Role of metabolism of the mating pheromone in sexual differentiation of the heterobasidiomycete Rhodosporidium toruloides.

A trypsin-type endopeptidase (Kamiya et al., Biochem. Biophys. Res. Commun. 94:855-860, 1980) responsible for the metabolism of rhodotorucine A, the farnesyl undecapeptide mating pheromone secreted by mating type A cells of Rhodosporidium toruloides, was biologically characterized. Metabolic activity was found to be present exclusively on the cell surface of the pheromone target cell. The activity was highly specific to the pheromone, and a biologically inactive analog which has the complete amino acid sequence of rhodotorucine A but lacks the farnesyl residue was not metabolized by intact cells. Pheromone metabolism was inhibited by trypsin substrates such as tosyl-L-arginine methyl ester. The presence of tosyl-L-arginine methyl ester strongly inhibited the sexual differentiation induced by the pheromone at a concentration which did not affect the vegetative growth of R. toruloides. Pheromone-induced sexual differentiation was also strongly inhibited by a metabolizable analog, rhodotorucine A S-oxide, but not by a non-metabolizable one. In mutants defective in early processes of mating, the decrease in the pheromone metabolic activity correlated well with the extent of loss of sensitivity to the pheromone. Both the pheromone metabolism and the capacity for sexual differentiation of a sterile mutant were restored concomitantly with reversion from the sterile to the fertile phenotype. These results suggested that metabolism of the mating pheromone plays an essential role in the process of sexual differentiation in R. toruloides.     

Modification of the erythrocyte membrane by sulfhydryl group reagents

Summary In this study, the consequences of modification of human erythrocyte membrane sulfhydryl groups by N-ethyl maleimide (NEM), 5,5dithiobis-(2-nitrobenzoic acid) (DTNB) andp-hydroxymercuriphenyl sulfonate (PHMPS) were investigated. These reagents differ in chemical reactivity, membrane penetrability and charge characteristics.Results of sulfhydryl modification were analyzed in terms of inhibitory effects on activities of five membrane enzymes; Mg⁺⁺- and Na⁺, K⁺-ATPase, K⁺-dependent and independentp-nitrophenyl phosphatase (NPPase) and DPNase. Structural considerations involved in the sulfhydryl-mediated inhibition were evaluated by studying the changes in susceptibility to sulfhydryl alteration produced by shearing membranes into microvesicles and by the addition of the membrane modifiers, Mg⁺⁺ and ATP.Conclusions from the data suggest that the effects of NEM appeared to result from modification of a single class of sulfhydryls; DTNB interacted with two different sulfhydryl classes. Increasing concentrations of PHMPS resulted in the sequential modification of many types of sulfhydryls, presumably as a result of increasing membrane structural disruption. DTNB and PHMPS caused solubilization of about 15% of membrane protein at concentrations giving maximal enzyme inhibition.In contrast to the usually observed parallels between Na⁺, K⁺-ATPase and K⁺-dependent NPPase, activities of Mg⁺⁺-ATPase, Na⁺, K⁺-ATPase and K⁺-dependent NPPase varied independently as a result of sulfhydryl modification. We suggest complex structural and functional relationships exist among these components of the membrane ATP-hydrolyzing system.Our studies indicate that the effects of sulfhydryl group reagents on these membrane systems should not be ascribed to sulfhydryl modificationper se, but rather to the resulting structural perturbations. These effects depend upon the structural characteristics of the particular membrane preparation studied and on the chemical characteristics of the sulfhydryl group reagent used.     

Transient increase of Ca2+ uptake as a signal for mating pheromone-induced differentiation in the heterobasidiomycetous yeast Rhodosporidium toruloides.

The role of Ca2+ for the signaling of rhodotorucine A, a mating pheromone of Rhodosporidium toruloides, was investigated. The efficiency with which the target cells responded to the mating pheromone was dependent on the Ca2+ concentration in the medium. The pheromone induced a very rapid and transient increase of Ca2+ uptake in the recipient cell. We concluded that the transient increase in the intracellular Ca2+ concentration could play an essential role in the control of differentiation by the pheromone.     

The oxidation-reduction state of serum proteins in multiple sclerosis patients: effect of interferon beta-1b.

The concentration of reduction equivalents in serum was studied in a cohort of healthy individuals, in a group of multiple sclerosis (MS) patients undergoing treatment with interferon beta-1b and another group of MS patients who refused treatment with interferon beta-1b. Two classes of sulfhydryl groups were detectable in serum: (1) the uncovered sulfhydryls, accessible to the oxidation-reduction substrate 5,5-dithiobis-(-2-nitrobenzoic acid) (DTNB); and (2) the hidden sulfhydryls that required previous heat denaturation of serum proteins to become accessible to DTNB. The concentration of the reduced form of both the uncovered- and hidden-type of sulfhydryls was higher in the serum of MS patients than in healthy individuals. Interferon beta-1b lowered the plasma concentration of the uncovered reduced sulfhydryls after 3 months of treatment. This was in contrast to a minor effect of interferon beta-1b in the hidden-form of sulfhydryl groups. The results suggest that the concentration of reduced sulfhydryls is a biochemical marker of the in vivo oxidation/reduction reactions in MS.     

Reactions of the sulfhydryl groups of alanyl-tRNA synthetase

The six sulfhydryl groups in each subunit of the alanyl-tRNA synthetase of Escherichia coli react with sulfhydryl reagents with at least four different rates. One reacts very rapidly with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and a second reacts somewhat less rapidly with this reagent. These two groups are required for transfer activity, which is lost in proportion to the extent of derivatization. Two other groups react more slowly, with a consequent loss of exchange activity. The remaining two sulfhydryl groups do not react with DTNB until the protein is denatured. The inactivations are reversed by dithiothreitol. Two sulfhydryl groups react with N-ethylmaleimide (NEM) and with a spin-label derivative of NEM. These reactions resemble the modification of two sulfhydryl groups with DTNB, in that they also inactivate the transfer reaction but not the ATP:PP_i exchange. The two spin labels are incorporated at similar rates but are in very different environments, one highly exposed and one highly immobilized. These groups do not interact with Mn²⁺, which is bound to the enzyme in the absence of ATP.     

Detection and quantification of free sulfhydryls in monoclonal antibodies using maleimide labeling and mass spectrometry

The detection of free sulfhydryls in proteins can reveal incomplete disulfide bond formation, indicate cysteine residues available for conjugation, and offer insights into protein stability and structure. Traditional spectroscopic methods of free sulfhydryl detection, such as Ellman’s reagent, generally require a relatively large amount of sample, preventing their use for the analysis of biotherapeutics early in the development cycle. These spectroscopic methods also cannot accurately determine the location of the free sulfhydryl, further limiting their utility. Mass spectrometry was used to detect free sulfhydryl residues in intact proteins after labeling with Maleimide-PEG₂-Biotin. As little as 2% cysteine residues with free sulfhydryls (0.02 mol SH per mol protein) could be detected by this method. Following reduction, the free sulfhydryl abundance on antibody heavy and light chains could be measured. To determine free sulfhydryl location at peptide-level resolution, free sulfhydryls and cysteines involved in disulfide bonds were differentially labeled with N-ethylmaleimide and d₅-N-ethylmaleimide, respectively. Following enzymatic digestion and nanoLC-MS, the abundance of free sulfhydryls at individual cysteine residues was quantified down to 2%. The method was optimized to avoid non-specific labeling, disulfide bond scrambling, and maleimide exchange and hydrolysis. This new workflow for free sulfhydryl analysis was used to measure the abundance and location of free sulfhydryls in 3 commercially available monoclonal antibody standards (NIST Monoclonal Antibody Reference Material (NIST), SILu?Lite SigmaMAb Universal Antibody Standard (Sigma-Aldrich) and Intact mAb Mass Check Standard (Waters)) and 1 small protein standard (β-Lactoglobulin A).     

Light modulation of enzyme activity: activation of the light effect mediators by reduction and modulation of enzyme activity by thiol-disulfide exchange

Light and dark modulation experiments with pea (Pisum sativum L.) chloroplast stromal fractions pretreated with dithiothreitol (to reduce protein disulfide bonds) or with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (to block sulfhydryl groups) suggest that light modulation involves thiol-disulfide exchange on the modulatable stromal enzyme protein. Light-dependent reduction of DTNB involves a photosynthetic electron transport chain component located on the reducing side of photosystem I prior to ferredoxin; DTNB may be acting as a light effect mediator substitute. The thylakoid-bound light effect mediator system, then, in its light-activated reduced form probably catalyzes thiol-disulfide exchange reactions on stromal enzymes.     

Effects of sulfhydryl reagents on phagocytosis and exocytosis in rabbit polymorphonuclear leukocytes

The effect of sulfhydryl reagents on phagocytosis and concomitant enzyme release and on ionophore A 23187 + Ca2+-induced exocytosis in rabbit polymorphonuclear leukocytes (PMN's) was studied. Membrane-penetrating sulfhydryl reagents such as cytochalasin A and N-naphthylmaleimide in micromolar concentrations inhibit both phagocytosis and exocytosis. Poorly penetrating reagents such as p-chloromercuribenzene sulfonate (pCMBS) and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), inhibit only in high concentrations (pCMBS), or they are ineffective as inhibitors (DTNB). Inhibition by pCMBS is not reversed by glutathione or dithiothreitol; this suggests that some pCMBS probably enters the cell. Specific intracellular sulfhydryl compounds appear to be essential in the cellular apparatus involved in phagocytosis and exocytosis; various possibilities are considered. A concentration of N-naphthylmaleimide which completely inhibits phagocytosis and exocytosis leaves cellular ATPase activity intact.     

Sulfhydryls of tubulin. A probe to detect conformational changes of tubulin.

The 20 cysteine residues of tubulin are heterogeneously distributed throughout its three-dimensional structure. In the present work, we have used the reactivity of these cysteine residues with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a probe to detect the global conformational changes of tubulin under different experimental conditions. The 20 sulfhydryl groups can be classified into two categories: fast and slow reacting. Colchicine binding causes a dramatic decrease in the reactivity of the cysteine residues and causes complete protection of 1.4 cysteine residues. Similarly, other colchicine analogs that bind reversibly initially decrease the rate of reaction; but unlike colchicine they do not cause complete protection of any sulfhydryl groups. Interestingly, in all cases we find that all the slow reacting sulfhydryl groups are affected to the same extent, that is, have a single rate constant. Glycerol has a major inhibitory effect on all these slow reacting sulfhydryls, suggesting that the reaction of slow reacting cysteines takes place from an open state at equilibrium with the native. Ageing of tubulin at 37 degrees C leads to loss of self-assembly and colchicine binding activity. Using DTNB kinetics, we have shown that ageing leads to complete protection of some of the sulfhydryl groups and increased reaction rate for other slow reacting sulfhydryl groups. Ageing at 37 degrees C also causes aggregation of tubulin as indicated by HPLC analysis. The protection of some sulfhydryl groups may be a consequence of aggregation, whereas the increased rate of reaction of other slow reacting sulfhydryls may be a result of changes in global dynamics. CD spectra and acrylamide quenching support such a notion. Binding of 8-anilino-1-naphthalenesulfonate (ANS) and bis-ANS by tubulin cause complete protection of some cysteine residues as indicated by the DTNB reaction, but has little effect on the other slow reacting cysteines, suggesting local effects.     

Antibacterial action of lactoperoxidase-thiocyanate-hydrogen peroxide on Streptococcus agalactiae.

Antibacterial activity of lactoperoxidase (LP)-thiocyanate (SCN)-hydrogen peroxide (H2O2) on Streptococcus agalactiae requires that the three reactants must be in contact with the cells simultaneously. Small but assayable amounts of LP adsorb to the cell surface and are not removed by washing. A diffusible antibacterial product of LP-SCN-H2O2 reaction was not found under our experimental conditions. Incubation of S. agalactiae cells with LP-H2O2 and 14C-labeled sodium SCN resulted in the incorporation of SCN into the bacterial protein. Most of the LP-catalyzed, incorporated SCN was released from the bacterial protein. Most of the LP-catalyzed, incorporated SCN was released from the bacterial protein with dithiothreitol. Cells that had their membrane permeability changed by treatment with Cetab or 80% ethanol incorporated more SCN than did untreated cells, i.e., approximately 1 mol of SCN for each mol of sulfhydryl group present in the reaction mixture. Alteration of membrane permeability caused protein sulfhydryls, normally protected by the cytoplasmic membrane, to become exposed to oxidation. The results suggest the LP-H2O2-catalyzed incorporation of SCN into the proteins of S. agalactiae by a mechanism similar to that reported for bovine serum albumin. Removal of reactive protein sulfhydryls from a functional role in membrane transport and in glucolysis in a likely cause of the antibacterial effect for S. agalactiae.     

Antibacterial action of lactoperoxidase-thiocyanate-hydrogen peroxide on Streptococcus agalactiae.

Antibacterial activity of lactoperoxidase (LP)-thiocyanate (SCN)-hydrogen peroxide (H2O2) on Streptococcus agalactiae requires that the three reactants must be in contact with the cells simultaneously. Small but assayable amounts of LP adsorb to the cell surface and are not removed by washing. A diffusible antibacterial product of LP-SCN-H2O2 reaction was not found under our experimental conditions. Incubation of S. agalactiae cells with LP-H2O2 and 14C-labeled sodium SCN resulted in the incorporation of SCN into the bacterial protein. Most of the LP-catalyzed, incorporated SCN was released from the bacterial protein. Most of the LP-catalyzed, incorporated SCN was released from the bacterial protein with dithiothreitol. Cells that had their membrane permeability changed by treatment with Cetab or 80% ethanol incorporated more SCN than did untreated cells, i.e., approximately 1 mol of SCN for each mol of sulfhydryl group present in the reaction mixture. Alteration of membrane permeability caused protein sulfhydryls, normally protected by the cytoplasmic membrane, to become exposed to oxidation. The results suggest the LP-H2O2-catalyzed incorporation of SCN into the proteins of S. agalactiae by a mechanism similar to that reported for bovine serum albumin. Removal of reactive protein sulfhydryls from a functional role in membrane transport and in glucolysis in a likely cause of the antibacterial effect for S. agalactiae.     

Inactivation of Escherichia coli glycerol kinase by 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide: evidence for nucleotide regulatory binding sites

Glycerol kinase (EC 2.7.1.30, ATP:glycerol 3-phosphotransferase) from Escherichia coli is inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and by N-ethylmaleimide (NEM) in 0.1 M triethanolamine at pH 7 and 25 degrees C. The inactivation by DTNB is reversed by dithiothreitol. In the cases of both reagents, the kinetics of activity loss are pseudo first order. The dependencies of the rate constants on reagent concentration show that while the inactivation by NEM obeys second-order kinetics (k2app = 0.3 M-1 s-1), DTNB binds to the enzyme prior to the inactivation reaction; i.e., the pseudo-first-order rate constant shows a hyperbolic dependence on DTNB concentration. Complete inactivation by each reagent apparently involves the modification of two sulfhydryl groups per enzyme subunit. However, analysis of the kinetics of DTNB modification, as measured by the release of 2-nitro-5-thiobenzoate, shows that the inactivation is due to the modification of one sulfhydryl group per subunit, while two other groups are modified 6 and 15 times more slowly. The enzyme is protected from inactivation by the ligands glycerol, propane-1,2-diol, ATP, ADP, AMP, and cAMP but not by Mg2+, fructose 1,6-bisphosphate, or propane-1,3-diol. The protection afforded by ATP or AMP is not dependent on Mg2+. The kinetics of DTNB modification are different in the presence of glycerol or ATP, despite the observation that the degree of protection afforded by both of these ligands is the same.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple oxidation products of sulfhydryl groups near the active site of thiolase I from porcine heart

The inactivation of porcine heart thiolase I with the disulfide reagents 5,5'-dithiobis(2-nitrobenzoate) (DTNB) and 2,2- and 4,4-dithiopyridine in 0.2 M phosphate buffer, pH 7.5, follows second-order kinetics with rate constants of 2.2 X 10(2), 25 X 10(2), and 5.8 X 10(2) M-1 min-1, respectively. Stoichiometric concentrations of the thiol-oxidizing reagent diethyl azodicarboxylate inactivate thiolase in less than 1 min at pH 7.5. The presence of saturating concentrations of the substrate acetoacetyl coenzyme A or the formation of the acetyl enzyme (a normal catalytic intermediate) results in a significant protection against the inactivation of thiolase by DTNB, 2,2-dithiopyridine, and diethyl azodicarboxylate. All five sulfhydryl residues of native thiolase react with either of the dipyridyl disulfides, but only the equivalent of 3.2 residues react with DTNB even at high concentrations and prolonged incubation times. The reaction of thiolase with DTNB leads to the formation of 1.0-1.4 mol of intrachain disulfide and 0.65 mol of mixed disulfides. After inactivation of thiolase with an equimolar concentration of diethyl azodicarboxylate, 1.2 mol of intrachain disulfide per subunit is found. No cross-linking between the subunits occurs as a result of the reaction of thiolase with DTNB or diethyl azodicarboxylate. The DTNB-inactivated enzyme can be reactivated with excess dithiothreitol while the diethyl azodicarboxylate inactivated enzyme is totally resistant to reactivation by dithiothreitol. There appear to be at least two different ways of forming inactive, oxidized enzyme products depending on the oxidant used, suggesting the possibility of multiple sulfhydryl groups at or near the active site.     

Modification of cellular protein sulfhydryl groups by activated soluble immune response suppressor

Soluble immune response suppressor (SIRS), a product of murine Ly-2+ T lymphocytes, is activated to SIRSox by H2O2 produced by macrophages: SIRSox directly inhibits cell division by normal and neoplastic cells and antibody secretion by B lymphocytes. To examine the mechanism of SIRSox-mediated inhibition, a variety of cellular functions were measured after treatment of cells with SIRSox. These included respiration, glucose transport, microtubule content, glutathione content, production of H2O2 or superoxide anion, and the activities of a variety of different enzymes. Several cellular activities or measurements were inhibited or lowered after SIRSox-treatment, including cell division, microtubule content, glutathione reductase activity, and thioredoxin reductase activity; inhibition was partially reversed by the sulfhydryl reducing agent dithiothreitol. Protein sulfhydryl content of P815 mastocytoma cells and several other cell types was lowered by 35 to 45% after exposure to SIRSox. Protein sulfhydryl loss was also partially restored after incubation with dithiothreitol. Sulfhydryl loss was not due to cell lysis. In addition, treatment of crude cellular particulate fractions with SIRSox resulted in protein sulfhydryl loss and formation of protein sulfenyl derivatives. A comparison of the amount of SIRS and H2O2 present to the number of protein sulfhydryls lost or sulfenyl derivatives formed suggests that SIRSox acts catalytically, serves as a co-factor in protein sulfhydryl oxidation, or that it activates a second pathway that is directly responsible for sulfhydryl oxidation.     

Topographical localization and characterization of microsomal glucose-6-phosphatase binding sites accessible to 4,4'-diazidostilbene 2,2'-disulfonic acid

The effect of the photoactivated reagent 4,4'-diazidostilbene 2,2'-disulfonic acid (DASS) on rat liver microsomal glucose-6-phosphatase has been investigated in order to analyze the accessibility and the chemical nature of functional sites of the integral enzyme protein. The following results were obtained. (i) When native rat liver microsomes are irradiated with the photoactive reagent, the activity of glucose-6-phosphatase is progressively inhibited. However, complete reactivation is obtained by modification of the DASS-labeled microsomes with Triton X-114. (ii) Inhibition of glucose-6-phosphatase is also reversed when the DASS-labeled microsomes are treated with p-mercuribenzoate or dithiothreitol. (iii) When native microsomes are labeled with DASS an intensely fluorescent adduct is formed whose emission and excitation maximum corresponds with those obtained when cysteine or 3-mercaptopropionic acid are irradiated in the presence of the photolabile reagent. (iv) The data from fluorescence measurements show that p-mercuribenzoate and dithiothreitol reduce fluorescence labeling of the microsomes whereas Triton modification of the DASS-labeled membranes does not affect the DASS-induced fluorescence. (v) Glucose 6-phosphate hydrolysis of the partially purified glucose-6-phosphatase is also inhibited as observed with native microsomes. The DASS-induced inhibition is reversed and prevented by p-mercuribenzoate; however, the partially purified enzyme cannot be reactivated by Triton X-114. (vi) When glucose-6-phosphatase is partially purified from the DASS-labeled microsomes this enzyme preparation is fluorescence labeled and inhibited. From these results we conclude that DASS directly reacts with the integral phosphohydrolase mainly by chemical modification of essential sulfhydryl groups of the enzyme protein accessible from the cytoplasmic surface of the native microsomal membrane. The Triton-induced reactivation of the glucose-6-phosphatase of DASS-labeled microsomes is explained in terms of conformational changes of the integral protein elicited during modification of the surrounding membrane by detergent.     

Peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Coincident nitration of enzyme tyrosyl residues has minimal impact on catalytic activity.

Tryptophan hydroxylase, the initial and rate-limiting enzyme in serotonin biosynthesis, is inactivated by peroxynitrite in a concentration-dependent manner. This effect is prevented by molecules that react directly with peroxynitrite such as dithiothreitol, cysteine, glutathione, methionine, tryptophan, and uric acid but not by scavengers of superoxide (superoxide dismutase), hydroxyl radical (Me(2)SO, mannitol), and hydrogen peroxide (catalase). Assuming simple competition kinetics between peroxynitrite scavengers and the enzyme, a second-order rate constant of 3.4 x 10(4) M(-1) s(-1) at 25 degrees C and pH 7.4 was estimated. The peroxynitrite-induced loss of enzyme activity was accompanied by a concentration-dependent oxidation of protein sulfhydryl groups. Peroxynitrite-modified tryptophan hydroxylase was resistant to reduction by arsenite, borohydride, and dithiothreitol, suggesting that sulfhydryls were oxidized beyond sulfenic acid. Peroxynitrite also caused the nitration of tyrosyl residues in tryptophan hydroxylase, with a maximal modification of 3.8 tyrosines/monomer. Sodium bicarbonate protected tryptophan hydroxylase from peroxynitrite-induced inactivation and lessened the extent of sulfhydryl oxidation while causing a 2-fold increase in tyrosine nitration. Tetranitromethane, which oxidizes sulfhydryls at pH 6 or 8, but which nitrates tyrosyl residues at pH 8 only, inhibited tryptophan hydroxylase equally at either pH. Acetylation of tyrosyl residues with N-acetylimidazole did not alter tryptophan hydroxylase activity. These data suggest that peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Modification of tyrosyl residues by peroxynitrite plays a relatively minor role in the inhibition of tryptophan hydroxylase catalytic activity.     

Modulation of receptor dynamics by the regulator of G protein signaling Sst2

G protein–coupled receptor (GPCR) signaling is fundamental to physiological processes such as vision, the immune response, and wound healing. In the budding yeast Saccharomyces cerevisiae, GPCRs detect and respond to gradients of pheromone during mating. After pheromone stimulation, the GPCR Ste2 is removed from the cell membrane, and new receptors are delivered to the growing edge. The regulator of G protein signaling (RGS) protein Sst2 acts by accelerating GTP hydrolysis and facilitating pathway desensitization. Sst2 is also known to interact with the receptor Ste2. Here we show that Sst2 is required for proper receptor recovery at the growing edge of pheromone-stimulated cells. Mathematical modeling suggested pheromone-induced synthesis of Sst2 together with its interaction with the receptor function to reestablish a receptor pool at the site of polarized growth. To validate the model, we used targeted genetic perturbations to selectively disrupt key properties of Sst2 and its induction by pheromone. Together our results reveal that a regulator of G protein signaling can also regulate the G protein–coupled receptor. Whereas Sst2 negatively regulates G protein signaling, it acts in a positive manner to promote receptor retention at the growing edge.     

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.     

Interaction of photosynthetic pigments with various organic solvents. Magnetic circular dichroism approach and application to chlorosomes

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.