Interspecies interaction based on transfer of a thioredoxin-like compound in anaerobic chitin-degrading mixed cultures

Abstract Fermentation of chitin by mixed cultures of the chitinolytic Clostridium sp. strain 9.1 and various non-chitinolytic bacteria proceeded up to eight times faster than in pure cultures. The addition of spent media of such mixed cultures also resulted in a marked stimulation of chitinolysis in pure cultures of strain 9.1. Pure cultures fermented chitin much faster if supplemented with either spent media or cell-free extracts of the non-chitinolytic bacteria. The compound responsible for this stimulation was thermostable (10 min at 85° C) and could not be removed by passage over Sephadex G-25, indicating a molecular weight of more than 1500. The heat stable enzyme thioredoxin (from Saccharomyces cerevisiae ) was shown to stimulate the chitin fermentation in a similar manner. Alkylation of this enzyme reduced its stimulatory action significantly indicating its (di)thiol: disulfide interchanging activity.
It is hypothesized that essential sulfhydryl groups in the chitinolytic system of strain 9.1 are reduced by thioredoxin and/or similar thiol: disulfide transhydrogenases present in the cell-free extracts and spent media, resulting in an acceleration of chitin hydrolysis and fermentation. This stimulation may thus be the result of a new type of interspecies interaction in anaerobic mixed cultures.     

Essential Sulfhydryl Group in the Transport-catalyzing Protein of the Hexose-Proton Cotransport System of Chlorella

The polyene antibiotic nystatin transforms the sugar-proton contransport system of Chlorella to a mere facilitated diffusion system. This experimental condition was used to test the sugar-translocating unit of the active uptake system for possible essential sulfhydryl groups. It could be shown that the catalyzed translocation of sugar is sensitive to the sulfhydryl-reactive compound N-ethylmaleimide. Sugar flow by passive leak as induced by the detergent Triton X-100 is not affected by sulfhydryl reagents. These results show that the sugar-translocating carrier protein possesses a sulfhydryl group, which is essential for its function.     

Effect of sulfhydryl reagents on nucleoside transport in cultured mammalian cells

Incubation of Novikoff rat hepatoma cells; mouse L929, P388 and L1210 cells; and Chinese hamster ovary cells with sulfhydryl reagents, such as p-hydroxymercuribenzoate or p-hydroxymercuribenzenesulfonate, reduced the zero-trans influx of uridine in a concentration-dependent manner. The sensitivity of uridine transport to inhibition varied somewhat for the cell lines, Chinese hamster ovary cells being the most sensitive. Maximum inhibition by p-hydroxymercuribenzoate occurred in 10–20 min of incubation at 37 °C, and was associated with a decrease in maximum transport velocity without significant change in substrate affinity of the carrier. The development of inhibition of uridine influx correlated with binding of [¹⁴C]p-hydroxymercuribenzoate to the cells. Inhibition of transport also roughly correlated with a decreased binding of 6-nitrobenzylthioinosine to high-affinity binding sites on the cells (presumably representing the nucleoside transporter) without affecting binding affinity. Treatment of cells with p-hydroxymercuribenzenesulfonate reduced uridine influx and efflux to a similar extent. Inhibition of uridine transport and binding of [¹⁴C]p-hydroxymercuribenzoate were readily reversed by incubation of the cells with dithiothreitol. The results indicate that sulfhydryl groups are essential for the functioning of the nucleoside transporter, perhaps for the binding of substrate. Blockage of the sulfhydryl groups results in a reversible inactivation of the carrier. Treatment of the cells with the sulfhydryl reagents also caused a concentration-dependent increase in cell volume, which was readily reversed by incubation of the cells with dithiothreitol but seemed unrelated to the inhibition of nucleoside transport.     

Inhibition of lectin-induced lymphocyte activation by diamide and other sulfhydryl reagents

Inhibition of lectin-induced lymphocyte activation by five reagents capable of combining with or oxidizing free sulfhydryl groups was examined. Each of the reagents tested was capable of inhibiting [methyl-³H]thymidine or [¹⁴C]uridine incorporation into trichloroacetic acid-insoluble material. Four of these reagents, iodoacetamide and N-ethylmaleimide (alkylating agents) and 5,5′-dithiobis (2-nitrobenzoic acid) and p-hydroxymercuriphenylsulfonic acid (sulfhydryl binding agents), inhibited activation when added to lymphocyte cultures together with lectin or at any time thereafter through 48 hr. In contrast, the sulfhydryl oxidizing agent diazine dicarboxylic acid bis[N,N-dimethylamide] (diamide) was effective only when added within 30–60 min of lectin or when added after 24 hr. This inhibition of lymphocyte activation was not due to decreased intracellular levels of reduced glutathione or to inhibition of binding of lectin to the lymphocyte. These results suggest that maintenance of free sulfhydryl groups is important during the early induction of lymphocyte activation and suggest that an obligatory step or steps in the activation sequence may involve sulfhydryl interactions.     

Comparative inhibition studies of the phosphotransferase and glycerophosphate acylation systems in membrane vesicles of Escherichia coli

Purified membrane vesicles were treated with various reagents specific for different amino acid side-chains. Titration of sulfhydryl groups with specific reagents shows that the sulfhydryl content of membrane vesicles as estimated directly is similar to that found by treating spheroplasts or cells and then isolating the membrane vesicles. The blocking of sulfhydryl groups specifically inhibits the α-methylglucoside transport system (phosphotransferase system), whereas the glycerophosphate acylation system is not affected. The kinetics of inhibition of the first system show that a high reactivity of the sulfhydryl groups is involved. Inhibition of the acyltransferase activity by sulfhydryl reagents occurs only on partial denaturation of the membranes induced by mild sonication, heat or toluene treatment. The Inhibition is at the level of the glycerol 3-phosphate:acyl thioester acyltransferase.The effects of sonication and/or sulfhydryl reagents were measured by sulfhydryl titration, by assays of NADH oxidase and d-lactate dehydrogenase activities, as well as by 1-anilino-8-naphthalene sulfonate binding. The results support the hypothesis that the acyltransferase system is embedded within the membrane and that the readily accessible permease system is closer to (or at) the surface of the membrane.     

Microsomal sn-glycerol 3-phosphate and dihydroxyacetone phosphate acyltransferase activities from liver and other tissues. Evidence for a single enzyme catalizing both reactions.

Liver microsomal cytochrome P-448 purified from 3-methylcholanthrene-treated rats or rabbits contained seven free sulfhydryl groups per mole of enzyme as determined by amino acid analysis or by spectrophotometric titrations with 5,5′-dithiobis(2-nitroben-zoic acid), 4,4′-dipyridinedisulfide, 2-nitro-5-thiocyanobenzoic acid, and p-mercuribenzoate. The rat cytochrome P-448-catalyzed hydroxylation of benzo[a]pyrene was inhibited 70% after modification of the enzyme with 5,5′-dithiobis(2-nitrobenzoic acid) but was unaffected after titration of the enzyme with other sulfhydryl reagents, suggesting that the sulfhydryl groups may not be essential for catalysis. On the other hand, the rabbit cytochrome P-448-catalyzed hydroxylation of benzo[a]pyrene was inhibited following the modification of this enzyme with all of the sulfhydryl reagents listed above. Whether the loss in catalytic activity in this case is due to the essential role of the sulfhydryl groups in catalysis or to the steric hindrance or conformational change due to the substituent is uncertain.     

The effect of sulfhydryl group reagents on the permeation of mitochondrial aspartate aminotransferase into mitochondria in vitro.

When mitochondria are incubated with radioactively labeled mitochondrial aspartate aminotransferase (EC 2.6.1.1), the enzyme is taken up into the organelles. Mersalyl and p-hydroxymercuriphenyl sulfonic acid, but not N-ethylmaleimide or ethacrynic acid, decrease the extent of this uptake. Inhibition of the uptake by low concentrations of mercurial reagents is due to blockage of a single sulfhydryl group per monomer of the enzyme. Blockage of mitochondrial thiols does not inhibit uptake of the enzyme. A single sulfhydryl group out of a total of six per monomer of the native enzyme reacts with 5,5′-dithiobis-(2-nitrobenzoic acid). This is the same sulfhydryl group that reacts with low levels of mercurial reagents with consequent inhibition of uptake of the enzyme into mitochondria but without effect on the catalytic activity. N-Ethylmaleimide does not react with this group. N-Ethylmaleimide reacts with a different sulfhydryl group with concomitant decrease in enzymic activity but with no effect on uptake of the enzyme into mitochondria. High levels of mercurial reagents similarly decrease enzymic activity. Unlike the effect on uptake into mitochondria, the inhibition by mercurial reagents of enzymic activity is not reversed by treatment with cysteine. The significance of these observations with respect to the mechanism of uptake of aspartate aminotransferase into mitochondria is discussed, and comparisons are made between the reactivities of sulfhydryl groups in rat liver aspartate aminotransferase and in the enzymes from other animals.     

Modulation of activity of human alkaline phosphatases by Mg2+ and thiol compounds

Interaction of purified human liver and placental alkaline phosphatases (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) with sulfhydryl groups, sulfhydryl reagents, and Mg2+ were studied. L-Cysteine (0.1 mmol/l) or Mg2+ activated the liver enzyme 4-5-fold and the placental enzyme 2-3-fold, with optimal pH 7.5-8.0; these activations were not additive. L-Cysteine (2 mmol/l) inhibited both enzymes maximally at pH greater than 9.0; phosphate protected the enzymes. S-Methylcysteine had little effect, with or without Mg2+. Inhibition by sulfur-containing compounds paralleled their ability to bind Zn2+. Fluoresceine mercury acetate (specific for sulfhydryl groups) inhibited the isoenzymes, whereas iodoacetic acid, iodoacetamide, dithionitrobenzoic acid, and p-chloromercuribenzoate had little effect. The inhibition was reversed by L-cysteine and only slightly protected by inorganic phosphate. Thus, there are two sites on human liver and placental alkaline phosphatase that interact with L-cysteine; a Mg2+-binding site, which results in activation, and a site that involves one or both of the bound Zn2+ ions and results in inactivation. Both enzymes have a protected essential thiol group.     

Comparative effects of sulfhydryl reagents on membrane-bound and solubilized UDP-glucose:ceramide glucosyltransferase from Golgi membranes. Evidence for partial involvement of a thiol group in the nucleotide sugar binding site of the solubilized enzyme

We have studied the inactivation of membrane-bound and solubilized UDP-glucose:ceramide glucosyltransferase from Golgi membranes by various types of sulfhydryl reagents. The strong inhibition of the membrane-bound form by the non-penetrant mercurial-type reagents clearly corroborated the fact that in sealed and right-side-out Golgi vesicles the ceramide glucosyltransferase is located on the cytoplasmic face. No significant differences in the susceptibility to the various sulfhydryl reagents were noted when solubilized enzyme was assayed, showing that solubilization does not reveal other critical SH groups. The different results obtained must be interpreted with regard to several thiol groups, essential for enzyme activity. No protection by the substrate UDP-glucose against mercurial-type reagents was obtained indicating that these thiol groups were not located in the nucleotide sugar binding domain. A more thorough investigation of the thiol inactivation mechanism was undertaken with NEM (N-ethylmaleimide), an irreversible reagent. The time dependent inactivation followed first order kinetics and provided evidence for the binding of 1 mol NEM per mol of enzyme. UDP-Glucose protected partially against NEM inactivation, indicating that the thiol groups may be situated in or near the substrate binding domain. Inactivation experiments with disulfide reagents showed that increased hydrophobicity led to more internal essential SH groups which are not obviously protected by the substrate UDP-glucose, thus not implicated in the substrate binding domain, but rather related to conformational changes of the enzyme during the catalytic process.Abbreviations Chaps 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate - Mops 4-morpholinepropanesulfonic acid - PC phosphatidylcholine - NEM N-ethylmaleimide - CPDS carboxypyridine disulfide (dithio-6,6-dinicotinic acid) - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - DTP dithiodipyridine - p-HMB para-hydroxymercuribenzoate - DTT dithiothreitol - BAL British anti-Lewisite (dimercaptopropanol) - Zw 3–14 Zwittergent 3–14     

Modification of three enzymes of the β-ketoadipate pathway by N-methyl-N′-nitro-N-mtrosoguanidine

The sensitivities of three enzymes of the β-ketoadipate pathway to inactivation by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) were determined in vivo and in vitro under conditions compatible with mutagenesis.One enzyme, β-ketoadipate enol-lactone hydrolase, is very sensitive to inactivation by low concentrations of MNNG. This enzyme is also sensitive to inactivation by N-ethylmaleimide and mercurial reagents. The free sulfhydryl content of native enol-lactone hydrolase was determined to be two moles free sulfhydryl per mole of enzyme. A 95% inactivation of enol-lactone hydrolase by MNNG results in a masking of slightly more than one mole sulfhydryl per mole enzyme.Muconate lactonizing enzyme is moderately sensitive to inactivation by low concentrations of MNNG, but is not inactivated by sulfhydryl reagents. Muconolactone isomerase is resistant to inactivation by low concentrations of MNNG and is not inactivated by sulfhydryl reagents. Upon exposure to high concentrations of MNNG, muconolactone isomerase is rapidly inactivated. Spectrophotometric evidence indicates the lysine residues are nitroguanidinated proportionally with a loss in the enzymatic activity.These data indicate that the exposure of cells to low concentrations of MNNG should affect the activity of enzymes with essential sulfhydryl groups.     

Reactive sulfhydryl groups of coenzyme B12-dependent diol dehydrase: Differential modification of essential and nonessential ones

The apoenzyme of diol dehydrase was inactivated by four sulfhydryl-modifying reagents, p-chloromercuribenzoate, 5,5′-dithiobis(2-nitrobenzoate) (DTNB), iodoacetamide, and N-ethylmaleimide. In each case pseudo-first-order kinetics was observed. p-Chloromercuribenzoate modified two sulfhydryl groups per enzyme molecule and modification of the first one resulted in complete inactivation of the enzyme. DTNB also modified two sulfhydryl groups, but modification of the second one essentially corresponded to the inactivation. In both cases, the inactivation was reversed by incubation with dithiothreitol. Cyanocobalamin, a potent competitive inhibitor of adenosylcobalamin, protected the essential residue, but not the nonessential one, against the modification by these reagents. By resolving the sulfhydryl-modified cyanocobalamin-enzyme complex, the enzyme activity was recovered, irrespective of treatment with dithiothreitol. From these results, we can conclude that diol dehydrase has two reactive sulfhydryl groups, one of which is essential for catalytic activity and located at or in close proximity to the coenzyme binding site. The other is nonessential for activity. Neitherp-chloromercuribenzoate- nor DTNB-modified apoenzyme was able to bind cyanocobalamin, whereas the iodoacetamide- and N-ethylmaleimide-modified apoenzyme only partially lost the ability to bind cyanocobalamin. The inactivation of diol dehydrase by p-chloromercuribenzoate and DTNB did not bring about dissociation of the enzyme into subunits. Total number of the sulfhydryl groups of this enzyme was 14 when determined in the presence of 6 m guanidine hydrochloride. No disulfide bond was detected.     

The role of enzyme I in the unmasking of an essential thiol of the membrane-bound enzyme II of the phosphoenolpyruvate-glucose phosphotransferase system of Escherichia coli

The membrane-bound component of the phosphotransferase system of Escherichia coli, responsible for the phosphorylative uptake of methyl-α-d-glucoside has an essential thiol group which becomes available to inactivation by thiol reagents in the presents of the phosphate-accepting sugar or when phosphoenolpyruvate synthesis is inhibited. The form resistant to the thiol reagent requires not only the absence of sugar and an intact phosphoenol-pyruvate generating system, but also an intact system generating phosphorylated Hpr which is impaired by heating of a thermosensitive enzyme I mutant.     

Interaction between catalytic and regulatory sites of the pyruvate dehydrogenase from Escherichia coli studied by the ESR technique

The accessibility of sulfhydryl groups at the pyruvate dehydrogenase component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was reinvestigated. Hydrophobic interactions appear to control the reactivity of an essential cysteine residue at the active site with thiol reagents. This explains why the essential cysteine residue reacts only with thiol reagents of minor polarity, like p-hydroxymercuribenzoate or phenylmercuric nitrate, but not with Ellman's reagent or jodoacetamide. The pyruvate dehydrogenase component was modified with a nitroxide derivative of p-hydroxymercuribenzoate. The ESR spectrum of the spin-labelled enzyme changed dramatically upon addition of the cofactors thiamine diphosphate and Mg2+. Obviously spin-spin interaction occurs under these conditions caused by a transition of an inactive to an active state of the enzyme. The same conformational change is observed when the allosteric activator AMP instead of the cofactors was bound to the enzyme. The implications of these results for the allosteric regulation of the pyruvate dehydrogenase complex are discussed.     

Chloroplast sulfhydryl groups and the light activation of fructose-1,6-bisphosphatase

Studies of isolated intact spinach (Spinacia oleracea L.) chloroplasts reveal that most of the available sulfhydryl groups are associated with stromal protein as opposed to a thylakoid membrane fraction under non-denaturing conditions. Increases in sulfhydryl content of approximately 50% occurred with illumination and could be correlated kinetically with a reductive activation of fructose-1,6-bisphosphatase during CO₂-assimilation. Inhibition of linear electron flow with 3-(3,4-dichlorophenyl)-1,1-dimethylurea prevented light driven increases in both fructose-1,6-bisphosphatase activity and the relative sulfhydryl number. These results provide evidence for the operation of a reductive enzyme activating system in vivo.     

Entamoeba histolytica and E. invadens: Sulfhydryl-Dependent Proteolytic Activity1

Sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME) activated proteolytic enzymes present in extracts of Entamoeba histolytica and E. invadens; SDS (0.5%) and 2-ME (1.4 and 715 mM) doubled the enzymatic activity when assayed on a stained insoluble substrate. Urea (4 M) did not reduce this activity, suggesting that amebic proteases are stable in the above denaturant conditions. Specific reagents for sulfhydryl (-SH) groups completely inhibited proteolytic activity regardless of pH. Inhibition with alkylating agents, such as N-ethylmaleimide and iodoacetamide, was reversed with 715 mM 2-ME as was also observed with papain. We conclude from these results that the main proteolytic enzymes contained in extracts of E. histolytica and E. invadens are dependent on free thiol groups.     

Evidence for the Existence of Two Essential and Proximal Cysteinyl Residues in NADP-Malic Enzyme from Maize Leaves

Incubation of maize (Zea mays) leaf NADP-malic enzyme with monofunctional and bifunctional N-substituted maleimides results in an irreversible inactivation of the enzyme. Inactivation by the monofunctional reagents, N-ethylmaleimide (NEM) and N-phenylmaleimide, followed pseudo-first-order kinetics. The maximum inactivation rate constant for phenylmaleimide was 10-fold higher than that for NEM, suggesting a possible hydrophobic microenvironment of the residue(s) involved in the modification of the enzyme. In contrast, the inactivation kinetics with the bifunctional maleimides, ortho-, meta-, and para-phenylenebismaleimide, were biphasic, probably due to different reactivities of the groups reacting with the two heads of these bifunctional reagents, with a possible cross-linking of two sulfhydryl groups. The inactivation by mono and bifunctional maleimides was partially prevented by Mg²⁺ and l-malate, and NADP prevented the inactivation almost totally. Determination of the number of reactive sulfhydryl groups of the native enzyme with [³H]NEM in the absence or presence of NADP showed that inactivation occurred concomitantly with the modification of two cysteinyl residues per enzyme monomer. The presence of these two essential residues was confirmed by titration of sulfhydryl groups with [³H]NEM in the enzyme previously modified by o-phenylenebismaleimide in the absence or presence of NADP.     

Fat metabolism in higher plants. The elongation of saturated and unsaturated acyl-CoAs by a stromal system from isolated spinach chloroplasts.

A soluble enzyme system from the stroma of chloroplasts isolated from Spinacease oleraceae elongated various long chain acyl-CoAs using acetyl-CoA as a two-carbon donor. Partial purification of the system was achieved by ammonium sulfate fractionation and molecular sieve chromatography. The elongation system required NADPH and NADH for the reduction steps. Several nucleoside triphosphates markedly stimulated elongation. Inhibition occurred with several thiol binding reagents and with free CoA. The possible significance of elongation via acyl-CoAs in chloroplasts is discussed.     

Derivatization of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi as probe of the interrelationship between the catalytic sites and the dimer interface

In the interface of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), one cysteine of each monomer forms part of the intersubunit contacts. The relatively slow derivatization of these cysteines by sulfhydryl reagents induces progressive structural alterations and abolition of catalysis [Garza-Ramos et al. (1998) Eur. J. Biochem. 253, 684-691]. Derivatization of the interface cysteine by 5, 5-dithiobis(2-nitrobenzoate) (DTNB) and methylmethane thiosulfonate (MMTS) was used to probe if events at the catalytic site are transmitted to the dimer interface. It was found that enzymes in the active catalytic state are significantly less sensitive to the thiol reagents than in the resting state. Maximal protection against derivatization of the interface cysteine by thiol reagents was obtained at near-saturating substrate concentrations. Continuous recording of derivatization by DTNB showed that catalysis hinders the reaction of sulfhydryl reagents with the interface cysteine. Therefore, in addition to intrinsic structural barriers, catalysis imposes additional impediments to the action of thiol reagents on the interface cysteine. In TcTIM, the substrate analogue phosphoglycolate protected strongly against DTNB action, and to a lesser extent against MMTS action; in TbTIM, phosphoglycolate protected against the effect of DTNB, but not against the action of MMTS. This indicates that barriers of different magnitude to the reaction of thiol reagents with the interface cysteine are induced by the events at the catalytic site. Studies with a Cys14Ser mutant of TbTIM confirmed that all the described effects of sulfhydryl reagents on the trypanosomal enzymes are a consequence of derivatization of the interface cysteine.     

Phosphate transport protein of rat heart mitochondria: location of its SH-groups and exploration of their environment

(1) The properties of the SH groups of the phosphate transport protein of rat heart mitochondria were investigated on the basis of inhibition caused by SH reagents under different conditions. (2) The essential thiol groups are located near the external surface, as they are accessible to impermeable reagents from the external space. (3) The environment of the sulfhydryl groups influences their reactivity, as alteration of the external pH affects adversely their reactions with ionizable and non-ionizable SH reagents. (4) Intramitochondrial pH exerts a transmembrane effect: alkalinization augments and acidification diminishes the reaction rate of the sulfhydryl groups on the opposite surface of the membrane. (5) Changes of the concentration of the transported substrate occurring exclusively in the extramitochondrial space do not influence the reactivity of the essential SH groups. (6) It is concluded that in transport studies the phosphate transport protein of heart and liver mitochondria show basic similarity. It is suggested that the amino-acid sequence around the NEM-reactive cysteine (i.e., Lys-41 - Cys-42 - Arg-43) does not participate in substrate binding.     

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.