces cyclooxygenase and peroxidase activities by 80-90%. In the present work, we investigated the effect of these Cys-to-Ser mutations on the sensitivity of the enzyme to inhibition by cyclooxygenase inhibitors, the ability of the enzyme to form homodimers, the extent of glycosylation of the enzyme, and the sensitivity of the enzyme to maleimide enzyme inhibitors. No significant differences were observed between native and mutant enzymes in any of these parameters. The results suggest that the loss of activity observed in the mutant enzymes is not due to major differences in protein folding or aggregation. Most surprising was the finding that the sensitivity of prostaglandin H synthase-1 to maleimide-containing inhibitors was not affected by mutating any of the Cys to Ser. This indicates that the inhibition of cyclooxygenase activity affected by N-ethylmaleimide and N-carboxyheptylmaleimide is not due to modification of a cysteine residue.
Several fluorescent sulfhydryl reagents were tested as probes for assessing substrate-induced conformational change of the human erythrocyte glucose carrier. Of these, 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (Mal-ANS) inhibited 3-O-methylglucose transport most strongly and specifically labeled a previously characterized exofacial sulfhydryl on the glucose carrier. Analysis of equilibrium cytochalasin B binding in cells treated with Mal-ANS suggested that the inhibition of transport was due to a partial channel-blocking effect, and not to competition for the substrate binding site or to hindrance of carrier conformational change. In purified glucose carrier prepared from cells labeled on the exofacial sulfhydryl with Mal-ANS, a blue shift in the peak of fluorescence indicated that the fluorophore was in a relatively hydrophobic environment. Mal-ANS fluorescence in such preparations was quenched by ligands with affinity for the outward-facing carrier (ethylidene glucose, D-glucose, and maltose), but not by inhibitors considered to bind to the inward-facing carrier conformation (cytochalasin B or phenyl beta-D-glucoside). The effect of ethylidene glucose appeared to be related to an interaction with the glucose carrier, since the concentration dependence of ethylidene glucose-induced quench correlated well with the ability of the sugar analog to inhibit cytochalasin B binding to intact cells. The hydrophilic quenchers iodide and acrylamide decreased carrier-bound Mal-ANS fluorescence, resulting in downward-curving Stern-Volmer plots. Whereas ethylidene glucose enhanced iodide-induced quench, it had no effect on that of acrylamide.(ABSTRACT TRUNCATED AT 250 WORDS)
To test the role of cysteines in the function of GLUT1 glucose transporter, site-directed mutagenesis was used to replace all six GLUT1 cysteines with serine residues. When the individual and combined Cys-->Ser mutants were expressed in Xenopus laevis oocytes, zero-trans uptake of 3-O-methylglucose was comparable to that seen in native GLUT1. The "cysteineless" construct also retained the kinetic features of GLUT1, including an asymmetric transport mechanism and similar substrate and inhibitor affinities. Whereas GLUT1 transport was inhibited by sulfhydryl reagents, that of the "cysteineless" construct was not. These results show that cysteines are not required for GLUT1 function or oligomer formation. The "cysteineless" construct may therefore serve as a template for reintroducing cysteines back into GLUT1 at sites useful for testing transporter structure and function.
10-Formyltetrahydrofolate dehydrogenase (10-FTH-FDH: EC 126.96.36.199) catalyzes the NADP(+)-dependent oxidation of 10-formyltetrahydrofolate (10-HCO-H4PteGlu) to tetrahydrofolate (H4PteGlu) and CO2 and the NADP(+)-independent hydrolytic cleavage of 10-HCO-H4PteGlu to H4PteGlu and formate. 10-FTHFDH has a 485 amino acid domain at the C-terminus which is 46% identical to aldehyde dehydrogenase (ALDH: EC 188.8.131.52) and contains a conserved active site cysteine (Cys-707). 10-FTHFDH catalyzed NADP(+)-dependent oxidation of propanal and the hydrolysis of p-nitrophenyl acetate (pNPA) in a similar fashion to ALDH. Initial rate studies gave Km values of 46 and 636 microM, respectively, for NADP+ and propanal, while pNPA had a Km of 220 microM. Propanal was able to compete with 10-HCO-H4PteGlu for NADP(+)-dependent oxidation but had no effect on the NADP(+)-independent hydrolase reaction. N-Ethylmaleimide inhibited NADP(+)-dependent 10-HCO-H4PteGlu oxidation but only partially inhibited (65%) hydrolase activity. Disulfiram, a potent inhibitor of cytosolic ALDH, inhibited NADP(+)-dependent propanal oxidation by 10-FTHFDH. We propose that the dehydrogenase reaction of 10-FTHFDH has a mechanism which proceeds through thiohemiacetal and thioester intermediates, similar to that described for aldehyde dehydrogenase. 10-FTHFDH hydrolase activity was dependent on 2-mercaptoethanol and is probably an artifact of the assay system. The N-terminal domain of 10-FTHFDH shows identity to glycinamide ribonucleotide transformylase (EC 184.108.40.206) and contains a putative 10-HCO-H4PteGlu binding site but shows no GAR-TF activity. NADP(+)-dependent oxidation of 10-HCO-H4PteGlu by 10-FTHFDH was inhibited by the folate anti-metabolite, 5,10-dideazatetrahydrofolate, a known GAR-TF inhibitor.
The sulfhydryl reagent 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) was used to study the functional role of an exofacial sulfhydryl group on the human erythrocyte hexose carrier. Above 1 mM DTNB rapidly inhibited erythrocyte 3-O-methylglucose influx, but only to about half of control rates. Efflux was also inhibited, but to a lesser extent. Uptake inhibition was completely reversed by incubation and washing with 10 mM cysteine, whereas it was only partially reduced by washing in buffer alone, suggesting both covalent and noncovalent interactions. The covalent thiol-reversible reaction of DTNB occurred on the exofacial carrier, since (i) penetration of DTNB into cells was minimal, (ii) blockade of potential uptake via the anion transporter did not affect DTNB-induced hexose transport inhibition, and (iii) DTNB protected from transport inhibition by the impermeant sulfhydryl reagent glutathione-maleimide-I. Maltose at 120 mM accelerated the covalent transport inhibition induced by DTNB, whereas 6.5 microM cytochalasin B had the opposite effect, indicating under the one-site carrier model that the reactive sulfhydryl is on the outward-facing carrier but not in the substrate-binding site. In contrast to glutathione-maleimide-I, however, DTNB did not restrict the ability of the carrier to reorient inwardly, since it did not affect equilibrium cytochalasin B binding. Thus, carrier conformation determines exposure of the exofacial carrier sulfhydryl, but reaction of this group may not always "lock" the carrier in an outward-facing conformation.