Identification of 30 kDa protein for Ca(2+) releasing action of myotoxin a with a mechanism common to DIDS in skeletal muscle sarcoplasmic reticulum.

The molecular mechanism of Ca(2+) release by myotoxin a (MTYX), a polypeptide toxin isolated from the venom of prairie rattlesnakes (Crotalus viridis viridis), was investigated in the heavy fraction of sarcoplasmic reticulum (HSR) of rabbit skeletal muscles. [(125)I]MYTX bound to four HSR proteins (106, 74, 53 and 30 kDa) on polyvinylidene difluoride (PVDF) membrane. DIDS, 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, bound predominantly to 30 kDa protein on the PVDF membrane, the molecular weight of which was similar to one of the MYTX binding proteins. The maximum (45)Ca(2+) release induced by caffeine (30 mM) was further increased in the presence of MYTX (10 microM) or DIDS (30 microM), whereas that induced by DIDS (30 microM) was not affected by MYTX (10 microM). MYTX inhibited [(3)H]DIDS binding to HSR in a concentration-dependent manner. Furthermore, [(125)I]MYTX binding to 30 kDa protein was inhibited by DIDS in a concentration-dependent manner. These results suggest that MYTX and DIDS release Ca(2+) from HSR in a common mechanism. The 30 kDa protein may be a target protein for the Ca(2+) releasing action of MYTX and DIDS.     

Binding of vanadate to human erythrocyte ghosts and subsequent events

Spectroscopic techniques were used to investigate the interaction between vanadate and human erythrocyte ghosts. Direct evidence from ⁵¹V nuclear magnetic resonance (NMR) studies suggested that the monomeric and polymeric vanadate species may bind to the anion binding sites of band 3 protein of the erythrocyte membrane. The results of ⁵¹V NMR studies and the quenching effect of vanadate on the intrinsic fluorescence of the membrane proteins indicated that in the low concentration range of vanadate (<0.6 mm), monomeric vanadate binds mostly to the anion sites of band 3 protein with the dissociation constant close to 0.23 mm. The experiments of sulfhydryl content titration by the method of Ellman and residue sulfhydryl-labeled fluorescence spectroscopies clearly displayed that vanadate reacts directly with sulfhydryl groups. The appearance of the anisotropic election spin resonance (ESR) signal of vanadyl suggests that a small (c. 3%) amount of vanadate was reduced by sulfhydryl groups of membrane proteins. The fluidity and order of intact ghost membrane were reduced by the reaction with vanadate, as shown by the ESR studies employing the protein- and lipid-specific spin labels. It was concluded that although vanadates mainly bind to band 3 protein, a minor part of vanadate may oxidize the residue sulfhydryl groups of membrane proteins, and thus decrease the fluidity of erythrocyte membrane.     

Influence of liposome charge and composition on their interaction with human blood serum proteins

The roles of sulfhydryl and disulfide groups in the specific binding of synthetic cannabinoid CP-55,940 to the cannabinoid receptor in membrane preparations from the rat cerebral cortex have been examined. Various sulfhydryl blocking reagents including p-chloromercuribenzoic acid (p-CMB), N-ethylmaleimide (NEM), o-iodosobenzoic acid (o-ISB), and methyl methanethiosulfonate (MMTS) inhibited the specific binding of [³H]CP-55,940 to the cannabinoid receptor in a dose-dependent manner. About 80–95% inhibition was obtained at a 0.1 mM concentration of these reagents. Scatchard analysis of saturation experiments indicates that most of these sulfhydryl modifying reagents reduce both the binding affinity (K_d) and capacity (B_max). On the other hand, DL-dithiothreitol (DTT), a disulfide reducing agent, also irreversibly inhibited the specific binding of [³H]CP-55,940 to the receptor and about 50% inhibition was obtained at a 5 mM concentration. Furthermore, 5mM DTT was abelt to dissociate 50% of the bound ligand from the ligand-receptor complex. The marked inhibition of [³H]CP-55,940 binding by sulfhydryl reagents suggests that at least one free sulfhydryl group is essential to the binding of the ligand to the receptor. In addition, the inhibition of the binding by DTT implies that besides free sulfhydryl group(s), the integrity of a disulfide bridge is also important for [³H]CP-55,940 binding to the cannabinoid receptor.     

Inhibition of adenylate cyclase by arsenite and cadmium: evidence for a vicinal dithiol requirement.

The effect of sodium arsenite and cadmium chloride on adenylate cyclase activity was examined in turkey erythrocyte membranes. Sodium arsenite was a weak inhibitor of adenylate cyclase -7mM produced only 60% inhibition. Its effect, however, was greatly potentiated by equimolar 2,3 dimercaprol- wherein 0.7 mM sodium arsenite inhibited 100% with an apparent Ki of 0.1 mM. Equimolar mercaptoethanol was less effective in potentiating sodium arsenite inhibition. Thus 0.7mM sodium arsenite in the presence of equimolar mercaptoethanol inhibited adenylate cyclase 56%. Excess 2,3 dimercaprol reversed inhibition by sodium arsenite or cadmium chloride. Sodium arsenite or cadmium chloride inhibited all forms of adenylate cyclase activity tested, including nonhormonal stimulation. Equimolar sodium arsenite and dimercaprol, at concentrations that caused 100% inhibition of adenylate cyclase activity, reduced the binding of the beta-receptor specific ligand iodohydroxybenzylpindolol by less than 15%. These results suggest that turkey erythrocyte membranes contain closely juxtaposed thiol groups and that interaction of such groups with arsenate interferes with the catalytic function of adenulate cyclase.     

Arsenite and cadmium(II) as probes of glucocorticoid receptor structure and function

Low concentrations of arsenite, but not arsenate, and Cd2+ blocked steroid binding to the glucocorticoid receptors of HTC cells. Inhibition by arsenite was faster and occurred at lower concentrations than for Cd2+. Half-maximal inhibition of [3H]dexamethasone binding was seen after a 30-min preincubation with approximately 7 microM arsenite. The effect of arsenite and of Cd2+ appears to be mediated by a reaction with vicinal dithiols of the receptor as shown by (a) the reversal of arsenite inhibition by much lower concentrations of dithiothreitol (approximately 0.1 mM) than of beta-mercaptoethanol (approximately 10 mM); (b) the ability of both arsenite and Cd2+ to block [3H]dexamethasone 21-mesylate labeling of receptors but not of other thiol-containing proteins; and (c) the known selectivity of arsenite and of Cd2+ for reactions with vicinal dithiols. Arsenite forms a tight complex with these vicinal dithiols since the removal of loosely associated arsenite by gel exclusion chromatography did not reverse the inhibition of steroid binding. The effect of other ions on steroid binding was also examined. Half-maximal inhibition of binding occurred with approximately 5 microM selenite, whereas up to 300 microM Zn2+ was without effect. Much higher concentrations of arsenite were required for effects on unactivated and activated complexes. Arsenite slowly induced a loss of unactivated complexes but rapidly inhibited a portion of the DNA binding of activated complexes. Any effect on activation occurred at arsenite concentrations equal to or higher than those that inhibited DNA binding. In contrast, Cd2+ concentrations similar to those that block steroid binding caused a biphasic loss of unactivated complexes and a marginal loss of activated complexes. This is the first report of effects of arsenite on glucocorticoid receptors. These results confirm directly our earlier hypothesis that steroid binding to rat glucocorticoid receptors involves a vicinal dithiol (Miller, N. R., and Simons, S. S., Jr. (1988) J. Biol. Chem. 263, 15217-15225) and show that arsenite is a potent new reagent for probing receptor structure and function.     

The vanadate complex of the calcium-transport ATPase of the sarcoplasmic reticulum, its formation and dissociation

Vanadate binding to different sarcoplasmic reticulum membrane preparations was determined by measuring bound vanadate colorimetrically and by phosphorylating the vanadate-free enzyme fraction with [gamma-32P] ATP. Colorimetry allowed the study of the dependence of equilibrium vanadate binding on ionized magnesium and the displacing effect of ionized calcium at vanadate concentrations greater than 0.1 mM only. At saturating magnesium concentration the enzyme binds 6-8 nmol vanadate/mg protein and half-maximum saturation is reached at 40 microM. Vanadate is displaced from the enzyme when its high-affinity calcium-binding sites are saturated and conversely calcium is solely displaced from its high-affinity binding sites by vanadate. The phosphorylation procedure allowed the measurement of equilibrium binding as well as the kinetics of vanadate binding and release at vanadate concentrations below 0.1 mM. Half-times of 30s and 3s were observed for vanadate release induced by 0.1 mM and 1 mM calcium respectively. Millimolar concentrations of ATP are required for vanadate displacement. Under equilibrium conditions the enzyme displays an affinity for vanadate of 1.6 X 10(6) M-1. The dependence on the concentration of vanadate of the rate of vanadate binding yielded an affinity of only 1 X 10(4) M-1. Closed vesicles bind vanadate much more slowly than calcium-permeable preparations. The initial rate of calcium-induced vanadate dissociation is accelerated considerably when the vesicles are made calcium permeable. The rate of vanadate dissociation from calcium-permeable vesicles reaches half-maximum values at 1-2 mM calcium indicating that the internal low-affinity calcium-binding sites must first be occupied in order to release bound vanadate. The results suggest that vanadate binding leads to a transition of the external high to internal low-affinity calcium-binding sites.     

New evidence for the essential role of arginine residues in anion transport across the red blood cell membrane

2,3-Butanedione, in the dark and in the presence of borate, reacts rapidly to inactivate the sulfate equilibrium exchange across the human red cell membrane. Reactivation occurs spontaneously after the removal of borate, indicating the reaction of butanedione with essential arginine residues. The inactivation of the transport system depends on the concentration of the reagent, on the incubation time and exhibits pseudo-first-order kinetics. Chloride ions are able to protect the transport system against inactivation with the reagent. This would suggest the participation of the modified residue in the substrate binding site. When the transport system is inhibited to 50-60% by butanedione, the transporter can still bind covalently the anion transport inhibitor 2H2DIDS up to 85 +/- 12% of its total binding capacity. 3H2DIDS concentration was either 3.15, 10 or 20 microM. Modification of resealed ghosts with 50 mM butanedione under conditions where the transport system is to more than 75% inhibited, causes a reduction of only about 30% of the reversibly bound 3H2DIDS.     

A1 H spin echo and 51V NMR study of the interaction of vanadate with intact erythrocytes

 The action of vanadate on intact human erythrocytes was studied by ¹H spin echo and ⁵¹V NMR spectroscopy as a model for the behaviour of vanadium(V) complexes in experimental diabetes. Vanadate is reduced by the intact erythrocyte at the expense of intracellular glutathione which rapidly depletes from the intracellular volume. Using the blocking agent 4,4′-diisothio-cyanatostilbene-2,2′-disulfonic acid (DIDS), which specifically blocks the anion transporter, vanadate reduction could be inhibited and glutathione depletion arrested. Thus, for the reaction with the intact cell to occur, vanadium(V) must cross the cell wall, possibly via the anion transporter. Nitrofurantoin was used to inhibit glutathione reductase in the erythrocyte suspensions. Under these conditions, treatment of the cells with vanadate induced glutathione oxidation prior to depletion. A study of the reaction of vanadate with haemolysate indicates that, without the influence of the membrane, rapid oxidation of glutathione to glutathione disulfide by the vanadyl cation occurs with no glutathione depletion, and that under these conditions vanadate reduction is incomplete. This study generates a model for the behaviour of vanadium complexes in vivo, providing a basis for the rational design and synthesis of new vanadium-based agents as insulin mimics. In essence, vanadium is transported across the membrane as vanadate(V), is reduced in situ by glutathione, and becomes complexed to a wide range of intracellular binding sites. Exchange reactions between glutathione and sulfhydryl groups present on haemoglobin and membrane lead to the depletion of glutathione from the cytosol. Received: 12 June 1996 / Accepted: 20 January 1997     

Factors determining the conformation and quaternary structure of isolated human erythrocyte band 3 in detergent solution.

Fluorescence spectroscopy was used to follow the kinetics of covalent binding of DIDS (4,4'-diisothiocyanato-2,2'-stilbenedisulfonate) to isolated band 3 in C12E8. We have discovered a dilution-induced loss in the ability of band 3 monomer to form a covalent adduct with DIDS. The loss in DIDS reactivity with dilution followed a 50:50 biphasic time course despite the use of a homogeneous preparation of band 3 oligomers. The loss in reactivity generally correlated with the association of band 3 dimers and tetramers to higher oligomeric structures. The final aggregated product was capable of binding BADS (4-benzamido-4'-amino-2,2'-stilbenedisulfonate) reversibly, but with an affinity nearly 30-fold lower than that of the starting material. Removal of the cytoplasmic domain of band 3 slowed the conformational interconversion of the integral domain by about 5-fold and inhibited the aggregation process. The conformational interconversion was slowed in the presence of 150 mM chloride but not in 90 mM sulfate. Covalent binding of DIDS inhibited the aggregation of band 3. Addition of 250 microM lipid inhibited both the loss of DIDS reactivity and the protein aggregation process. While several types of lipid offer protection, phosphatidic acid accelerated the decay process by eliminating the biphasicity. We conclude that the conformation of the integral domain of band 3 can be modulated allosterically by the addition of ligands, including various lipids. The results offer direct evidence for cooperative interactions between band 3 subunits during loss of activity, and they show that the cytoplasmic domain participates in the control of this transition.     

Remodulation of central carbon metabolic pathway in response to arsenite exposure in Rhodococcus sp. strain NAU‐1

Arsenite‐tolerant bacteria were isolated from an organic farm of Navsari Agricultural University (NAU), Gujarat, India (Latitude: 20°55′39.04″N; Longitude: 72°54′6.34″E). One of the isolates, NAU‐1 (aerobic, Gram‐positive, non‐motile, coccobacilli), was hyper‐tolerant to arsenite (As^III, 23 mM) and arsenate (As^V, 180 mM). 16S rRNA gene of NAU‐1 was 99% similar to the 16S rRNA genes of Rhodococcus (Accession No. HQ659188). Assays confirmed the presence of membrane bound arsenite oxidase and cytoplasmic arsenate reductase in NAU‐1. Genes for arsenite transporters (arsB and ACR3(1)) and arsenite oxidase gene (aoxB) were confirmed by PCR. Arsenite oxidation and arsenite efflux genes help the bacteria to tolerate arsenite. Specific activities of antioxidant enzymes (catalase, ascorbate peroxidase, superoxide dismutase and glutathione S‐transferase) increased in dose‐dependent manner with arsenite, whereas glutathione reductase activity decreased with increase in As^III concentration. Metabolic studies revealed that Rhodococcus NAU‐1 produces excess of gluconic and succinic acids, and also activities of glucose dehydrogenase, phosphoenol pyruvate carboxylase and isocitrate lyase were increased, to cope with the inhibited activities of glucose‐6‐phosphate dehydrogenase, pyruvate dehydrogenase and α‐ketoglutarate dehydrogenase enzymes respectively, in the presence of As^III. Enzyme assays revealed the increase in direct oxidative and glyoxylate pathway in Rhodococcus NAU‐1 in the presence of As^III.     

Solubilization and reconstitution of the renal phosphate transporter

Proteins from brush-border membrane vesicles of rabbit kidney cortex were solubilized with 1% octylglucoside (protein to detergent ratio, 1:4 (w/w). The solubilized proteins (80.2 +/- 2.3% of the original brush-border proteins, n = 10, mean +/- S.E.) were reconstituted into artificial lipid vesicles or liposomes prepared from purified egg yolk phosphatidylcholine (80%) and cholesterol (20%). Transport of Pi into the proteoliposomes was measured by rapid filtration in the presence of a Na+ or a K+ gradient (out greater than in). In the presence of a Na+ gradient, the uptake of Pi was significantly faster than in the presence of a K+ gradient. Na+ dependency of Pi uptake was not observed when the liposomes were reconstituted with proteins extracted from brush-border membrane vesicles which had been previously treated with papain, a procedure that destroys Pi transport activity. Measurement of Pi uptake in media containing increasing amounts of sucrose indicated that Pi was transported into an intravesicular (osmotically sensitive) space, although about 70% of the Pi uptake appeared to be the result of adsorption or binding of Pi. However, this binding of Pi was not dependent upon the presence of Na+. Both Na+-dependent transport and the Na+-independent binding of Pi were inhibited by arsenate. The initial Na+-dependent Pi transport rate in control liposomes of 0.354 nmol Pi/mg protein per min was reduced to 0.108 and 0 nmol Pi/mg protein per min in the presence of 1 and 10 mM arsenate, respectively. Future studies on reconstitution of Pi transport systems must analyze and correct for the binding of Pi by the lipids used in the formation of the proteoliposomes.     

Existence of high- and low-affinity vanadate-binding sites on Ca(2+)-ATPase of the sarcoplasmic reticulum.

The binding of vanadate to isolated sarcoplasmic reticulum (SR) membranes was measured colorimetrically by equilibrium sedimentation and ion exchange column filtration. The concentration dependence of vanadate binding exhibited a biphasic curve with two phases of equal amplitude. A similar biphasic curve of the vanadate dependence was observed with the purified Ca(2+)-ATPase prepared by deoxycholate extraction. Sites of vanadate binding could be classified into two distinct species based on apparent affinity; the high-affinity binding sites have a dissociation constant below 0.1 microM, and the low-affinity sites one of 36 microM. The maximum amount of vanadate bound to each of the high- or low-affinity sites was estimated to be 2.6-3.6 nmol/mg SR protein, which corresponds to approximately 0.5 mol of vanadate bound per mol of Ca(2+)-ATPase. These results indicate that 1 mol of Ca(2+)-ATPase contains 0.5 mol of high-affinity vanadate-binding sites as well as 0.5 mol of low-affinity vanadate-binding sites. Vanadate binding to the low-affinity sites was competitively inhibited by inorganic phosphate, while vanadate binding to the high-affinity sites resulted in a non-competitive inhibition of the phosphoenzyme formation from inorganic phosphate. When SR membrane were solubilized with polyoxy-ethylene-9-laurylether (C12E9), the vanadate binding exhibited a monophasic concentration dependency curve with a dissociation constant of 13 microM. The number of vanadate-binding sites was estimated to be 7.2 nmol/mg SR protein which represents about 1 mol of site per mol of Ca(2+)-ATPase. Vanadate binding to the solubilized Ca(2+)-ATPase was competitively inhibited by inorganic phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

51V NMR study of vanadate binding to myosin and its subfragment 1

The binding of various forms of vanadate to myosin and myosin subfragment 1 (S-1) was studied by 51V NMR at increasing vanadate concentrations between 0.06 and 1.0 mM. The distribution of the various forms of vanadate in the solution depended on the total concentration of vanadate. At low concentrations, the predominant vanadate form was monomeric, while at high concentration, it was tetrameric. The presence of myosin or S-1 in the solution produced a significant broadening of the signal of each form of vanadate, indicating that all of them bind to the protein. Addition of ATP, which does not affect the 51V NMR spectra in the absence of proteins, causes their significant alteration in the presence of myosin or S-1. The changes, which include the broadening of the signal of the monomeric and the narrowing of the signal of the oligomeric vanadate forms, indicate that more monomeric and less oligomeric vanadate binds to the proteins in the presence than in the absence of ATP. Irradiation by near-UV light in the presence of vanadate cleaves S-1 at three specific sites--at 23, 31, and 74 kDa from the N-terminus. The cleavages at 23 and 31 kDa are specifically inhibited by the addition of ATP. The vanadate-associated photocleavage of S-1 also depends on the total concentration of vanadate; it is observed only when the concentration of vanadate is at least 0.2 mM. This was also the lowest concentration at which oligomeric vanadate was detected in the 51V NMR spectra. From the parallel concentration dependence of the photocleavage and the appearance of the tetrameric vanadate, it is concluded that photocleavage occurs only when tetrameric vanadate binds to S-1.     

Mechanism of inhibition of transducin guanosine triphosphatase activity by vanadate

The visual excitation system of the retinal rod outer segments and the hormone-sensitive adenylate cyclase complex are regulated through guanine nucleotide-binding proteins, transducin in the former and inhibitory and stimulatory regulatory components, Gi and Gs, in the latter. These proteins are functionally and structurally similar; all are heterotrimers composed of alpha, beta, and gamma subunits and exhibit guanosine triphosphatase activity stimulated by light-activated rhodopsin or the agonist-receptor complex. Adenylate cyclase can be stimulated by vanadate, which, like NaF, probably acts through Gs. Effects of vanadate on the function of a guanine nucleotide-binding protein were investigated in a reconstituted model system consisting of purified transducin subunits (T alpha, T beta gamma) and rhodopsin in phosphatidylcholine vesicles. Vanadate (decameric) inhibited [3H]GTP binding to T alpha and noncompetitively inhibited GTP hydrolysis in a concentration-dependent manner with maximal inhibition of approximately 90% at 3-5 mM. Vanadate also inhibited release of bound GDP but did not affect the rate of hydrolysis of bound GTP (single turnover rate), indicating that vanadate did not interfere with the intrinsic GTPase activity of T alpha. Binding of T alpha to rhodopsin and the ADP-ribosylation of T alpha by pertussis toxin, both of which are enhanced in the presence of T beta gamma, were inhibited by vanadate. These findings are consistent with the conclusion that vanadate can cause the dissociation of T alpha from T beta gamma, resulting in the inhibition of GDP-GTP exchange and thereby GTP hydrolysis. Adenylate cyclase activation could result from a similar effect of vanadate on Gs.     

Insights into the structure, solvation, and mechanism of ArsC arsenate reductase, a novel arsenic detoxification enzyme.

BACKGROUND: In Escherichia coli bearing the plasmid R773, resistance to arsenite, arsenate, antimonite, and tellurite is conferred by the arsRDABC plasmid operon that codes for an ATP-dependent anion pump. The product of the arsC gene, arsenate reductase (ArsC), is required to efficiently catalyze the reduction of arsenate to arsenite prior to extrusion. RESULTS: Here, we report the first X-ray crystal structures of ArsC at 1.65 A and of ArsC complexed with arsenate and arsenite at 1.26 A resolution. The overall fold is unique. The native structure shows sulfate and sulfite ions binding in the active site as analogs of arsenate and arsenite. The covalent adduct of arsenate with Cys-12 in the active site of ArsC, which was analyzed in a difference map, shows tetrahedral geometry with a sulfur-arsenic distance of 2.18 A. However, the corresponding adduct with arsenite binds as a hitherto unseen thiarsahydroxy adduct. Finally, the number of bound waters (385) in this highly ordered crystal structure approaches twice the number expected at this resolution for a structure of 138 ordered residues. CONCLUSIONS: Structural information from the adduct of ArsC with its substrate (arsenate) and with its product (arsenite) together with functional information from mutational and biochemical studies on ArsC suggest a plausible mechanism for the reaction. The exceptionally well-defined water structure indicates that this crystal system has precise long-range order within the crystal and that the upper limit for the number of bound waters in crystal structures is underestimated by the structures in the Protein Data Bank.     

Effects of arsenite stress on growth and proteome of Klebsiella pneumoniae

In the present study an arsenite, As(III), tolerating bacterium, MR4, was isolated from Mulla River Pune, India, capable of reducing arsenate to arsenite and identified as Klebsiella pneumoniae (HQ857583). Comparative proteomic analysis using two-dimensional gel electrophoresis (2-DGE) and matrix assisted laser desorption ionization-time of flight-time of flight (MALDI-TOF/TOF) was used to monitor the proteins undergoing changes in expression levels under 2.5 mM As(III) stress. The 2-DGE proteome map has shown that 60 proteins were differentially expressed under As(III) stress, of which 39 proteins were successfully identified with a MASCOT score greater than 70 (p<0.05). Among the identified proteins, membrane transport/binding proteins, porins, and amino acid metabolism enzymes were down-regulated while stress responsive proteins and antioxidant enzymes were up-regulated. Proteins involved in carbohydrate metabolism, particularly those in pentose phosphate pathway were also up-regulated while those involved in pyruvate metabolism were down-regulated. However, proteins involved in glycolysis and tricarboxylic acid cycle showed a mixed regulation response. These findings provide new insights into the probable mechanisms by which K. pneumoniae (HQ857583) could be adapting to As(III) stress.     

Uptake and toxicity of arsenite and arsenate in cultured brain astrocytes

Inorganic arsenicals are environmental toxins that have been connected with neuropathies and impaired cognitive functions. To investigate whether such substances accumulate in brain astrocytes and affect their viability and glutathione metabolism, we have exposed cultured primary astrocytes to arsenite or arsenate. Both arsenicals compromised the cell viability of astrocytes in a time- and concentration-dependent manner. However, the early onset of cell toxicity in arsenite-treated astrocytes revealed the higher toxic potential of arsenite compared with arsenate. The concentrations of arsenite and arsenate that caused within 24 h half-maximal release of the cytosolic enzyme lactate dehydrogenase were around 0.3 mM and 10 mM, respectively. The cellular arsenic contents of astrocytes increased rapidly upon exposure to arsenite or arsenate and reached after 4 h of incubation almost constant steady state levels. These levels were about 3-times higher in astrocytes that had been exposed to a given concentration of arsenite compared with the respective arsenate condition. Analysis of the intracellular arsenic species revealed that almost exclusively arsenite was present in viable astrocytes that had been exposed to either arsenate or arsenite. The emerging toxicity of arsenite 4 h after exposure was accompanied by a loss in cellular total glutathione and by an increase in the cellular glutathione disulfide content. These data suggest that the high arsenite content of astrocytes that had been exposed to inorganic arsenicals causes an increase in the ratio of glutathione disulfide to glutathione which contributes to the toxic potential of these substances.