Cysteine, γ-glutamyl-cysteine and glutathione contents of spinach leaves as affected by darkness and application of excess sulfur. II. Glutathione accumulation in detached leaves exposed to H2S in the absence of light is stimulated by the supply of glycine to the petiole

When illuminated leaf discs and detached leaves of spinach ( Spinacia oleracea L. cv. Estivato) were exposed to 0.4 and 0.25 μl 1^-1 H₂S, respectively, pool sizes of cysteine and glutathione increased. In the dark, apart from these compounds, the level of γ-glutamyl-cysteine also increased. Incubation of leaf discs with 1.0 m M buthionine sulfoximine (BSO) resulted in the accumulation of cysteine only, both in the light and in darkness. When glycine was supplied to the petioles of detached leaves exposed to H₂S in the dark, the accumulation of glutathione was stimulated, while γ-glutamyl-cysteine accumulation was prevented completely. Glycolate and glyoxylate, precursors of glycine in the glycolate pathway, had nearly the same effect as glycine. Although other amino acids were apparently taken up equally well as glycine when supplied to the petiole, they were much less effective, or not effective at all, in restoring glutathione synthesis in the dark. These results provide evidence, that H₂S-induced glutathione accumulation in spinach leaves in the dark is limited by the availability of glycine, giving rise to the accumulation of the metabolic precursor γ-glutamyl-cysteine.     

The effect of short-term H2S fumigation on water-soluble sulphydryl and glutathione levels in spinach

Abstract. Short-term fumigation of Spinacia oleracea with 380 μg m⁻³ H₂S (250 ppb) resulted in a rapid accumulation of water-soluble SH-compounds in the shoots. After 1 h exposure a substantial increase in the SH-content was already detectable and maximal accumulation, three- to four-fold that in control plants, was observed after 24 h of exposure. Irradiation during H₂S exposure only slightly affected the rate and level of SH-accumulation. H₂S fumigation did not affect the water-soluble SH-content of the roots. Glutathione was the sole water-soluble SH-compound accumulating upon exposure to H₂S. It was calculated that during the first hour of exposure to 380 μg m⁻³ H₂S 39% of the possible absorbed H₂S was converted into glutathione. The SH-content of the water-soluble proteins of the shoots was not affected by H₂S exposure. When fumigation was stopped, a rapid decrease in glutathione content was observed and after 48 h the content was comparable to that of the control plants. Contrary to H₂S, SO₂ fumigation did not result in a rapid accumulation of glutathione in spinach shoots. The possible role of glutathione accumulation during H₂S fumigation is discussed.     

Hydrogen sulfide and the vasculature: a novel vasculoprotective entity and regulator of nitric oxide bioavailability?

Hydrogen sulfide (H₂S) is a well known and pungent toxic gas that has recently been shown to be synthesised in man from the amino acids cystathionine, homocysteine and cysteine by at least two distinct enzymes; cystathionine-γ-lyase and cystathionine-β-synthase. In the past few years, H₂S has emerged as a novel and increasingly important mediator in the cardiovascular system but delineating the precise physiology and pathophysiology of H₂S is proving to be complex and difficult to unravel with disparate findings reported with cell types, tissue types and animal species reported. Therefore, in this review we summarize the mechanisms by which H₂S has been proposed to regulate blood pressure and cardiac function, discuss the mechanistic discrepancies reported in the literature as well as the therapeutic potential of H₂S. We also examine the methods of H₂S detection in biological fluids, processes for H₂S removal and discuss the reported blood levels of H₂S in man and animal models of cardiovascular pathology. We also highlight the complex interaction of H₂S with nitric oxide in regulating cardiovascular function in health and disease.     

Plant responses to H2S and SO2 fumigation. II. Differences in metabolism of H2S and SO2 in spinach

Fumigation of spinach (Spinacia oleracea L. cvs Estivato and Monosa) with H₂S or SO, for 1 to 6 days resulted in accumulation of sulfhydryl (SH) compounds in the shoots of both H₂S- and SO₂-exposed plants. The sulfate concentration in shoots of SO₂-exposed plants increased linearly with time. SH accumulation showed saturation kinetics as a function of time as well as H₂S concentration, ascribed to the internal H₂S concentration in the plant and the availability of substrates for glutathione synthesis, respectively. SH compounds accumulated more at lower exposure temperatures, whereas sulfate accumulation was more pronounced at higher temperatures. These results are discussed in relation to the possible foliar uptake of H₂S and SO₂, the temperature dependence of uptake and the water solubility of these gases. The possibility of SO₂-induced H₂S emission rather than sulfate accumulation as a source for SH accumulation is also discussed. Cessation of fumigation resulted in a decrease in SH compounds and sulfate content that could be accounted for by sulfur metabolism and growth, respectively.     

Light activates H2 15O flow in rice: Detailed monitoring using a positron-emitting tracer imaging system (PETIS)

Water (H₂ ¹⁵O) translocation from the roots to the top of rice plants ( Oryza saliva L. cv. Nipponbare) was visualized over time by a positron-emitting tracer imaging system (PETIS). H₂ ¹⁵O flow was activated 8 min after plants were exposed to bright light (1 500 μmol m⁻² s⁻¹). When the light was subsequently removed, the flow gradually slowed and completely stopped after 12 min. In plants exposed to low light (500 μmol m⁻² s⁻¹), H₂ ¹⁵O flow was activated more slowly, and a higher translocation rate of H₂ ¹⁵O was observed in the same low light at the end of the next dark period. NaCl (80 m M ) and methylmercury (1 m M ) directly suppressed absorption of H₂ ¹⁵O by the roots, while methionine sulfoximine (1 m M ), abscisic acid (10 μ M ) and carbonyl cyanide m -chlorophenylhydrazone (10 m M ) were transported to the leaves and enhanced stomatal closure, reducing H₂ ¹⁵O translocation.     

Production of the gaseous signal molecule hydrogen sulfide in mouse tissues

The gaseous molecule hydrogen sulfide (H₂S) has been proposed as an endogenous signal molecule and neuromodulator in mammals. Using a newly developed method, we report here for the first time the ability of intact and living brain and colonic tissue in the mouse to generate and release H₂S. This production occurs through the activity of two enzymes, cystathionine-γ-lyase and cystathionine-β-synthase. The quantitative expression of messenger RNA and protein localization for both enzymes are described in the liver, brain, and colon. Expression levels of the enzymes vary between tissues and are differentially distributed. The observation that, tissues that respond to exogenously applied H₂S can endogenously generate the gas, strongly supports its role as an endogenous signal molecule.     

Competition for sulfide among colorless and purple sulfur bacteria in cyanobacterial mats

Abstract The vertical zonation of light, O₂, H₂S, pH, and sulfur bacteria was studied in two benthic cyanobacterial mats from hypersaline ponds at Guerrero Negro, baja California, Mexico. The physical-chemical gradients were analyzed in the upper few mm at ≥ 100 μm spatial resolution by microelectrodes and by a fiber optic microprobe. In mats, where oxygen produced by photosynthesis diffused far below the depth of the photic zone, colorless sulfur bacteria ( Beggiatoa sp.) were the dominant sulfide oxidizing organisms. In a mat, where the O₂–H₂S interface was close to the photic zone, but yet received no significant visible light, purple sulfur bacteria ( Chromatium sp.) were the dominant sulfide oxidizers. Analysis of the spectral light distribution heare showed that the penetration of only 1% of the incident near-IR light (800–900 nm) into the sulfide zone was sufficient for the development of Chromatium in a narrow band of 300 μm thickness. The balance betweem O₂ and light penetration down into the sulfide zone thus deterined in mcro-scale which type of sulfur bacteria becamed dominant.     

Content of low-molecular-weight thiols during the imbibition of Pea seeds

The metabolism of low-molecular-weight thiols was investigated in seeds of Pisum sativum L. cv. Kleine Rheinländerin during imbibition in water for 14 h. The amount of oxidized glutathione (GSSG) decreased from 319 nmol (g dry weight)⁻¹ in dry seeds to 38 nmol (g dry weight)⁻¹ within the first 14 h of imbibition. The decrease may have been due to the reduction of GSSG to reduced glutathione (GSH), catalyzed by the enzyme glutathione reductase (GR; EC 1.6.4.2). The enzyme activity was high in dry seeds [25 nkat (g dry weight)⁻¹] and decreased to 20 nkat (g dry weight)⁻¹ within 14 h of imbibition. The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) decreased from 100 nkat (g dry weight)⁻¹ in dry seeds to 67 nkat (g dry weight)⁻¹ after 14 h of imbibition. Within 14 h the amount of γ-glutamyl-cysteine (γ-GC) decreased from 135 to 38 nmol (g dry weight)⁻¹, whereas the cysteine content rose from 81 nmol (g dry weight)⁻¹ in dry seeds to a maximum of 170 nmol (g dry weight)⁻¹ after 12 h of imbibition, which may be due to the degradation of γ-GC into cysteine.     

Chloroplast glutathione reductase: Purification and properties

Glutathione reductase was partially purified from isolated pea chloroplasts ( Pisum sativum L. cv. Progress #9). A 1600-fold purification was obtained and the purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)⁻¹ min⁻¹. The enzyme had a native molecular weight of approximately 156 kdalton and consisted of two each of two subunits of about 41 and 42 kdalton. The K_m for oxidized glutathione was 11 μ M and the K_m for NADPH was 1.7 μ M . Enzyme activity was affected by the ionic strength of the assay medium, and maximum activity was observed at an ionic strength of between 60 and 100 m M . The enzyme was inactivated by sulfhydryl modifying reagents and the presence of either oxidized glutathione or NADPH affected the extent of inactivation. Chloroplast glutathione reductase probably serves in the removal of photosynthetically derived H₂O₂ by reducing dehydroascorbate for ascorbate-linked reduction of H₂O₂. Intermediates of this reaction sequence, dehydroascorbate, ascorbate, reduced glutathione, and NADPH had no effect on enzymic activity.     

Isolation of sulfite reductase variants of a commercial wine yeast with significantly reduced hydrogen sulfide production

The production of hydrogen sulfide (H₂S) during fermentation is a common and significant problem in the global wine industry as it imparts undesirable off-flavors at low concentrations. The yeast Saccharomyces cerevisiae plays a crucial role in the production of volatile sulfur compounds in wine. In this respect, H₂S is a necessary intermediate in the assimilation of sulfur by yeast through the sulfate reduction sequence with the key enzyme being sulfite reductase. In this study, we used a classical mutagenesis method to develop and isolate a series of strains, derived from a commercial diploid wine yeast (PDM), which showed a drastic reduction in H₂S production in both synthetic and grape juice fermentations. Specific mutations in the MET10 and MET5 genes, which encode the catalytic α- and β-subunits of the sulfite reductase enzyme, respectively, were identified in six of the isolated strains. Fermentations with these strains indicated that, in comparison with the parent strain, H₂S production was reduced by 50–99%, depending on the strain. Further analysis of the wines made with the selected strains indicated that basic chemical parameters were similar to the parent strain except for total sulfite production, which was much higher in some of the mutant strains.     

Enzymes of assimilatory sulfate reduction in leaves of Pisum sativum: Activity changes during ontogeny and in vivo regulation by H2S and cyst(e)ine

Enzyme activities of assimilatory sulfate reduction were measured in leaves of Pisum sativum L., cv. Vatters Frühbusch, during their ontogenetic development, and during treatment with H₂S and cyst(e)ine. Ribulose bisphosphate (RuBP) carboxylase (EC 4.1.1.39) and ferredoxin-dependent nitrite reductase (Fd-NiR, EC 1.7.7.1) were measured for comparison. In etiolated pea leaves, ATP-sulfurylase (ATPase, EC 2.7.7.4), adenosine 5'-phosphosulfate sulfotransferase (APSSTase), ferredoxin-dependent sulfite reductase (Fd-SiR, EC 1.8.7.1) and O-acetyl-L-serine sulfhydrylase (OASSase, EC 4.2.99.8) activities were measured in appreciable rates, while neither RuBP carboxylase nor Fd-NiR activities could be detected.
During the first 2–7 days after transfer into the light all enzyme activities increased. After reaching maximal activities, ATPase, APSSTase, and Fd-SiR activities decreased in all leaves to low or indetectable levels during the following 3–6 days. RuBP carboxylase, Fd-NiR and OASSase, on the other hand, decreased slowly and were still at high levels of activity at the end of the experiment.
Fumigation of pea plants with 1.5 μl l⁻¹ H₂S delayed the initial increase and the subsequent decrease of ATPase activity by 1–3 days. APSSTase activity decreased for 1–2 days, increased rapidly during the next 4–6 days and retained a high level of activity until the end of the experiment as did Fd-SiR. One to two days after the beginning of fumigation the leaves started to accumulate high amounts of cyst(e)ine.
When pea plants with excised roots were placed on a nutrient solution containing cyst(e)ine, APSSTase activity decreased more on 0.2 and 0.5 m M than on 1.0 m M. Fd-SiR activity was only slightly decreased on 1.0 m M cyst(e)ine. Neither Fd-NiR nor RuBP carboxylase activities were affected.     

Cysteine-induced H2S emission, ATP depletion, and membrane electrical responses in oat coleoptiles

Incubation of oat ( Avena sativa L. cv. Victory) coleoptile segments in 4 m M L-cysteine reduced the tissue ATP level to 42 nmol ( g fresh weight)⁻¹ (35% of normal) over a 2 h period. Emissions of H₂S accompanied this depletion in ATP suggesting an H₂S production by desulfhydration of cysteine similar to that reported in other plants. Additions of exogenous H₂S to the sections also caused ATP depletion. Aminooxyacetate, an inhibitor of cysteine desulfhydrase (EC 4.4.1.1), eliminated the cysteine-induced H₂S emission and the ATP depletion. Prolonged exposure to cysteine depressed the electrical polarity of the cell membrane from – 116 mV to –85 mV. That and other electrical responses appear to reflect a reduced capacity for ATP-dependent H⁺ extrusion. These effects should be taken into account whenever cysteine is used in physiological experiments.     

Studies on a Capillary-Rich Fraction Isolated from Brain: Histaminic Components and Characterization of the Histamine Receptors Linked to Adenylate Cyclase

Abstract: A fraction enriched in capillaries has been prepared from the guinea pig cerebral cortex. The purity of this fraction was checked by light- and electron-microscopic examination and by its high enrichment in alkaline phosphatase and γ-glutamyl transpeptidase. In the capillary-rich fraction, the endogenous level of histamine was 1.9%'of that measured in the initial hornogenate. The histamine-synthesizing enzyme, I-histidine decarboxylase, and the metabolizing enzyme, histamine-N-methyltransferase, were barely detectable. In addition, histamine elicits a twofold stimulation in the accumulation of cyclic AMP in this capillary fraction with an EC₅₀ of 5 γM. Agonists and antagonists of the two types of histamine receptors (H₁ and H₂) were used for the characterization of the receptors mediating this action: H₂-receptor agonists were able to activate the adenylate cyclase with "relative potencies" similar to that found on typical H₂-receptors, and cimetidine, a specific H₂-receptor antagonist, competitively inhibited the response to histamine with a K₁ value reflecting its interaction with a single population of H₂-receptors. On the contrary, data obtained with H₁-receptor agonists and antagonists reflect their interaction with H₂-receptors rather than H₁-receptors. Thus H₂-receptors are involved in the activation of adenylate cyclase of the capillary fraction.     

Glutathione is an important antioxidant molecule in the yeast Saccharomyces cerevisiae

Abstract The tripeptide γ-l-glutamyl-l-cystinylglycine (glutathione) is one of the major antioxidant molecules of cells and is thought to play a vital role in buffering the cell against reactive oxygen species and toxic electrophiles. We wished to determine the role of glutathione in the protection of the yeast Saccharomyces cerevisiae against oxidative stress. This study shows that glutathione is an important antioxidant molecule in yeast, with γ-glutamylcysteine synthetase ( gshI ) mutants, deficient in glutathione synthesis, being hypersensitive to H₂O₂ and Superoxide anions in both exponential- and stationary-phase cultures. Despite this, these mutants are still able to induce adaptive stress responses to oxidants.     

Implications of water stress-induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings

Vigna cutjang Endl. cv. Pusa Barsati seedlings, subjected to increasing degrees of water stress (−0.5, −1.0, −1,5 MPa), produced an approximately proportional increase in glycolate oxidase activity, hydrogen peroxide (H₂O₂) and proline content but a decrease in catalase activity, ascorbic acid and protein content. Leaf water potential (leaf ψ) and relative water content (RWC) were also lowered with increasing stress. Pretreatment with l -cysteine and reduced glutathione (10-3 M) decreased glycolate oxidase activity, H₂O₂ content, ascorbic acid oxidase activity, proline content and also slightly improved the water status of leaves stressed (−1.0 MPa) for 2 days. Pretreatment of non-stressed seedlings with these antioxidants had little or no effect. These studies indicate that treatment with antioxidants makes the plant tolerant against water stress by modulating the endogenous levels of H₂O₂ and ascorbic acid in stressed tissue.     

OPTIMIZATION OF CULTURE CONDITIONS FOR ENUMERATION OF H2S BACTERIA IN THE FLESH OF SEAFISH

H₂S bacteria of seafish flesh are weakly halophilic and require on average 1.68% NaCl according to statistical studies. Enumeration is optimal on PCA-H₂S(a PCA medium supplemented with sulfur sources and increased NaCl concentrations) incubated at 25C. Total aerobic bacteria can be counted simultaneously on this medium. The proportion of H₂S bacteria relative to total aerobic bacteria increased slightly during prolonged storage of the fish, but was highly variable. Models relating H₂S bacterial counts to spoilage of fish are sigmoidal and showed that when the count exceeds 10,000 CFU/g, whole or filleted fish stored in ice at 0C are unfit for consumption. Shewanella putrefaciens accounted for 69% of the H₂S bacteria at the fifth day of storage and 100% at the fifteenth.     

The role of glycine in determining the rate of glutathione synthesis in poplar. Possible implications for glutathione production during stress

The terminal step of glutathione (GSH) synthesis is the condensation of γ-glutamyl-cysteine (γ-EC) with glycine. Relatively little information exists concerning the importance of photorespiratory glycine in determining the rate of conversion of γ-EC to GSH. Consequently, the effect of exogenous glycine and of illumination on foliar contents of γ-EC and GSH was studied in excised leaves and leaf discs from untransformed poplar ( Populus tremula × P. alba ) and poplar overexpressing γ-glutamylcysteine synthetase (γ-ECS; EC 6.3.2.2). Poplars strongly overexpressing γ-ECS (ggs28) had enhanced levels of γ-EC and GSH compared to untransformed poplars. The relationship between γ-EC and GSH contents in ggs28 was light dependent. In illuminated leaves, GSH contents were up to 50-fold higher than γ-EC. On darkening, γ-EC accumulated markedly and GSH declined, so that the GSH:γ-EC ratio was close to 1. These dark-induced changes were prevented by supplying glycine through the petiole or by incubation of leaf discs on glycine. Dark accumulation of γ-EC in leaf discs from untransformed poplar was also prevented by supplying glycine. Supplying cysteine in the dark to discs from untransformed poplar and ggs28 increased γ-EC levels markedly but GSH levels only slightly. Subsequent illumination caused γ-EC to decrease and GSH to increase. Supplying glycine in concert with cysteine had similar effects to illumination. The data suggest that photorespiratory glycine is essential for GSH synthesis, especially under stress conditions, where increased amounts of GSH are required.     

H2S-induced pancreatic acinar cell apoptosis is mediated via JNK and p38 MAP kinase

Treatment of pancreatic acinar cells by hydrogen sulphide has been shown to induce apoptosis. However, a potential role of mitogen-activated protein kinases (MAPKs) in this apoptotic pathway remains unknown. The present study examined the role of MAPKs in H₂S-induced apoptosis in mouse pancreatic acinar cells. Pancreatic acinar cells were treated with 10 μM NaHS (a donor of H₂S) for 3 hrs. For the evaluation of the role of MAPKs, PD98059, SP600125 and SB203580 were used as MAPKs inhibitors for ERK1/2, JNK1/2 and p38 MAPK, respectively. We observed activation of ERK1/2, JNK1/2 and p38 when pancreatic acini were exposed to H₂S. Moreover, H₂S-induced ERK1/2, JNK1/2 and p38 activation were blocked by pre-treatment with their corresponding inhibitor in a dose-dependent manner. H₂S-induced apoptosis led to an increase in caspase 3 activity and this activity was attenuated when caspase 3 inhibitor were used. Also, the cleavage of caspase 3 correlated with that of poly-(ADP-ribose)-polymerase (PARP) cleavage. H₂S treatment induced the release of cytochrome c , smac from mitochondria into the cytoplasm, translocation of Bax into mitochondria and decreased the protein level of Bcl-2. Inhibition of ERK1/2 using PD98059 caused further enhancement of apoptosis as evidenced by annexin V staining, while SP600125 and SB203580 abrogated H₂S-induced apoptosis. Taken together, the data suggest that activation of ERKs promotes cell survival, whereas activation of JNKs and p38 MAP kinase leads to H₂S-induced apoptosis.