Isethionate as a product from taurine during nitrogen-limited growth of <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> TauN1

Klebsiella oxytoca TauN1 represents a group of isolates which utilise taurine (2-aminoethanesulfonate) quantitatively as a sole source of combined nitrogen for aerobic growth. During growth, a compound is excreted, which has now been identified as isethionate (2-hydroxyethanesulfonate). An ion-chromatographic separation of isethionate was developed to quantify the putative isethionate, whose identity was confirmed by matrix-assisted, laser-desorption ionisation time-of-flight mass spectrometry. Strain TauN1 utilised taurine (and excreted isethionate) concomitantly with growth. Cell-free extracts contained inducible taurine transaminase, which yielded sulfoacetaldehyde. A soluble, NADP-dependent isethionate dehydrogenase converted sulfoacetaldehyde to isethionate. The enzyme was partially purified and it apparently belonged to the family of short-chain alcohol dehydrogenases.We hope that the Leader of the Sulfur Department, Norbert Pfennig LSD, will be amused by the biology involving some of the compounds from his domain.     

An improved sulphur assay applied to a problem of isethionate metabolism in squid axon and other nerves

An assay has ken developed for total sulphur which is based on a wet oxidation and measurement with a spectrophotofluorometer of light scattering by barium sulphate. The method has been adapted to the measurement of isethionate in squid nerve and blood, in other cephalopod nerve, and in the nerve tissue of other species including mammals. A correlation has been found between isethionate contents and the activity in the same tissues of one kind of DFP-hydrolysing enzyme, the highest levels of both being in squid nerve. Squid nerve also took up cysteine rapidly and metabolized it predominantly to hypotaurine but not to isethionate. We speculate that a hypotaurine derivative is a reserve form of isethionate, and that the so-called DFPase is involved in the release of hypotaurine and its metabolism to isethionate as needed.     

Hydrogen sulfide as a precursor for the synthesis of isethionate in the squid giant axon.

Cysteine is taken up by the squid giant axon to about 200% of equivalent distribution, whereas sulfide is taken up (probably as hydrogen sulfide) to about 40% of equivalence. Thereafter, the squid axon synthesizes its major anion, isethionate, in about equal amounts from the sulfide, or from the sulfur of cysteine, but not at all from the carbons of cysteine. Squid nerve also contains rhodanese, an enzyme which transfers the outer (sulfane) sulfur of thiosulfate to cyanide to produce thiocyanate. It is speculated that, instead of “detoxifying cyanide,” as the reaction involving rhodanese is commonly described, the physiological role of this enzyme is the formation of a carbon-sulfur bond, leading finally, in the squid, to the formation of isethionate. This is the first evidence concerning the pathway for the synthesis of isethionate in squid nerve where this compound is normally present at a concentration of 150 mm.     

Purification of a DFP-hydrolyzing enzyme from squid head ganglion

An enzyme which hydrolyzes DFP and similar organophosphorus compounds has been purified 1300-fold from squid head ganglion. This enzyme which we term squid nerve DFPase is markedly different from previously reported DFPases from mammalian and microbial sources. The enzyme shows marked selectivity with respect to substrates, permitting some speculation about the nature of its active site. A striking feature of squid nerve DFPase is its relative limitation to cephalopod nerve. Available evidence suggests that a similar distribution may exist for isethionate, the major anion in squid nerve. This tentative parallel between DFPase and isethionate raises the possibility of a function for squid nerve DFPase.     

Role of the decrease in ionized calcium in the inhibition of insulin release by chloride-free solutions

Replacement of extracellular Cl- by isethionate or sulfate during stimulation with glucose or tolbutamide reversibly inhibited insulin release by perifused mouse islets. The concentration of ionized Ca2+ was decreased by 30 and 55% in isethionate and sulfate solutions, respectively. If this fall was prevented, the inhibition of release was only slightly affected (isethionate) or substantially attenuated (sulfate). In conclusion, the inhibition of insulin release occurring in Cl(-)-free solutions cannot be completely ascribed to a decrease in ionized Ca2+ in the medium. The contribution of this latter depends on the Cl- substitute.     

The metabolism of taurine to isethionate

The biosynthesis of isethionate from taurine in mammalian tissue has been reexamined. In vivo metabolism of taurine to isethionate was demonstrated but it was shown that a number of acyltaurine metabolites also behave like isethionate on the conventional dual column ion-exchange chromatographic analytical system. Hydrolysis of these column effluates coupled with high-voltage electrophoresis resolves this ambiguity. In vivo formation of isethionate from taurine in mammals seems to occur from gut microorganism metabolism since: (a) germ-free mice could not convert taurine to isethionate, (b) gut anaerobes were able to metabolize taurine, (c) in vitro rat and mouse tissue failed to metabolize taurine to isethionate. These findings are in conflict with earlier reports.     

Comparative aspects of utilization of sulfonate and other sulfur sources by Escherichia coli K12

Selected biochemical features of sulfonate assimilation in Escherichia coli K-12 were studied in detail. Competition between sulfonate-sulfur and sulfur sources with different oxidation states, such as cysteine, sulfite and sulfate, was examined. The ability of the enzyme sulfite reductase to attack the C-S linkage of sulfonates was directly examined. Intact cells formed sulfite from sulfonate-sulfur. In cysteine-grown cells, when cysteine was present with either cysteate or sulfate, assimilation of both of the more oxidized sulfur sources was substantially inhibited. In contrast, none of three sulfonates had a competitive effect on sulfate assimilation. In studies of competition between different sulfonates, the presence of taurine resulted in a decrease in cysteate uptake by one-half, while in the presence of isethionate, cysteate uptake was almost completely inhibited. In sulfite-grown cells, sulfonates had no competitive effect on sulfite utilization. An E. coli mutant lacking sulfite reductase and unable to utilize isethionate as the sole source of sulfur formed significant amounts of sulfite from isethionate. In cell extracts, sulfite reductase itself did not utilize sulfonate-sulfur as an electron acceptor. These findings indicate that sulfonate utilization may share some intermediates (e.g. sulfite) and regulatory features (repression by cysteine) of the assimilatory sulfate reductive pathway, but sulfonates do not exert regulatory effects on sulfate utilization. Other results suggest that unrecognized aspects of sulfonate metabolism, such as specific transport mechanisms for sulfonates and different regulatory features, may exist.     

Osmotic regulation of prolactin secretion. Possible role of chloride

The role of osmotic pressure in the exocytosis of prolactin from rat pituitary tumor (GH) cells in culture was investigated. Reducing the osmotic strength of the medium from 300 mosm to 150 mosm by removal of NaCl did not alter basal secretion of prolactin but inhibited secretion stimulated by thyrotropin-releasing hormone (TRH) and forskolin. Both basal and stimulated secretion of prolactin were inhibited by increasing the osmotic strength of the medium with NaCl (IC50 at approximately 500 mosm). The stimulated release of hormone from GH-cells was independent of sodium and unaffected by replacement of sodium ion with tetramethylammonium or choline, or by addition of 500 nM tetrodotoxin. Secretagogue-stimulated release was, however, dependent upon chloride. Exchange of medium chloride with benzoate or isethionate significantly inhibited the stimulated release of prolactin (IC50 at approximately 60 mM exchange) regardless of the secretagogue utilized (phorbol ester, forskolin, depolarization plus BAY K8644, or TRH). Exchange of medium chloride with either isethionate or benzoate reduced cell volume by 10% compared to 60% for sucrose and mannitol, suggesting that inhibition of secretion by isethionate exchange was not a result of increased intracellular osmotic pressure. Complete exchange of medium chloride with isethionate did not alter equilibrium [3H]methyl-TRH binding, resting internal [Ca2+], or the [Ca2+]i response to depolarization and TRH as measured with intracellularly trapped Fura 2. Chloride removal did not change resting internal pH and recovery from an acid load as measured by the intracellular pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The stimulated secretion of prolactin was also inhibited by exchange of chloride with isethionate in normal pituitary cells in primary culture and the ability of normal cells to respond to the dopamine agonist bromocryptine was not affected by the exchange. These results suggest that exocytosis of prolactin from GH-cells and normal pituitary cells in culture is an osmotically driven process that is chloride-dependent. Stimulated release is more chloride-dependent than constitutive release. The inhibitory effect of isethionate substitution occurs after signal transduction and is distinct from the site of dopamine inhibition of prolactin release.     

COMPARATIVE STUDIES ON THE DEGRADATION OF GABA and TAURINE IN THE BRAIN

Abstract— The degradation of taurine and GABA in mammalian brain was studied in vivo and in vitro. Small amounts of [³⁵S]isethionate (10–20 pmol/g brain wet weight) and [³⁵S]sulphate (about 2 pmol/g) were detected in mouse brain after intramuscular injection of [³⁵S]taurine. Taurine also produced isethionate in rat brain homogenates (about 20 nmol/h/g protein) and subcellular fractions (about 40 nmol/h/g protein in synaptosomes and about 300 nmol/h/g in mitochondria), but the reaction was not stimulated either by external electrical pulses or by the addition of various cofactors (NAD and NADP in both oxidized and reduced forms, riboflavin, glutathione. pyridoxal-5'-phosphate, ATP) to the incubation medium. [¹⁴C]GABA was readily metabolized to [¹⁴C]succinate both in vivo and in vitro. Isethionate formation activity was concentrated in the mitochondrial fraction, as was also GABA-T activity. Partially purified GABA-T from calf brain also slightly catalysed the formation of [³⁵S]isethionate (about 1.3 μmol/min/g protein) from [³⁵S]taurine. It appears that the slight formation of isethionate from taurine is coupled to GABA-T activity. The formation of isethionate from taurine is so small, that it apparently has no role in the control of the brain taurine pool.     

Probing an open CFTR pore with organic anion blockers

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that conducts Cl- current. We explored the CFTR pore by studying voltage-dependent blockade of the channel by two organic anions: glibenclamide and isethionate. To simplify the kinetic analysis, a CFTR mutant, K1250A-CFTR, was used because this mutant channel, once opened, can remain open for minutes. Dose-response relationships of both blockers follow a simple Michaelis-Menten function with K(d) values that differ by three orders of magnitude. Glibenclamide blocks CFTR from the intracellular side of the membrane with slow kinetics. Both the on and off rates of glibenclamide block are voltage dependent. Removing external Cl- increases affinity of glibenclamide due to a decrease of the off rate and an increase of the on rate, suggesting the presence of a Cl- binding site external to the glibenclamide binding site. Isethionate blocks the channel from the cytoplasmic side with fast kinetics, but has no measurable effect when applied extracellularly. Increasing the internal Cl- concentration reduces isethionate block without affecting its voltage dependence, suggesting that Cl- and isethionate compete for a binding site in the pore. The voltage dependence and external Cl- concentration dependence of isethionate block are nearly identical to those of glibenclamide block, suggesting that these two blockers may bind to a common binding site, an idea further supported by kinetic studies of blocking with glibenclamide/isethionate mixtures. By comparing the physical and chemical natures of these two blockers, we propose that CFTR channel has an asymmetric pore with a wide internal entrance and a deeply embedded blocker binding site where local charges as well as hydrophobic components determine the affinity of the blockers.     

Cloning and Characterization of a Sulfonate/α-Ketoglutarate Dioxygenase from Saccharomyces cerevisiae

The Saccharomyces cerevisiae open reading frame YLL057c is predicted to encode a gene product with 31.5% amino acid sequence identity to Escherichia coli taurine/alpha-ketoglutarate dioxygenase and 27% identity to Ralstonia eutropha TfdA, a herbicide-degrading enzyme. Purified recombinant yeast protein is shown to be an Fe(II)-dependent sulfonate/alpha-ketoglutarate dioxygenase. Although taurine is a poor substrate, a variety of other sulfonates are utilized, with the best natural substrates being isethionate and taurocholate. Disruption of the gene encoding this enzyme negatively affects the use of isethionate and taurine as sulfur sources by S. cerevisiae, providing strong evidence that YLL057c plays a role in sulfonate catabolism.     

Sulfonates as terminal electron acceptors for growth of sulfite-reducing bacteria (Desulfitobacterium spp.) and sulfate-reducing bacteria: effects of inhibitors of sulfidogenesis.

This study demonstrates the ability of Desulfitobacterium spp. to utilize aliphatic sulfonates as terminal electron acceptors (TEA) for growth. Isethionate (2-hydroxyethanesulfonate) reduction by Desulfitobacterium hafniense resulted in acetate as well as sulfide accumulation in accordance with the expectation that the carbon portion of isethionate was oxidized to acetate and the sulfur was reduced to sulfide. The presence of a polypeptide, approximately 97 kDa, was evident in isethionate-grown cells of Desulfitobacterium hafniense, Desulfitobacterium sp. strain PCE 1, and the two sulfate-reducing bacteria (SRB)-Desulfovibrio desulfuricans IC1 (T. J. Lie, J. R. Leadbetter, and E. R. Leadbetter, Geomicrobiol. J. 15:135-149, 1998) and Desulfomicrobium norvegicum; this polypeptide was not detected when these bacteria were grown on TEA other than isethionate, suggesting involvement in its metabolism. The sulfate analogs molybdate and tungstate, effective in inhibiting sulfate reduction by SRB, were examined for their effects on sulfonate reduction. Molybdate effectively inhibited sulfonate reduction by strain IC1 and selectively inhibited isethionate (but not cysteate) reduction by Desulfitobacterium dehalogenans and Desulfitobacterium sp. strain PCE 1. Desulfitobacterium hafniense, however, grew with both isethionate and cysteate in the presence of molybdate. In contrast, tungstate only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium spp. Similarly, another inhibitor of sulfate reduction, 1,8-dihydroxyanthraquinone, effectively inhibited sulfate reduction by SRB but only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium hafniense.     

Effects of Cl- deficiency on the membrane potential in mouse pancreatic beta-cells

The membrane potential of mouse pancreatic beta-cells was measured with microelectrodes. In the resting cell (3 mM D-glucose), the membrane potential was -63 +/- 3 mV (mean +/- S.E. for four experiments). In the presence of 3 mM D-glucose, total Cl- substitution by isethionate induced a depolarization by 3-4 mV, and readmission of Cl- induced a hyperpolarization by 3-5 mV. At 10 mM glucose, reduction of Cl- to 12 mM by substituting isethionate for Cl- reversibly shifted the repolarization potential by 6-9 mV in the positive direction and stimulated the burst activity during the initial 2-3 min by increasing the fraction of plateau phase. This was followed by a gradual inhibition of electrical activity, including decrease in fraction of plateau phase and slow wave amplitude. Total substitution of Cl- by isethionate or methyl sulphate reversibly shifted the repolarization potential by 3-4 mV in the positive direction and rapidly inhibited the electrical burst pattern without any initial stimulation. Glucose-induced (10 mM) insulin release (15 min) and 45Ca2+ uptake (3 min) were strongly inhibited by reducing the Cl- concentration to 10 mM (isethionate as substitute) and were further inhibited by further reduction of the Cl- concentration. It is suggested that beta-cells are equipped with on electrogenic Cl- flux, which can affect the burst pattern of electrical activity. The inhibitory effects of Cl- substitution may be explained by an influence of Cl- on the voltage-controlled Ca2+ channels.     

Oxidative phosphorylation. The effect of anions on the inhibition by triethyltin of various mitochondrial functions, and the relationship between this inhibition and binding of triethyltin

1. The binding of triethyltin to rat liver mitochondria is unaffected by the nature of the predominant anion in the incubation medium. 2. With chloride, bromide or iodide as the predominant anion, ATP synthesis linked to the oxidation of pyruvate or succinate and ATP hydrolysis stimulated by 2,4-dinitrophenol are much more sensitive to triethyltin than they are when nitrate or isethionate is the predominant anion. 3. When nitrate or isethionate is the predominant anion, oxygen uptake stimulated by 2,4-dinitrophenol is not inhibited by triethyltin. 4. In the presence of nitrate or isethionate anions, inhibition of ATP synthesis is directly related to the binding of triethyltin to mitochondria. 5. The relationship of the above effects to the anion–hydroxide ion exchange mediated by triethyltin and the relevance of this to published arrangements for coupling of electron transport to ATP synthesis are discussed.     

Mammalian hypotaurine aminotransferase: isethionate is not a product

We report that mammalian tissues posses hypotaurine (2-aminoethane- sulfinate) aminotransferase activity. One product of transamination, sulfinoacetaldehyde could theoretically undergo internal oxidation-reduction leading to isethionate (2-hydroxyethanesulfonate). This hypothesis was examined. No isethionate was formed $\text{in vitro}$.     

Sulfonates as Terminal Electron Acceptors for Growth of Sulfite-Reducing Bacteria (Desulfitobacterium spp.) and Sulfate-Reducing Bacteria: Effects of Inhibitors of Sulfidogenesis

This study demonstrates the ability of Desulfitobacterium spp. to utilize aliphatic sulfonates as terminal electron acceptors (TEA) for growth. Isethionate (2-hydroxyethanesulfonate) reduction by Desulfitobacterium hafniense resulted in acetate as well as sulfide accumulation in accordance with the expectation that the carbon portion of isethionate was oxidized to acetate and the sulfur was reduced to sulfide. The presence of a polypeptide, approximately 97 kDa, was evident in isethionate-grown cells of Desulfitobacterium hafniense, Desulfitobacterium sp. strain PCE 1, and the two sulfate-reducing bacteria (SRB)—Desulfovibrio desulfuricans IC1 (T. J. Lie, J. R. Leadbetter, and E. R. Leadbetter, Geomicrobiol. J. 15:135–149, 1998) and Desulfomicrobium norvegicum; this polypeptide was not detected when these bacteria were grown on TEA other than isethionate, suggesting involvement in its metabolism. The sulfate analogs molybdate and tungstate, effective in inhibiting sulfate reduction by SRB, were examined for their effects on sulfonate reduction. Molybdate effectively inhibited sulfonate reduction by strain IC1 and selectively inhibited isethionate (but not cysteate) reduction by Desulfitobacterium dehalogenans and Desulfitobacterium sp. strain PCE 1. Desulfitobacterium hafniense, however, grew with both isethionate and cysteate in the presence of molybdate. In contrast, tungstate only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium spp. Similarly, another inhibitor of sulfate reduction, 1,8-dihydroxyanthraquinone, effectively inhibited sulfate reduction by SRB but only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium hafniense.     

The utilization of organosulphonates by soil and freshwater bacteria

Utilization of the biogenic aliphatic organosulphonates taurine, isethionate, sulphoacetaldehyde and sulphoacetate was investigated in 100 soil and freshwater bacteria isolated on modified complete mineral salts medium. More than 90% could use all the compounds as sole sulphur sources, and some 10% used taurine and isethionate as sole carbon and energy, or sole carbon, energy and sulphur sources. None could mineralize sulphoacetaldehyde or sulphoacetate; however, two isolates capable of growth on sulphoacetate as sole carbon, energy and sulphur source were obtained by enrichment culture. The results suggest that in the majority of environmental bacteria the pathways of organosulphonate biodegradation may be independently controlled by the supply of carbon and sulphur to the cell, and that a number of routes may exist for cleavage of the organosulphonate C–S bond.     

Anion secretion by the isolated rabbit pancreas

The isolated rabbit pancreas secretes a fluid containing chloride and bicarbonate in about equal concentrations. Replacement of bicarbonate by acetate, phosphate or isethionate, replacement of Na⁺ by Li⁺ and addition of ouabain to the bathing medium of the pancreas inhibit the secretion of fluid, chloride and bicarbonate in a similar fashion and by maximally 100%. Replacement of chloride by isethionate inhibits fluid secretion by maximally 50%, chloride secretion by 90% and bicarbonate secretion by 20%. It is concluded that fluid secretion is based on a Na⁺-gradient-dependent bicarbonate influx or proton efflux in the ductular cell, and that the secretion of chloride is secondary to that of bicarbonate.     

Effect of amidine derivatives on nitric oxide production by Leishmania amazonensis promastigotes and axenic amastigotes.

The effects of pentamidine isethionate (reference drug) and N,N'-diphenyl-4-methoxy-benzamidine (test compound) on NO. production by Leishmania amazonensis promastigotes and axenic amastigotes were investigated by measuring nitrite, a by-product of nitric oxide released into culture supernatants. The NO. production by infective promastigotes was inhibited by OCH(3)-amidine in about 23.53% and by pentamidine in only 3.78%. In axenic amastigotes, the inhibition of NO. production by OCH(3)-amidine was significantly higher (52.94%; p=0.01) than that by pentamidine, which inhibited this radical production nonsignificantly (25.29%; p=0.1). The mechanism of amidine derivatives, as an antimicrobial agent, is unknown. However, other amidines, such as a diamidine (pentamidine), contain chemical structures shared by the guanidino group of the nitric oxide synthase substrate L-arginine, suggesting the possibility of an interaction with this enzyme or electronic factors (substituent constant) that alter physical and chemical properties significant for biological activity.