Competitive inhibition of hexokinase isoenzymes by mercurials.

A difference in the mode of inhibition of hexokinase [EC 2.7.1.1] isoenzymes by p-chloromercuribenzenesulfonate was confirmed with respect to glucose between two Type I isoenzyme preparations purified from the kidney and spleen of rat. Essentially the same difference was observed when galactose was used as the substrate in place of glucose, as the kidney Type I isoenzyme was inhibited in a competitive manner while the spleen counterpart was inhibited in a non-competitive manner by sulfhydryl inhibitor. Both the Type I isoenzymes, however, were competitively inhibited by other mercurial sulfhydryl inhibitors, methyl and butyl mercuric chlorides. On the other hand, the Type II hexokinase isoenzymes purified from the muscle, heart, and spleen were all inhibited competitively by p-chloromercuribenzenesulfonate with respect to glucose. The mechanism of competitive inhibition of the hexokinase isoenzymes by sulfhydryl inhibitors was discussed in view of the difference in the mode of action of the mercurials with different isoenzymes.     

Nitrobenzylthioinosine-insensitive uridine transport in human lymphoblastoid and murine leukemia cells

Both human lymphoblastoid (RPMI 6410) and murine leukemia (L1210) cells were found to have a component of uridine transport which is insensitive to the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). In both cell lines NBMPR-insensitive uridine transport is inhibited by other nucleosides and by the sulfhydryl reagent p-chloromercuribenzenesulfonate. In RPMI 6410 cells NBMPR-insensitive transport accounts for only 2% of the initial rate of uridine transport. In contrast, 20% of the initial rate of transport of L1210 cells is insensitive to NBMPR, and uridine uptake over longer periods (10 min) is completely insensitive to NBMPR.     

Control of red cell urea and water permeability by sulfhydryl reagents

The binding constant for pCMBS (p-chloromercuribenzenesulfonate) inhibition of human red cell water transport has been determined to be 160 +/- 30 microM and that for urea transport inhibition to be 0.09 +/- 0.06 microM, indicating that there are separate sites for the two inhibition processes. The reaction kinetics show that both processes consist of a bimolecular association between pCMBS and the membrane site followed by a conformational change. Both processes are very slow and the on rate constant for the water inhibition process is about 10(5) times slower than usual for inhibitor binding to membrane transport proteins. pCMBS binding to the water transport inhibition site can be reversed by cysteine while that to the urea transport inhibition site can not be reversed. The specific stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamidostilbene-2,2'-disulfonate) causes a significant change in the time-course of pCMBS inhibition of water transport, consistent with a linkage between anion exchange and water transport. Consideration of available sulfhydryl groups on band 3 suggests that the urea transport inhibition site is on band 3, but is not a sulfhydryl group, and that, if the water transport inhibition site is a sulfhydryl group, it is located on another protein complexed to band 3, possibly band 4.5.     

Charcot-Leyden crystal protein (galectin-10) is not a dual function galectin with lysophospholipase activity but binds a lysophospholipase inhibitor in a novel structural fashion

Charcot-Leyden crystal (CLC) protein, initially reported to possess weak lysophospholipase activity, is still considered to be the eosinophil's lysophospholipase, but it shows no sequence similarities to any known lysophospholipases. In contrast, CLC protein has moderate sequence similarity, conserved genomic organization, and near structural identity to members of the galectin superfamily, and it has been designated galectin-10. To definitively determine whether or not CLC protein is a lysophospholipase, we reassessed its enzymatic activity in peripheral blood eosinophils and an eosinophil myelocyte cell line (AML14.3D10). Antibody affinity chromatography was used to fully deplete CLC protein from eosinophil lysates. The CLC-depleted lysates retained their full lysophospholipase activity, and this activity could be blocked by sulfhydryl group-reactive inhibitors, N-ethylmaleimide and p-chloromercuribenzenesulfonate, previously reported to inhibit the eosinophil enzyme. In contrast, the affinity-purified CLC protein lacked significant lysophospholipase activity. X-ray crystallographic structures of CLC protein in complex with the inhibitors showed that p-chloromercuribenzenesulfonate bound CLC protein via disulfide bonds with Cys(29) and with Cys(57) near the carbohydrate recognition domain (CRD), whereas N-ethylmaleimide bound to the galectin-10 CRD via ring stacking interactions with Trp(72), in a manner highly analogous to mannose binding to this CRD. Antibodies to rat pancreatic lysophospholipase identified a protein in eosinophil and AML14.3D10 cell lysates, comparable in size with human pancreatic lysophospholipase, which co-purifies in small quantities with CLC protein. Ligand blotting of human and murine eosinophil lysates with CLC protein as probe showed that it binds proteins also recognized by antibodies to pancreatic lysophospholipase. Our results definitively show that CLC protein is not one of the eosinophil's lysophospholipases but that it does interact with eosinophil lysophospholipases and known inhibitors of this lipolytic activity.     

The redox state of cysteines 201 and 317 of the erythrocyte anion exchanger is critical for ankyrin binding

Previous studies have demonstrated that modification of erythrocyte membrane cysteine residues via disulfide cross-briding or direct derivatization with thiol reagents promotes massive morphological, rheological, and structural changes in the cell. To determine whether disruption of the band 3-ankyrin interaction, the major membrane-cytoskeletal linkage, might contribute to the above lesions, we quantitatively measured the band 3-ankyrin interaction following modification of Cys-201 and/or Cys-317 of the cytoplasmic domain of band 3. It was observed that irreversible alkylating agents (e.g. N-ethylmaleimide or iodoacetamide and its derivatives), reversible derivatizing compounds (.e.g. p-chloromercuribenzenesulfonate or glutathione), and native disulfide bond formation all blocked the ankyrin interaction. Comparison of the extent of sulfhydryl modification with the degree of inhibition of ankyrin binding further confirmed that cysteine modification was directly responsible for the inhibition. However, analysis of the site of sulfhydryl derivatization revealed that inhibition of ankyrin binding could be initiated in some cases with derivatization of Cys-201, while in other cases obstruction of Cys-317 appeared to be essential. This apparent discrepancy was resolved by demonstrating that Cys-201 of one strand of the cytoplasmic domain of band 3 dimer could disulfide bond with Cys-317 of the opposite strand, thus demonstrating that all four cysteines of the band 3 dimer are clustered at the interface between subunits. We argue that derivatization or disulfide cross-linking of these cysteines can block ankyrin binding by both conformational and steric mechanisms.     

The diversity of sulfhydryl groups in the human erythrocyte membrane

Human bank blood erythrocytes were exposed to the mercurials p-chloromercuribenzoate (PCMB), chlormerodrin (CM), p-chloromercuribenzenesulfonate (PCMBS), and 1-bromomercuri-2-hydroxypropane (BMHP) for different time intervals, at different concentrations and in combination with n-ethylmaleimide (NEM) added before, and 2-mercaptoethylguanidine (MEG) and reduced glutathione (GSH) added after the mercurial. Binding patterns of the mercurials to the cells and effects on permeability of the cells were measured. The results indicate that the erythrocyte membrane contains multiple classes of sulfhydryl groups, alteration of which has a variety of effects on cell permeability. PCMB, chlormerodrin and PCMBS react with at least three classes of sulfhydryls, two of which are associated with the sodium-potassium barrier and, when altered, result in potassium loss, sodium accumulation and hemolysis. BMHP reacts with at least two classes of sulfhydryls, one of which is associated with permeability, and, when altered, results in hemolysis in isotonic solutions of choline chloride or lactose. The results provide additional insight into the structure and function of the erythrocyte membrane.     

Cysteine scanning mutagenesis of helices 2 and 7 in GLUT1 identifies an exofacial cleft in both transmembrane segments

Cysteine scanning mutagenesis in conjunction with site-directed chemical modification of sulfhydryl groups by p-chloromercuribenzenesulfonate (pCMBS) or N-ethylmaleimide (NEM) was applied to putative transmembrane segments (TM) 2 and 7 of the cysteine-less glucose transporter GLUT1. Valid for both helices, the majority of cysteine substitution mutants functioned as active glucose transporters. The residues F72, G75, G76, G79, and S80 within helix 2 and G286 and N288 within helix 7 were irreplaceable because the mutant transporters displayed transport activities that were lower than 10% of Cys-less GLUT1. The indicated cluster of glycine residues within TM 2 is located on one face of the helix and may provide space for a bulky hydrophobic counterpart interacting with another transmembrane segment or lipid side chains. Characteristic for helix 7, three glutamine residues (Q279, Q282, and Q283) played an important role in transport activity of Cys-less GLUT1 because an individual replacement with cysteine reduced their transport rates by about 80%. ParaCMBS-sensitivity scanning of both transmembrane segments detected several membrane-harbored residues to be accessible to the extracellular aqueous solvent. The pCMBS-reactive sulfhydryl groups were located exclusively in the exofacial half of the plasma membrane and, when presented in a helical model, lie along one side of the helices. Taken together, transmembrane segments 2 and 7 form clefts accessible to the extracellular aqueous solvent. The lining residues are however excluded from interaction with intracellular solutes, as justified by microinjection of pCMBS into the cytoplasm of Xenopus oocytes.     

Functional reconstitution of the malate carrier of barley mesophyll vacuoles in liposomes

The malate carrier of barley (Hordeum vulgare L.) mesophyll vacuoles was highly purified by chromatography on hydroxyapatite followed by affinity-chromatography using 5-amino-1,2,3-benzenetricarboxylic acid as ligand. The carrier, reconstituted in asolectin liposomes, had properties similar to those described previously for the carrier in intact vacuoles (Martinoia, E., Flügge, U.I., Kaiser, G., Heber, U. and Heldt, H.W. (1985) Biochim. Biophys. Acta 806, 311-319). The apparent Km for malate uptake was 2-3 mM, and the uptake was inhibited by other carboxylic acids (preferentially tricarboxylic). The sulfhydryl reagent, p-chloromercuribenzenesulfonate, as well as the anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, also inhibited malate uptake. The transport was dependent on the membrane potential with an optimum at about 35 mV.     

Group-selective reagent modification of the sodium- and chloride-coupled glycine transporter under native and reconstituted conditions.

Glycine transporter from rat brain stem and spinal cord is inactivated by specific sulfhydryl reagents. Modification of lysine residues also promotes a decrease of the transporter activity but in a lesser extent than that promoted by thiol group reagents. Mercurials showed a more marked inhibitory effect than maleimide derivatives. SH groups display a similar reactivity for p-chloromercuribenzenesulfonate (pCMBS) and mersalyl in synaptosomal membrane vesicles and proteoliposomes reconstituted with the solubilized transporter. However, different reactivity is observed with N-ethylmaleimide (MalNEt), the greatest effect being attained in membrane vesicles. The rate of inactivation by pCMBS and MalNEt is pseudo-first-order showing time- and concentration-dependence. pCMBS and MalNEt decrease the Vmax for glycine transport and to a lesser extent act on the apparent Km. Treatment with dithiothreitol (DTT) of the transporter modified by pCMBS results in a complete restoration of transporter activity indicating that the effect exercised by the reagent is specific for cysteine residues on the protein. It is concluded that SH groups are involved in the glycine transporter function and that these critical residues are mostly located in a relatively hydrophilic environment of the protein.     

The reactivity of the gamma-aminobutyric acid transporter GAT-1 toward sulfhydryl reagents is conformationally sensitive. Identification of a major target residue.

The gamma-aminobutyric acid (GABA) transporter GAT-1 is a prototype of neurotransmitter transporters that maintain low synaptic levels of the transmitter. Transport by GAT-1 is sensitive to the polar sulfhydryl reagent 2-aminoethyl methanethiosulfonate. Following replacement of endogenous cysteines to other residues by site-directed mutagenesis, we have identified cysteine 399 as the major determinant of the sensitivity of the transporter to sulfhydryl modification. Cysteine-399 is located in the intracellular loop connecting putative transmembrane domains eight and nine. Binding of both sodium and chloride leads to a reduced sensitivity to sulfhydryl reagents, whereas subsequent binding of GABA increases it. Strikingly binding of the nontransportable GABA analogue SKF100330A gives rise to a marked protection against sulfhydryl modification. These effects were not observed in C399S transporters. Under standard conditions GAT-1 is almost insensitive toward the impermeant 2-(trimethylammonium)ethyl methanethiosulfonate. However, in a chloride-free medium, addition of SKF100330A renders wild type GAT-1, but not C399S, very sensitive to this impermeant reagent. These observations indicate that the accessibility of cysteine 399 is highly dependent on the conformation of GAT-1. Consequently, topological assignments based on accessibility of endogeneous or engineered cysteines to small polar sulfhydryl reagents need to be interpreted with extreme caution.     

Binding of DTNB to band 3 in the human red cell membrane

Inhibition of red cell water transport by the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) has been reported by Naccache and Sha'afi ((1974) J. Cell Physiol. 84, 449-456) but other investigators have not been able to confirm this observation. Brown et al. ((1975) Nature 254, 523-525) have shown that, under appropriate conditions, DTNB binds only to band 3 in the red cell membrane. We have made a detailed investigation of DTNB binding to red cell membranes that had been treated with the sulfhydryl reagent N-ethylmaleimide (NEM), and our results confirm the observation of Brown et al. Since this covalent binding site does not react with either N-ethylmaleimide or the sulfhydryl reagent pCMBS (p-chloromercuribenzenesulfonate), its presence has not previously been reported. This covalent site does not inhibit water transport nor does it affect any transport process we have studied. There is an additional low-affinity (non-covalent) DTNB site that Reithmeier ((1983) Biochim. Biophys. Acta 732, 122-125) has shown to inhibit anion transport. In N-ethylmaleimide-treated red cells, we have found that this binding site inhibits water transport and that the inhibition can be partially reversed by the specific stilbene anion exchange transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), thus linking water transport to anion exchange. DTNB binding to this low-affinity site also inhibits ethylene glycol and methyl urea transport with the same KI as that for water inhibition, thus linking these transport systems to that for water and anions. These results support the view that band 3 is a principal constituent of the red cell aqueous channel, through which urea and ethylene glycol also enter the cell.     

Novikoff hepatoma deoxyribonucleic acid polymerase. Sensitivity of the beta-polymerase to sulfhydryl blocking agents.

Unlike other beta-class eukaryotic DNA polymerases, the enzyme purified from the Novikoff hepatoma is inhibited by both sulfhydryl blocking agents N-ethylmaleimide (NEM) and p-hydroxymercuribenzoate (pHMB). The degree of sensitivity varies depending on the enzyme purity, pH of the reaction, and the presence of sulfhydryl reducing agents. Novikoff beta-polymerase activity is unaffected by the presence of 2-mercaptoethanol (2-Me) or dithiothreitol (DTT); however, the combination of 2-mercaptoethanol and NEM or pHMB acts to reverse the inhibition of the sulfhydryl blocking agent. The reversal of inhibition involves more than just a titration of NEM with 2-mercaptoethanol since a) the combination of these two reagents actually stimulates the DNA polymerase, and b) dithiothreitol did not reverse the inhibition. Binding of the polymerase to DNA did not affect the enzyme sensitivity to NEM.     

EPR method for the measurement of cellular sulfhydryl groups.

An EPR method that can measure the concentration of sulfhydryl groups in intact cells has been developed using a specially designed stable nitroxyl biradical. The biradical, RS-SR, contains a disulfide bond and readily undergoes thiol-disulfide exchange reactions with thiols resulting in a characteristic EPR spectrum which can be analyzed to provide a quantitative measure of sulfhydryl groups. The data obtained from the EPR method are in good agreement with those obtained from the conventional optical method using Ellman's reagent. The advantages of the EPR method are that the measurement can be carried out on intact cells or any other highly colored, absorbing and/or scattering solutions and the sensitivity is such that only a few cells (approximately 100) are needed for each quantitative measurement.     

A continuous fluorescence assay for sulfhydryl oxidase

Flavin-dependent sulfhydryl oxidases represent a newly discovered family of proteins with a range of cellular locations and putative roles. The avian and mammalian proteins can catalyze the direct oxidation of protein cysteine residues to disulfides with the reduction of dioxygen to hydrogen peroxide. Although thiols interfere with the peroxidase-mediated quantitation of hydrogen peroxide, a very sensitive, continuous fluorescence assay of the sulfhydryl oxidases can be devised with careful selection of thiol substrate concentration and fluorogen. Purified avian enzyme (or crude chicken egg white) was used for these experiments. Homovanillic acid was found to be a suitable fluorogen in the presence of 300 microM thiols from either dithiothreitol or reduced ribonuclease A. High concentrations of horseradish peroxidase minimized the effects of contaminating catalase in biological samples. Using fluorescence microcells, the assay could detect 15fmol of avian sulfhydryl oxidase and the rates were linearly dependent on enzyme concentration up to 6nM. Aspects of the interaction among thiols, homovanillic acid, and peroxidase are discussed which limit the sensitivity of the assay and require that care is exercised in the application of this new procedure. Finally, the assay is used to show that there is sulfhydryl oxidase activity in a number of secretory fluids including human tears.     

Site-specific alteration of cysteine 176 and cysteine 234 in the lactose carrier of Escherichia coli

In the present study, Cys-176 and Cys-234 in the lactose carrier have been modified to serine residues via site-specific mutagenesis. The resultant mutants have been characterized with regard to galactoside transport activity and sulfhydryl reagent sensitivity. The mutant proteins (in which Cys-176 or Cys-234 had been replaced with serine) are able to effectively transport galactosides, although the transport rates for lactose and methyl-beta-D-galactopyranoside are slightly reduced compared to the normal lactose carrier. In addition, both mutants are less sensitive than the wild-type to high concentrations of two different sulfhydryl reagents, N-ethylmaleimide and p-hydroxymercuribenzoate. Overall, the data are consistent with the idea that Cys-176 and Cys-234 are close to the substrate recognition site. However, neither residue appears to be essential for galactoside transport by providing an ionizable group near the active site or by forming a disulfide bond.     

Thiol modification of cysteine 327 in the eighth transmembrane domain of the light subunit xCT of the heteromeric cystine/glutamate antiporter suggests close proximity to the substrate binding site/permeation pathway

We measured sensitivity to thiol modification of the heteromeric glutamate/cystine transporter 4F2hc-xCT expressed in Xenopus oocytes. p-Chloromercuribenzoate (pCMB) and p-chloromercuribenzenesulfonate (pCMBS) rapidly blocked transport activity. Cys(327), located in the middle of the eighth transmembrane domain of the light subunit (xCT), was found to be the main target of inactivation. Cysteine, an impermeant reducing reagent, reversed pCMB and pCMBS effects only when applied from the extracellular medium. l-Glutamate and l-cystine, but not l-arginine, protected from the inactivation with an IC(50) similar to the K(m). Protection was not temperature-dependent, suggesting that it did not depend on large substrate-induced conformational changes. Mutation of Cys(327) to Ala and Ser slightly modified the K(m) and a C327L mutant abolished transport function without compromising transporter expression at the plasma membrane. The results indicate that Cys(327) is a functionally important residue accessible to the aqueous extracellular environment and is structurally linked to the permeation pathway and/or the substrate binding site.     

Cell density regulates prolyl 4-hydroxylase activity independent of mRNA levels

In embryonic avian tendon, cell density regulates collagen production. This control is propagated through the alpha-subunit of prolyl 4-hydroxylase where protein levels were previously shown to rise fivefold with increasing cell density. In contrast, mRNA levels are now shown not to change by both Northern and RNAse protection assays. This lack of increase contrasts with previous reports as does the mRNA length: this is 50% larger as confirmed by sequencing the 3' end. Alternative sites for cell density regulation of the enzyme could rely on its sensitivity to sulfhydryl groups. Using a fluorescent sulfhydryl probe as well as a sulfhydryl inhibitor, one observes a strong cell density response, supporting the hypothesis that cellular redox potential could alter protein stability.     

Synthesis of a hydrolase for the membrane-form variant surface glycoprotein is repressed during transformation of Trypanosoma brucei

A membrane-bound phospholipase C-like hydrolase present in lysates of bloodstream forms of Trypanosoma brucei rapidly converts the membrane form of the variant surface protein to the soluble form and 1,2-dimyristoylglycerol [(1985) M.A.J. Ferguson et al. J. Biol. Chem., 260, 4963-4968]. The hydrolase is inhibited by p-chloromercuribenzenesulfonate. The synthesis of the enzyme is rapidly repressed upon differentiation of bloodstream forms to procyclic cells and the enzyme activity declines to an undetectable level during subsequent growth of procyclic forms.     

Electrophoretic determination of sulfhydryl groups and its application to complex protein samples, in vitro protein synthesis mixtures, and cross-linked proteins

Without prior fractionation, the number of sulfhydryl groups of individual polypeptides in a protein mixture can be determined, provided their molecular weights and approximate isoelectric points are known. Urea-denatured protein samples are reacted with iodoacetamide and iodoacetate in a modified version of Creighton's procedure. After separation by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and isoelectric focusing, the number of sulfhydryl groups is determined by counting the protein bands which have additional negative charges. This method requires little material and provides an additional parameter, besides the molecular weight and isoelectric point, for the identification and characterization of a protein. The sensitivity may be enhanced for nonradioactive proteins by using 14C-labeled iodoacetamide and iodoacetate. The procedure has been applied to prokaryotic in vitro protein synthesis mixtures, bacterial membrane protein, and trypsin-cleaved or chemically cross-linked subunits of the F1 ATPase from Escherichia coli.     

Thiol-disulfide exchanges modulate aldo–keto reductase family 1 member B10 activity and sensitivity to inhibitors

The reversible thiol/disulfide exchange is an important regulatory mechanism of protein enzymatic activity. Many protein enzymes are susceptible to S-thiolation induced by reactive oxygen species (ROS); and the glutathione (GSH) and free amino acid cysteine (Cys) are critical cellular thiol anti-oxidants, protecting proteins from irreversible oxidative damage. In this study, we found that aldo–keto reductase family 1 member B10 (AKR1B10) contains 4 Cys residues, i.e., Cys45, Cys187, Cys200, and Cys299. Exposing AKR1B10 to ROS mixtures resulted in significant decrease of its free sulfhydryl groups, up to 40–50% in the presence of physiological thiol cysteine at 0.5 or 1.0 mM; and accordingly, AKR1B10 enzymatic activity was reversibly decreased, in parallel with the oxidation of the sulfhydryl groups. ROS-induced thiolation also affected the sensitivity of AKR1B10 to inhibitors EBPC, epalrestat, and statil. Together our results showed for the first time that AKR1B10's enzymatic activity and inhibitor sensitivity are modulated by thiol/disulfide exchanges.