Low Concentrations Of Dithiothreitol Research Articles

Evaluation and minimization of over-alkylation in proteomic sample preparation

Reduction and alkylation are essential steps in shotgun proteomic sample preparation, but over-alkylation can occur on peptide N-terminus and amino acid residues other than cysteine, which adversely affects protein identification and quantification. To date, different sample preparation protocols are used in different laboratories, but a systematic comparison of the published protocols has not been done yet in the aspect of over-alkylation. Here, we comprehensively evaluated various protocols using different denaturants, different digestion buffers and different concentrations of reduction and alkylation reagents. The suggested protocols from the manufacturers of RapiGest and ProteaseMAX induce the highest degree of over-alkylation compared with others. Carbamidomethylation at lysine increases over 4 times while using triethylammonium bicarbonate buffer compared with that using others, and it should be minimized for improving the labeling efficiency of stable isotope labeling reagents that label lysine residues. The use of digestion buffers at pH 6 reduces over-alkylation, but greatly introduces some other artificial modifications. Excessive iodoacetamide was proved to be the major cause of over-alkylation during trypsin digestion. Therefore, over-alkylation can be minimized by using low concentration of dithiothreitol and iodoacetamide for reduction and alkylation or quenching the excessive iodoacetamide before digestion.

Aeration mitigates endoplasmic reticulum stress in Saccharomyces cerevisiae even without mitochondrial respiration

Saccharomyces cerevisiae is a facultative anaerobic organism that grows well under both aerobic and hypoxic conditions in media containing abundant fermentable nutrients such as glucose. In order to deeply understand the physiological dependence of S. cerevisiae on aeration, we checked endoplasmic reticulum (ER)-stress status by monitoring the splicing of HAC1 mRNA, which is promoted by the ER stress-sensor protein, Ire1. HAC1-mRNA splicing that was caused by conventional ER-stressing agents, including low concentrations of dithiothreitol (DTT), was more potent in hypoxic cultures than in aerated cultures. Moreover, growth retardation was observed by adding low-dose DTT into hypoxic cultures of ire1Δ cells. Unexpectedly, aeration mitigated ER stress and DTT-induced impairment of ER oxidative protein folding even when mitochondrial respiration was halted by the ρo mutation. An ER-located protein Ero1 is known to directly consume molecular oxygen to initiate the ER protein oxidation cascade, which promotes oxidative protein folding of ER client proteins. Our further study using ero1-mutant strains suggested that, in addition to mitochondrial respiration, this Ero1-medaited reaction contributes to mitigation of ER stress by molecular oxygen. Taken together, here we demonstrate a scenario in which aeration acts beneficially on S. cerevisiae cells even under fermentative conditions.

Redox-responsive shell-sheddable micelles self-assembled from amphiphilic chondroitin sulfate-cholesterol conjugates for triggered intracellular drug release

Reduction-responsive shell-sheddable micelles based on disulfide-linked chondroitin sulfate-cholesterol conjugates (CS-ss-Chol) were developed for efficient encapsulation and triggered intracellular release of hydrophobic drug. CS-ss-Chol conjugates with different cholesterol content were synthesized and self-assembled into spherical micelles in aqueous medium with tunable sizes (124–237nm) and desirable critical micelle concentration (CMC) (5.8–3.6×10−2mg/L). These micelles were highly sensitive to intracellular reductive environments, which resulted in fast formation of large aggregates due to shedding of the CS shells through reductive cleavage of the intermediate disulfide bonds. Quercetin, a model hydrophobic drug, was efficiently loaded into the core of these micelles, with a drug loading efficiency of 23.4%. The in vitro release studies showed that quercetin release was minimal (<20% in 24h) at low concentrations of dithiothreitol (DTT, 10μM) mimicking the extracellular environment, but significantly enhanced (>50% in 24h) under tumor-relevant reductive condition (DTT, 20mM). MTT assays against HeLa cells and HSF cells indicated that these CS-ss-Chol micelles had negligible toxicity up to a conjugate concentration of 200μg/mL. These reduction-responsive shell-sheddable micelles may offer a promising and generic means to intracellularly delivery a wide variety of hydrophobic payloads in a selective and tunable way.

Characterization of Activity of a Potential Food-Grade Leucine Aminopeptidase from Kiwifruit

Aminopeptidase (AP) activity in ripe but firm fruit of Actinidia deliciosa was characterized using L-leucine-p-nitroanilide as a substrate. The enzyme activity was the highest under alkaline conditions and was thermolabile. EDTA, 1,10-phenanthroline, iodoacetamide, and Zn2+ had inhibitory effect while a low concentration of dithiothreitol (DTT) had stimulatory effect on kiwifruit AP activity. However, DTT was not essential for the enzyme activity. The results obtained indicated that the kiwifruit AP was a thiol-dependent metalloprotease. Its activity was the highest in the seeds, followed by the core and pericarp tissues of the fruit. The elution profile of the AP activity from a DEAE-cellulose column suggested that there were at least two AP isozymes in kiwifruit: one unadsorbed and one adsorbed fractions. It is concluded that useful food-grade aminopeptidases from kiwifruit could be revealed using more specific substrates.

Two Distinct Disulfide Bonds Formed in Human Heat Shock Transcription Factor 1 Act in Opposition To Regulate Its DNA Binding Activity

Under circumstances of heat stress, heat shock transcription factor 1 (HSF1) plays important roles in heat shock protein expression. In this study, an increasing concentration of dithiothreitol (DTT) was found to either enhance or inhibit the heat-induced trimerization of HSF1, suggesting the involvement of dual redox-dependent HSF1 activation mechanisms. Our in vitro experiments show that the heat-induced bonding between the cysteine C36 and C103 residues of HSF1 forms an intermolecular disulfide covalent bond (SS-I bond) and that it directly causes HSF1 to trimerize and bond to DNA. Gel filtration assays show that HSF1 can form intermolecular hydrophobic interaction-mediated (iHI-m) noncovalent oligomers. However, the lack of a trimerization domain prevents HSF1 activation, which suggests that iHI-m noncovalent trimerization is a precondition of SS-I bond formation. On the other hand, intramolecular SS-II bond (in which the C153, C373, and C378 residues of HSF1 participate) formation inhibits this iHI-m trimerization, thereby preventing SS-I bond formation and DNA binding. Thus, HSF1 activation is regulated positively by intermolecular SS-I bond formation and negatively by intramolecular SS-II bond formation. Importantly, these two SS bonds confer different DTT sensitivities (the SS-II bond is more sensitive). Therefore, a low concentration of DTT cleaves the SS-II bond but not the SS-I bond and thus improves DNA binding of HSF1, whereas a high concentration DTT cuts both SS bonds and inhibits HSF1 activation. We propose that these interesting effects further explain cellular HSF1 trimerization, DNA binding, and transcription when cells are under stress.

Thioredoxin-h1 Reduces and Reactivates the Oxidized Cytosolic Malate Dehydrogenase Dimer in Higher Plants

Cytosolic malate dehydrogenase (cytMDH) was captured by thioredoxin affinity chromatography as a possible target protein of cytosolic thioredoxin (Yamazaki, D., Motohashi, K., Kasama, T., Hara, Y., and Hisabori, T. (2004) Plant Cell Physiol. 45, 18-27). To further dissect this interaction, we aimed to determine whether cytMDH can interact with the cytosolic thioredoxin and whether its activity is redox-regulated. We obtained the active recombinant cytMDH that could be oxidized and rendered inactive. Inactivation was reversed by incubation with low concentrations of dithiothreitol in the presence of recombinant Arabidopsis thaliana thioredoxin-h1. Inactivation of cytMDH was found to result from formation of a homodimer. By cysteine mutant analysis and peptide mapping analysis, we were able to determine that the cytMDH homodimer occurs by formation of a disulfide bond via the Cys(330) residue. Moreover, we found this bond to be efficiently reduced by the reduced form of thioredoxin-h1. These results demonstrate that the oxidized form cytMDH dimer is a preferable target protein of the reduced form thioredoxin-h1 as suggested by thioredoxin affinity chromatography.

Redox modulation of GABA A receptors obscured by Zn 2+ complexation

Redox reagents are thought to modulate γ-Aminobutyric acid type A (GABA A) receptors by regulating the redox state of the N-terminal disulphide bridge. Examining the redox sensitivity of recombinant GABA A receptors in human embryonic kidney cells, using whole-cell patch clamp techniques, revealed that α1β2 H267A and α1β2γ2 receptors, which are both less sensitive to Zn 2+ and H + modulation, ablated the potentiating effect of the reducing agent, dithiothreitol (DTT) seen for α1β2 receptors. This effect could result from disruption to the redox signal transduction pathway or be due to DTT chelating Zn 2+ from its H267 inhibitory binding site, consequently potentiating GABA-activated currents in α1β2 but not α1β2 H267A or α1β2γ2 receptors. A Zn 2+ chelating agent, tricine, potentiated GABA currents for the αβ constructs and vertically displaced GABA dose–response curves, suggesting that these receptors are subject to some inhibition by basal Zn 2+. Tricine, did not affect the GABA currents of either α1β2 H267A or α1β2γ2 receptors but did prevent the potentiation by 2 mM DTT and reduced the potentiation caused by 10 mM DTT on α1β2 receptors. Thus, at low concentrations of DTT, a substantial component of the potentiation probably occurs via Zn 2+ chelation from H267 in the ion channel. In contrast, at higher DTT concentrations, it is more likely to be acting as a redox agent, which modulates both αβ and αβγ subunit receptors.

Two-phase unfolding pathway of ribonuclease A during denaturation induced by dithiothreitol.

The dynamics of the unfolding process of bovine pancreatic ribonuclease A (RNase A) unfolded by dithiothreitol (DTT) at a low concentration of 1:30 were investigated in alkaline phosphate-buffered saline solutions at 303K and 313K by using proton nuclear magnetic resonance ((1)H NMR) spectra. Three NMR spectral parameters including Shannon entropy, mutual information, and correlation coefficient were introduced into the analysis. The results show that the unfolding process of RNase A was slowed to the order of many hours, and the kinetics of the unfolding pathway described by the three parameters is best fit by a model of two consecutive first-order reactions. Temperature greatly influences the rate constants of the unfolding kinetics with different temperature effects observed for the fast and the slow processes. The consistencies and the differences between the three sets of parameters show that they reflect the same relative denaturation pathway but different spectra windows of the unfolding process of RNase A. The results suggest that the unfolding process of RNase A induced by low concentrations of DTT is a two-phase pathway containing fast and slow first-order reactions.

Effect of transition metals on binding of p53 protein to supercoiled DNA and to consensus sequence in DNA fragments.

Recently we have shown that wild-type human p53 protein binds preferentially to supercoiled (sc) DNA in vitro in both the presence and absence of the p53 consensus sequence (p53CON). This binding produces a ladder of retarded bands on an agarose gel. Using immunoblotting with the antibody DO-1, we show that the bands obtained correspond to ethidium-stained DNA, suggesting that each band of the ladder contains a DNA-p53 complex. The intensity and the number of these hands are decreased by physiological concentrations of zinc ions. At higher zinc concentrations, binding of p53 to scDNA is completely inhibited. The binding of additional zinc ions to p53 appears much weaker than the binding of the intrinsic zinc ion in the DNA binding site of the core domain. In contrast to previously published data suggesting that 100 microM zinc ions do not influence p53 binding to p53CON in a DNA oligonucleotide, we show that 5-20 microM zinc efficiently inhibits binding of p53 to p53CON in DNA fragments. We also show that relatively low concentrations of dithiothreitol but not of 2-mercaptoethanol decrease the concentration of free zinc ions, thereby preventing their inhibitory effect on binding of p53 to DNA. Nickel and cobalt ions inhibit binding of p53 to scDNA and to its consensus sequence in linear DNA fragments less efficiently than zinc; cobalt ions are least efficient, requiring >100 microM Co2+ for full inhibition of p53 binding. Modulation of binding of p53 to DNA by physiological concentrations of zinc might represent a novel pathway that regulates p53 activity in vivo.

A Novel Phospholipase A2Activity in Saliva of the Lone Star Tick,Amblyomma americanum(L.)

Saliva from female lone star ticks,Amblyomma americanum,contained a novel phospholipase A2(PLA2) activity that hydrolyzed14C-arachidonate from14C-arachidonyl phosphatidylcholine. The tick saliva PLA2(ts-PLA2) was active over a broad pH range (4.5–11.5) with two distinct pH optima of pH 5.5 and 9.5. Though extracellular PLA2s are reported to be activated by millimolar Ca2+, ts-PLA2was sensitive to submicromolar Ca2+and was half-maximally activated by 3.5μMCa2+. Tick saliva contains >500μMCa2+and the feeding lesion in the host is expected to contain millimolar Ca2+. Saliva exhibited a single peak of PLA2activity corresponding to a molecular weight of 55.7 ± 1.3 kDa by size exclusion chromatography. The ts-PLA2was unaffected by a variety of compounds known to inhibit either secreted or cytosolic PLA2s from other sources. However, ts-PLA2was inhibited by the substrate analog, oleyloxyethyl phosphorylcholine (IC50= 1.4 μM), and the end product, arachidonic acid (IC50= 38 μM). Low concentrations of dithiothreitol did not greatly affect ts-PLA2, but activity was reduced at higher concentrations. The PLA2activity found inA. americanumsalivary glands showed many similarities to ts-PLA2, but also some distinct differences. Secreted at the tick–host interface, ts-PLA2is thought to play an important, but unknown, role during the prolonged tick feeding.

Modulation of the arsenite-induced expression of stress proteins by reducing agents.

We examined the effects of reducing agents on the expression of heat shock protein 27 (hsp27), alpha B crystallin, and hsp70 in C6 rat glioma cells in response to stress. Cells were exposed to arsenite (100 microM for 1 h) in the presence of dithiothreitol at various concentrations (0.03-2 mM), and the accumulation of all three proteins was markedly stimulated in cells that had been exposed to arsenite in the presence of a low concentration (0.03-0.1 mM) of dithiothreitol. Stimulation of these arsenite-induced responses was also observed in the presence of 0.1 mM 2-mercaptoethanol or 0.05 mM dithioerythritol. The enhanced expression of mRNAs for hsp27, alpha B crystallin and hsp70, as well as the prolonged activation of heat shock transcription factor 1 (HSF1), were also observed in cells that had been treated with arsenite in the presence of 0.05 mM dithiothreitol. The arsenite-inducible expression of the three proteins was completely suppressed when dithiothreitol was present at concentrations above 1 mM during the stress period, although delayed activation of the binding to a heat shock element (HSE) by phosphorylated HSF was observed in these cells. Exposure of cells first to arsenite for 1 h and then to dithiothreitol resulted in a very effective suppression of the arsenite-inducible responses, and the responses were inhibited even by a low concentration of dithiothreitol. These results suggest that the signal transduction pathway for the arsenite-induced expression of hsps involves at least two redox-sensitive steps: (i) a process that is stimulated by mild reducing power during the stress period; and (ii) a process that is followed by the activation of HSF and is very sensitive to suppression by a reducing agent.

Ligand-Induced Recruitment and Phosphorylation of Reduced TGF-β Type I Receptor

Receptors I (TβR-I) and II (TβR-II) of transforming growth factor-β (TGF-β) are components of a heteromeric complex in which receptor I requires receptor II to bind ligand, and receptor II requires receptor I to signal. We show that pretreatment of cells with low concentrations of dithiothreitol, which is known to disrupt ligand binding to TβR-I, does not prevent interaction and complex formation between TβR-II and TβR-I. Nevertheless, our results demonstrate that ligand interaction with TβR-I is able to induce high-affinity convertion to the complex formed. We also demonstrate that transphosphorylation of TβR-I through bound TβR-II can occur independently of ligand binding to TβR-I.

Drosophila insulin receptor: lectin-binding properties and a role for oxidation-reduction of receptor thiols in activation

The glycosylation of the Drosophila insulin receptor (DIR) has been compared to that of the rat insulin receptor by examining the binding of receptors to the lectins wheat germ agglutinin, Concanavalin-A, and lentil lectin. Although rat insulin receptors bound and were specifically eluted from all three lectins, only a small fraction of the DIR (< 5%) was retained on wheat germ agglutinin. In contrast, the DIR bound strongly to Concanavalin-A and lentil lectin and was recovered from lentil lectin columns after elution with alpha-methyl-mannoside. The pattern of lectin binding indicates that glycosylation of the DIR and rat insulin receptors differs, with the DIR containing primarily high mannose-type oligosaccharides. After lectin chromatography, the DIR exhibited an elevated level of basal autophosphorylation and kinase activity, which could be restored to a low level by incubation with 0.5 mM dithiothreitol (DTT). DTT did not, however, affect ligand-stimulated kinase activity. The ability of low concentrations of DTT to deactivate the DIR kinase suggests that, like the mammalian receptor, beta-subunit thiols may be involved in regulation of conformational changes between activated and unactivated receptor states. Interestingly, DTT-induced deactivation of the DIR was blocked by preincubation with an antipeptide antibody against the carboxy-terminal domain of the DIR. This suggests that the DIR carboxyl terminus undergoes a conformational change during the activation-inactivation cycle of the kinase, which can be sterically hindered by the antibody. Conformational changes in this region of the mammalian receptor have been observed, and these data suggest that features of the insulin receptor activation mechanism have been substantially conserved during evolution.

Drosophila insulin receptor: lectin-binding properties and a role for oxidation-reduction of receptor thiols in activation.

The glycosylation of the Drosophila insulin receptor (DIR) has been compared to that of the rat insulin receptor by examining the binding of receptors to the lectins wheat germ agglutinin, Concanavalin-A, and lentil lectin. Although rat insulin receptors bound and were specifically eluted from all three lectins, only a small fraction of the DIR (< 5%) was retained on wheat germ agglutinin. In contrast, the DIR bound strongly to Concanavalin-A and lentil lectin and was recovered from lentil lectin columns after elution with alpha-methyl-mannoside. The pattern of lectin binding indicates that glycosylation of the DIR and rat insulin receptors differs, with the DIR containing primarily high mannose-type oligosaccharides. After lectin chromatography, the DIR exhibited an elevated level of basal autophosphorylation and kinase activity, which could be restored to a low level by incubation with 0.5 mM dithiothreitol (DTT). DTT did not, however, affect ligand-stimulated kinase activity. The ability of low concentrations of DTT to deactivate the DIR kinase suggests that, like the mammalian receptor, beta-subunit thiols may be involved in regulation of conformational changes between activated and unactivated receptor states. Interestingly, DTT-induced deactivation of the DIR was blocked by preincubation with an antipeptide antibody against the carboxy-terminal domain of the DIR. This suggests that the DIR carboxyl terminus undergoes a conformational change during the activation-inactivation cycle of the kinase, which can be sterically hindered by the antibody. Conformational changes in this region of the mammalian receptor have been observed, and these data suggest that features of the insulin receptor activation mechanism have been substantially conserved during evolution.

Intracellular Folding of Tissue-type Plasminogen Activator: EFFECTS OF DISULFIDE BOND FORMATION ON N-LINKED GLYCOSYLATION AND SECRETION

The addition of N-linked core oligosaccharides to membrane and secretory glycoproteins occurs co-translationally at asparagine residues in the tripeptide sequon Asn-Xaa-Ser/Thr soon after translocation of the nascent polypeptide into the lumen of the endoplasmic reticulum. However, the presence of the sequon does not automatically ensure core glycosylation, as many proteins contain sequons that remain either unglycosylated or glycosylated to a variable extent. To investigate whether intracellular protein folding can influence sequon utilization, we have expressed tissue-type plasminogen activator (t-PA) in cell culture in the presence of mild concentrations of the reducing agent dithiothreitol to prevent co-translational disulfide bond formation in the endoplasmic reticulum. We show that conditions that prevent disulfide bond formation lead to complete glycosylation of a sequon that otherwise undergoes variable glycosylation in untreated cells. This demonstrated that folding and disulfide bond formation of t-PA determines its extent of core N-linked glycosylation. When dithiothreitol was removed from the cells, the reduced and overglycosylated t-PA formed disulfide bonds, folded, and was secreted. We also show t-PA present within cells is more susceptible to reduction with low concentrations of dithiothreitol than secreted t-PA.

Unusual Insulin Binding to Cells Expressing an Insulin Receptor Mutated at Cysteine 524

The involvement of cysteine 524 of the insulin receptor in an intermolecular (class I) disulfide bond between the two α-subunits was investigated using site-directed mutagenesis. The oligomeric structure of the mutated receptor was partially disrupted, although a significant portion of the receptor remained in its heterotetrameric form. Interestingly, the mutated insulin receptor heterotetramer was more susceptible than the wildtype receptor to reduction to heterodimers by low concentrations of dithiothreitol. Insulin binding to solubilized mutant receptors was normal and the mutant receptors had normal affinity for insulin, but insulin binding to cells expressing mutant insulin receptors displayed positive cooperativity. Cysteine 524 is most likely involved in a class I disulfide bond and receptors mutated at this site displayed unusual insulin binding properties only in the cellular enviroment.

Fc site-specific labeling of immunoglobulins with calf intestinal alkaline phosphatase.

A strategy is described for the site-specific conjugation of alkaline phosphatase to the Fc region of immunoglobulins. Mild oxidation of immunoglobulins by sodium periodate followed by reductive amination with excess cystamine and sodium cyanoborohydride covalently attaches cystamine specifically to the oligosaccharide chains of the antibody's Fc region. The disulfide bonds of the conjugated cystamine moieties are then reduced to free thiols with a low concentration of dithiothreitol. The thiol-modified antibodies (typically containing three to four thiols per Fc region) are then brought into contact with maleimide-functionalized alkaline phosphatase to form antibody-alkaline phosphatase conjugates. The Fc site-specific conjugation methodology was optimized and successfully applied to a number of different monoclonal and polyclonal antibodies. The conjugates prepared show assay performances superior to those prepared by more conventional means. Evidence obtained indicates that this superior conjugate performance results from total retention of the antibody's antigen binding activity following the Fc site-specific conjugation procedure.

Extracellular Glutathione Suppresses Human Lung Fibroblast Proliferation

Alveolar epithelial lining fluid glutathione (GSH) is markedly decreased in patients with idiopathic pulmonary fibrosis (IPF). Because patients with IPF have exaggerated numbers of fibroblasts in their lower respiratory tract, we hypothesized that GSH can suppress lung fibroblast proliferation. To verify this hypothesis, we examined the ability of GSH to suppress human lung fibroblast (ATCC; HFL-1) proliferation in vitro in the presence of either IPF bronchoalveolar lavage fluid (BAL) or calf serum (CS). Both CS at a concentration of 10% and IPF BAL markedly increased fibroblast proliferation when compared to cells grown without CS or IPF BAL (10% CS = 93 +/- 4%, P less than 0.001; IPF BAL = 47 +/- 4%, P less than 0.001). In the presence of physiologic concentrations of GSH (0 to 500 microM), both CS- and IPF BAL-mediated fibroblast proliferation were markedly reduced, with 500 microM GSH inducing complete inhibition. Interestingly, glutathione disulfide (GSSH) and S-methylglutathione did not suppress proliferation, whereas various sulfhydryl-containing molecules (cysteine, N-acetylcysteine, 2-mercaptoethanol, and low concentrations of dithiothreitol) induced an inhibition of fibroblast proliferation similar to that observed with GSH. Most of the suppressive effect of GSH was mediated at the cell level since incubation of fibroblasts with 500 microM GSH for 1 h completely blocked the ability of the cells to subsequently proliferate in the presence of untreated 10% CS. Treatment of CS with 500 microM GSH for 1 h followed by removal of GSH by molecular sieve chromatography had no detectable effect on fibroblast proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxy-3-methylglutaryl coenzyme a reductase from the latex of Hevea brasiliensis

3-Hydroxy-3-methylglutaryl coenzyme A reductase (mevalonate: NADP + oxidoreductase acylating CoA; EC 1.1.1.34) was purified from fresh Hevea latex of clone RRIM 600. The latex was centrifuged and the sediment used for enzyme purification. The enzyme was solubilized by freeze-thawing in a buffer containing 1 % Brij W-1 and 20% glycerol. Affinity chromatography (HMG-CoA-Hexyl-Agarose) was used in the final purification step. The M r determined by SDS-PAGE was 44000 and that estimated from non-denaturing gel electrophoresis, 176000. The optimal pH was ca 7, with an apparent K m of 13 μM for (S)-HMG-CoA. A low concentration of dithiothreitol was required for maximal activity of the purified enzyme.

The Role of Insulin Receptor Sulphydryl Groups in Insulin Binding and Cellular Response in Rat Adipocytes

Phenylarsine oxide (PAO), an agent which reacts with vicinal sulphydryl groups and dithiothreitol (DTT), a disulphide reducing agent, inhibited insulin binding to intact adipocytes with half maximal inhibition occurring at 28 microM and 340 microM, respectively. Pretreatment of adipocytes with DTT (2mM) prevented insulin stimulation of glucose uptake by approximately 50%. The marked inhibition of insulin binding to adipocytes by PAO and DTT is consistent with the involvement of the receptor cysteine-rich region of hormone binding. Furthermore, DTT inhibition of insulin binding suggests that the integrity of disulphide bridges is critical for insulin binding. The inhibitory effect of DTT and PAO on insulin binding were not additive, instead addition of DTT to PAO-treated adipocytes effected 15% reversal of binding inhibition. The marked inhibition of insulin binding by addition of low concentrations of DTT (0.2-2.0mM) to intact adipocytes is in contrast to the previously reported biphasic response for the effect of DTT on insulin binding to isolated plasma membranes from rat adipocytes (Schweitzer et al. Proc. Natl. Acad. Sci. U.S.A. 77, 4692-4696, 1980). Scatchard plots for 125I-iodoinsulin binding to adipocytes in the basal state were linear. In contrast, Scatchard analysis of insulin binding to plasma membranes prepared from both basal and insulin-stimulated adipocytes yielded severely curvilinear plots. The data suggests that (i) fundamental differences exist between the receptor state in intact cells and isolated plasma membranes and (ii) that a disulphide-rich region within the insulin receptor, other than the previously reported class I and class II disulphide bridges, is critical for insulin binding and cellular response.