The transport of sulphate and sulphite in rat liver mitochondria

1. The mechanism of sulphite and sulphate permeation into rat liver mitochondria was investigated. 2. Extramitochondrial sulphite and sulphate elicit efflux of intramitochondrial phosphate, malate, succinate and malonate. The sulphate-dependent effluxes and the sulphite-dependent efflux of dicarboxylate anions are inhibited by butylmalonate, phenylsuccinate and mersalyl. Inhibition of the phosphate efflux produced by sulphite is caused by mersalyl alone and by N-ethylmaleimide and butylmalonate when present together. 3. External sulphite and sulphate cause efflux of intramitochondrial sulphate, and this is inhibited by butylmalonate, phenylsuccinate and mersalyl. 4. External sulphite and sulphate do not cause efflux of oxoglutarate or citrate. 5. Mitochondria swell when suspended in an iso-osmotic solution of ammonium sulphite; this is not inhibited by N-ethylmaleimide or mersalyl. 6. Low concentrations of sulphite, but not sulphate, produce mitochondrial swelling in iso-osmotic solutions of ammonium malate, succinate, malonate, sulphate, or phosphate in the presence of N-ethylmaleimide. 7. It is concluded that both sulphite and sulphate may be transported by the dicarboxylate carrier of rat liver mitochondria and also that sulphite may permeate by an additional mechanism; the latter may involve the permeation of sulphurous acid or SO(2) or an exchange of the sulphite anion for hydroxyl ion(s).     

Microbial metabolism of aryl sulphonates a re-assessment of colorimetric methods for the determination of sulphite and their use in measuring desulphonation of aryl and alkylbenzene sulphonates.

The reaction of 2,4-dinitroanilinomaleimide with sulphite which has been claimed as the basis of a suitable colorimetric assay for the anion was carefully re-examined. The sulphite-imide addition product provides a suitable and specific qualitative test for sulphite after separation by paper chromatography but the method as previously used is probably measuring the hydrolysis of the imide to 2,4-dinitroanilinomaleamic acid and cannot be used for sulphite determination either colorimetrically or in kinetic assays. A new colorimetric method for the determination of sulphite based on its reaction with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) is described and compared for sensitivity with the p-rosaniline-HCHO method. Both methods were used to show the formation of sulphite as the initial product of arylsulphonate metabolism by bacteria. The failure to find sulphite in similar cultures of a third organism was attributed to the very high activities of sulphite oxidase found in extracts. The Ellman reagent was examined as the basis of an indicator medium for the detection of sulphite-excreting colonies.     

Microbial metabolism of aryl sulphonates A re-assessment of colorimetric methods for the determination of sulphite and their use in measuring desulphonation of aryl and alkylbenzene sulphonates

The reaction of 2,4-dinitroanilinomaleimide with sulphite which has been claimed as the basis of a suitable colorimetric assay for the anion was carefully re-examined. The sulphite-imide addition product provides a suitable and specific qualitative test for sulphite after separation by paper chromatography but the method as previously used is probably measuring the hydrolysis of the imide to 2,4-dinitroanilinomaleamic acid and cannot be used for sulphite determination either colorimetrically or in kinetic assays. A new colorimetric method for the determination of sulphite based on its reaction with Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoic acid) is described and compared for sensitivity with thep-rosaniline-HCHO method. Both methods were used to show the formation of sulphite as the initial product of arylsulphonate metabolism by bacteria. The failure to find sulphite in similar cultures of a third organism was attributed to the very high activities of sulphite oxidase found in extracts. The Ellman reagent was examined as the basis of an indicator medium for the detection of sulphite-excreting colonies.     

Sulphite reductase and ATP sulfurylase in low- and high-sulphite forming wine yeasts

Sulphite reductase and ATP sulfurylase activities were compared in low- and high-sulphite forming wine yeasts grown in a synthetic medium. Reduced nicotinamide adenine dinucleotide phosphate-linked sulphite reductase activity was not detected in extracts from high-sulphite forming yeasts, although high activity was found in extracts from low-sulphite formers. High-sulphite forming yeasts had elevated ATP sulfurylase activity compared to the low-sulphite formers indicating derepression of enzyme synthesis. A high rate of activation and reduction of sulphate to sulphite was considered the main factor responsible for the accumulation of sulphite by high-sulphite forming wine yeasts.Over a 5-day fermentation period, sulphite accumulation in the growth medium by low-sulphite forming yeasts was correlated with ATP sulfurylase activity.Fellow of the National Research Advisory Council, Wellington, New Zealand     

Genotype-dependent sulphite tolerance of Australian Dekkera (Brettanomyces) bruxellensis wine isolates

Aims: The aim of this study was to determine sulphite tolerance for a large number of Dekkera bruxellensis isolates and evaluate the relationship between this phenotype and previously assigned genotype markers. Methods and Results: A published microplate‐based method for evaluation of yeast growth in the presence of sulphite was benchmarked against culturability following sulphite treatment, for the D. bruxellensis type strain (CBS 74) and a reference wine isolate (AWRI 1499). This method was used to estimate maximal sulphite tolerance for 41 D. bruxellensis isolates, which was found to vary over a fivefold range. Significant differences in sulphite tolerance were observed when isolates were grouped according to previously assigned genotypes and ribotypes. Conclusions: Variable sulphite tolerance for the wine spoilage yeast D. bruxellensis can be linked to genotype markers. Significance and Impact of the Study: Strategies to minimize risk of wine spoilage by D. bruxellensis must take into account at least a threefold range in effective sulphite concentration that is dependent upon the genotype group(s) present. The isolates characterized in this study will be a useful resource for establishing the mechanisms conferring sulphite tolerance for this industrially important yeast species.     

Toxic Effects of Sulphite in Combination with Peroxynitrite on Neuronal Cells

Abstract: Sulphite is widely used as a preservative and antioxidant in foods, beverages, and pharmaceuticals. Endogenous sulphite is generated during the normal metabolism of sulphur-containing amino acids, and alterations in sulphur amino acid metabolism occur in some neurodegenerative diseases. In particular, sulphite oxidase deficiency produces severe mental retardation, seizures, spastic quadriparesis, dislocated lenses, and early death. Exposure of a neuronal cell line (rat mesencephalic cells) to high levels of sulphite induced a time-dependent decrease in viability. Peroxynitrite was also toxic to this cell line, and sulphite affected the toxicity of ONOO⁻. Sulphite concentrations of ≤0.5 m M markedly potentiated cell damage induced by 200 µ M ONOO⁻. We propose that sulphite can act as a neurotoxic agent, especially in combination with peroxynitrite. Sulphite radicals may be involved in the neurotoxic effect.     

<Emphasis Type="Italic">k</Emphasis><Subscript>L</Subscript><Emphasis Type="Italic">a</Emphasis> determination: comparative study for a gas mass balance method

The determination of k(L) a by a gas balance method coupled with sulphite oxidation is compared for three kinds of processes (stirred tank, bubble column and fixed-bed column reactors) with a gassing-in and with a classical chemical sulphite oxidation method. The mathematical relations required for the determination of the k(L) a value are detailed. In coalescing gas-liquid conditions, the values calculated by the three methods are shown to be comparable. The gas balance method is more rapid than either the steady-state gassing-in or the chemical sulphite reaction rate measurement methods. It is also well adapted for three-phase systems (gas-liquid-solid) in which the non-coalescing effects of sulphite solution are reduced by solid interferences.     

The Wolinella succinogenes mcc gene cluster encodes an unconventional respiratory sulphite reduction system

Assimilatory and dissimilatory sulphite reductions are key reactions in the biogeochemical sulphur cycle and several distinct sirohaem-containing sulphite reductases have been characterized. Here, we describe that the Epsilonproteobacterium Wolinella succinogenes is able to grow by sulphite respiration (yielding sulphide) with formate as electron donor. Sulphite is reduced by MccA, a prototypical member of an emerging new class of periplasmic cytochrome c sulphite reductases that, phylogenetically, belongs to a multihaem cytochrome c superfamily whose members play crucial roles in the global sulphur and nitrogen cycles. Within this family, MccA represents an unconventional octahaem cytochrome c containing a special haem c group that is bound via two cysteine residues arranged in a unique CX(15)CH haem c binding motif. The phenotypes of numerous W.succinogenes mutants producing MccA variants underlined the structural importance of this motif. Several open reading frames of the mcc gene cluster were individually inactivated and characterization of the corresponding mutants indicated that the predicted iron-sulphur protein MccC, the putative quinol dehydrogenase MccD (a member of the NrfD/PsrC family) as well as a peptidyl-prolyl cis-trans isomerase, MccB, are essential for sulphite respiration. MccA synthesis in W. succinogenes was found to be induced by sulphite (but not by thiosulphate or sulphide) and repressed in the presence of fumarate or nitrate. Based on the results, a sophisticated model of respiratory sulphite reduction by the Mcc system is presented.     

Sulphite oxidation by mitochondria from green and etiolated peas

Sulphite oxidation by mitochondria prepared from green pea epicotyls had a higher K_m than did the sulphite oxidation of mitochondria prepared from etiolated pea epicotyls. Mitochondrial sulphite oxidation from green and etiolated tissues was inhibited by cyanide but not by azide, rotenone, antimycin A or oligomycin. Mitochondria from green and etiolated tissues were able to oxidize glyoxal-bisulphite, but not as effectively as sulphite.     

Evidence from electron-paramagnetic-resonance spectroscopy for a complex of sulphite ions with the molybdenum centre of sulphite oxidase.

Reduction of sulphite oxidase by sulphite at low pH values in Mes (4-morpholine-ethanesulphonic acid) buffer gives rise to a new molybdenum(V) electron-paramagnetic-resonance spectrum different from that obtained by photoreduction of the enzyme in the same medium. The spectrum is attributed to a sulphite complex of the enzyme, showing g-values of about 2.000, 1.972 and 1.963. The complex is analogous to that with the inhibitor phosphate in that it gives rise to no observable hyperfine coupling of Mo(V) to exchangeable protons.     

Transport of H+ in mitochondria induced by the uptake of sulfite, sulfate and thiosulfate

The addition of sulphite to rat-liver mitochondria (RLM) causes an uptake of H+ that is unaffected by NEM and butylmalonate. The uptake of H+ induced by sulphate or thiosulphate is abolished by NEM and butylmalonate in freshly isolated RLM, whereas it is inhibited only by butylmalonate in sulphite-pretreated mitochondria. The data suggest that sulphite is cotransported with H+, whereas the movement of H+ associated to the uptake of sulphate or thiosulphate by RLM is mediated by either phosphate or sulphite.     

Oxidation of thiosulphate and sulphite by Thiobacillus neapolitanus

Different bacterial cell fractions of Thiobacillus neapolitanus were examined in order to localize the active sites for thiosulphate and sulphite oxidation. Difference spectra of the fractions were made to determine the level at which electrons from sulphite and thiosulphate enter the respiratory chain. Active sites for thiosulphate are probably strictly connected with cell membranes. Thiosulphate and sulphite reduced cytochome b and c in bacterial cell extracts. It has been found that thiosulphate oxidation is accompanied by production of tetrathionate and trithionate.     

Chemical Modification of Rhodanese with Sulphite

The essential sulphydryl group of bovine liver rhodanese (thiosulphate: cyanide sulphurtrasferase, E.C. 2.8.1.1.) is modified by sulphite produced during the enzymatic reaction or added to the fully active enzyme. The enzyme treated with labelled reagent incorporates 1 equivalent of SO²₃^- and loses one -SH group with the formation of a S-sulphonate group at the active site. Mercaptoethanol is effective in both restoring enzyme activity and removing bound sulphite from protein. The inactivation process is dependent on the presence of oxygen and is antagonized by chelation of metal ions, that catalyze sulphite autoxidation, or by scavenging free radicals with mannitol or benzoate. Since the presence of superoxide dismutase and/or catalase protects the enzyme only to a small extent, the inactivation process should be attributed to sulphite radicals rather than intermediates of oxygen reduction.     

The cryptic nature of hepatic microsomal sulphite oxidase

1. The activity of rat liver microsomal sulphite oxidase (EC 1.8.3.1) was increased several-fold on aging of microsomes, on delipidation by extraction with acetone or on solubilization with deoxycholate, suggesting its existence in a cryptic state. 2. In rat liver most of the sulphite oxidase was present in the nuclear fraction and only a small portion in the microsomes. 3. Microsomal sulphite oxidase activity was low in the developing embryo and increased rapidly after birth.     

A medium for detecting sulphite-binding yeasts in meat products

The loss of free sulphite in meat products, and thereby preservative potential, is associated with an increase in bound sulphite resulting in part from acetaldehyde production by yeasts. A medium containing Schiff's reagent was devised to detect sulphite-binding yeasts. Yeast colonies that caused red colouration were recorded as acetaldehyde producers. Initially acetaldehyde-producing strains (as enumerated on the detection medium) represented 60% of the yeast flora of preserved minced lamb but, as the free sulphite level diminished, non-sulphite-binding yeasts increased in number.     

A generalization of kinetic data on sulphite oxidation systems

Kinetic data of the reaction between sulphite ions and dissolved oxygen depend on the purity of sulphite used. Thus it is adequate to determine them for each batch and concentration of sulphite used. Besides the dependence of reaction rate constants on catalyst concentration, it is usually also required to know their dependence on pH and on temperature. Both quantities can change to a great extent especially in absorption apparatus in which high sulphite conversion occurs. They profoundly influence the value of the reaction rate constant. To get a complete picture of the influence of these three quantities without repeating extensive kinetic measurements for every kind and concentration of sulphite used, a method for obtaining information on the influence of all three quoted quantities in the range of values of interest for application is presented.     

The role of microorganisms in transformations of sulphite in spruce forest soil

Transformations of sulphite and the participation of microorganisms were investigated in samples of fermentative (A₀₂) and humus (A₀₃) horizons from a spruce forest. About 80% of sulphite were almost immediately oxidized abiotically to sulphate, less than 1% persisted in the soil for several days and almost 20% were converted to a form not demonstrable as sulphite or sulphate. Microorganisms accelerated the disappearance of less than 10% of the added sulphite.     

Comparative study of paper sheets from olive tree wood pulp obtained by soda,sulphite or kraft pulping

Paper sheets from olive tree wood pulp obtained by soda, sulphite or kraft pulping were studied to examine the influence of pulp beating on properties of the paper sheets.Paper sheets from kraft and sulphite pulps exhibited the highest resistance, and sulphite pulp the highest brightness. Soda pulp required more intensive beating than did kraft or sulphite pulps; in fact, the PFI beater had be operated at a 40–50% higher number of beating revolutions to obtain soda pulp with 70–80° SR.The breaking length, stretch, burst index and tear index of paper sheets obtained from kraft pulp, beaten to a Shopper–Riegler index of 70–80° SR were 20–30%, 30–50%, 50–60% and 15–35% higher, respectively, than those of sheets obtained from soda pulp.     

The effect of sulphite on chlorophyll fluorescence and sucrose metabolism in poplar leaves

Sulphite at concentrations from 0.5 to 5.0 mM was supplied to illuminated, detached poplar (Populus deltoides Bartr. ex Marsh) leaves via the transpiration stream. Chlorophyll a fluorescence parameters, the contents of fructose-2,6-bisphosphate (Fru2,6BP) and starch, and extractable specific activity of sucrose-phosphate synthase (SPS), sucrose synthase (SuSy), acid invertase (AI), neutral invertase (NI), ATP-dependent fructose-6-phosphate 1-phosphotransferase (PFK) and pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase (PFP) were measured. Chlorophyll fluorescence parameters appeared to be unaffected by sulphite. Application of ≥ 1.0 mM sulphite led to an increase in the content of Fru2,6BP and starch. There was also a decline in the activity of SPS, NI and PFK. On the other hand, the influence of sulphite on the activity of AI and PFP was negligible. Specific activity of SuSy was inhibited by 1.0 and 2.5 mM but activated by 5.0 mM of sulphite. On the basis of the results obtained in the present study, we postulate that sulphite at concentrations ≥ 1.0 mM inhibits primarily sucrose synthesis, favours starch accumulation and has an indirect effect on the sucrolytic activities in poplar leaves.     

Determination of sulphite in mineral soils

Summary Two methods were evaluated to determine the concentration of sulphite in sandy loam soils. Use of p-rosaniline-formaldehyde reagent and sodium tetrachloromercurate as extractant proved effective only if a wide soil to extractant ratio was employed. Use of Ellman's reagent in conjunction with this extractant was however, unsatisfactory. Soils exposed to heavy atmospheric pollution differed in sulphite concentration only marginally compared with relatively unpolluted soils. The effect of soil water content on the stability of sulphite in soil was also determined.