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Identification of H2S3 and H2S produced by 3-mercaptopyruvate sulfurtransferase in the brain.

Kimura Y, Toyofuku Y, Koike S, Shibuya N, Nagahara N, Lefer D, Ogasawara Y, Kimura H - Sci Rep (2015)

Bottom Line: We recently found H2Sn in the brain.Purified recombinant 3MST and lysates of COS cells expressing 3MST produced H2S3 from 3 MP, while those expressing defective 3MST mutants did not.The present study provides new insights into the physiology of H2S3 and H2S, as well as novel therapeutic targets for diseases in which these molecules are involved.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan.

ABSTRACT
Hydrogen polysulfides (H2Sn) have a higher number of sulfane sulfur atoms than hydrogen sulfide (H2S), which has various physiological roles. We recently found H2Sn in the brain. H2Sn induced some responses previously attributed to H2S but with much greater potency than H2S. However, the number of sulfur atoms in H2Sn and its producing enzyme were unknown. Here, we detected H2S3 and H2S, which were produced from 3-mercaptopyruvate (3 MP) by 3-mercaptopyruvate sulfurtransferase (3MST), in the brain. High performance liquid chromatography with fluorescence detection (LC-FL) and tandem mass spectrometry (LC-MS/MS) analyses showed that H2S3 and H2S were produced from 3 MP in the brain cells of wild-type mice but not 3MST knockout (3MST-KO) mice. Purified recombinant 3MST and lysates of COS cells expressing 3MST produced H2S3 from 3 MP, while those expressing defective 3MST mutants did not. H2S3 was localized in the cytosol of cells. H2S3 was also produced from H2S by 3MST and rhodanese. H2S2 was identified as a minor H2Sn, and 3 MP did not affect the H2S5 level. The present study provides new insights into the physiology of H2S3 and H2S, as well as novel therapeutic targets for diseases in which these molecules are involved.

No MeSH data available.


Related in: MedlinePlus

H2S3 and H2S are produced by 3MST from 3 MP.(a,b) The production of H2S3 (a) and H2S (b) from 3 MP with lysates of COS cells expressing 3MST (●), rhodanese (○), or empty vector (○) as a source of the enzymes incubated for 15 min. (c,d) Determination of the 3MST genotype by polymerase chain reaction (PCR) (c). Western blot analysis (d) of 3MST in the brains of wild-type (+/+) and 3MST-KO (−/−) mice. (+/−): heterozygote. GAPDH was used as a control. (e,f) Concentrations of H2S3 (e) and H2S (f) in whole cells prepared from wild-type (open bar) and 3MST-KO-mice (filled bar). Suspensions of brain cells were exposed to 500 μM 3 MP (distilled water for a control) for 15 min (the intracellular 3 MP concentration reached 0.40 ± 0.09 μmol/g protein), and the concentrations of H2S3 (e) and H2S (f) were measured as monobromobimane adducts from cell lysates. ** and ##p < 0.01. **: the comparison with a value at 1 mM for (a,b). All data represent the mean ± standard error of the mean (SEM) of at least three experiments.
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f1: H2S3 and H2S are produced by 3MST from 3 MP.(a,b) The production of H2S3 (a) and H2S (b) from 3 MP with lysates of COS cells expressing 3MST (●), rhodanese (○), or empty vector (○) as a source of the enzymes incubated for 15 min. (c,d) Determination of the 3MST genotype by polymerase chain reaction (PCR) (c). Western blot analysis (d) of 3MST in the brains of wild-type (+/+) and 3MST-KO (−/−) mice. (+/−): heterozygote. GAPDH was used as a control. (e,f) Concentrations of H2S3 (e) and H2S (f) in whole cells prepared from wild-type (open bar) and 3MST-KO-mice (filled bar). Suspensions of brain cells were exposed to 500 μM 3 MP (distilled water for a control) for 15 min (the intracellular 3 MP concentration reached 0.40 ± 0.09 μmol/g protein), and the concentrations of H2S3 (e) and H2S (f) were measured as monobromobimane adducts from cell lysates. ** and ##p < 0.01. **: the comparison with a value at 1 mM for (a,b). All data represent the mean ± standard error of the mean (SEM) of at least three experiments.

Mentions: Because 3 MP, a substrate of 3MST, produces polysulfides, which bridge cysteine residues in the proteins1019, and cells expressing 3MST contain greater amounts of the form of polysulfides than do controls619, we hypothesized that 3MST may also produce diffusible polysulfides (H2Sn). To address this problem, we examined H2Sn production using lysates of COS cells expressing 3MST as a source of the enzyme. There are various methods to measure H2S including colorimetric methylene blue method, ion-selective or polarographic electrodes, gas chromatography, and monobromobimane assay20. The variations of endogenous levels of H2S in tissue and blood samples have been reported and are largely attributed to the contaminant of H2S released from acid-labile sulfur or bound sulfane sulfur mostly caused during the preparation of the samples rather than the methods21. The monobromobimane analysis with high performance liquid chromatography with fluorescence detection (LC-FL) was previously applied to the quantitative analysis of H2Sn13, and also to the analysis of H2S using both LC-FL and mass spectrometry22. In the present study LC-FL and LC-tandem mass spectrometry (LC-MS/MS) was used to analyze monobromobimane adducts of H2Sn and H2S. H2S3 and H2S were produced from 3 MP by 3MST in a concentration-dependent manner (Fig. 1a,b and Supplementary Fig. 1). Rhodanese, which is homologous to 3MST and may produce the bound form of polysulfides10, generated neither H2S3 nor H2S from 3 MP (Fig. 1a,b). Lysates of cells transfected with an empty vector did not produce either molecule (Fig. 1a,b). These observations suggest that 3MST produces H2S3 (as a major H2Sn) and H2S from 3 MP.


Identification of H2S3 and H2S produced by 3-mercaptopyruvate sulfurtransferase in the brain.

Kimura Y, Toyofuku Y, Koike S, Shibuya N, Nagahara N, Lefer D, Ogasawara Y, Kimura H - Sci Rep (2015)

H2S3 and H2S are produced by 3MST from 3 MP.(a,b) The production of H2S3 (a) and H2S (b) from 3 MP with lysates of COS cells expressing 3MST (●), rhodanese (○), or empty vector (○) as a source of the enzymes incubated for 15 min. (c,d) Determination of the 3MST genotype by polymerase chain reaction (PCR) (c). Western blot analysis (d) of 3MST in the brains of wild-type (+/+) and 3MST-KO (−/−) mice. (+/−): heterozygote. GAPDH was used as a control. (e,f) Concentrations of H2S3 (e) and H2S (f) in whole cells prepared from wild-type (open bar) and 3MST-KO-mice (filled bar). Suspensions of brain cells were exposed to 500 μM 3 MP (distilled water for a control) for 15 min (the intracellular 3 MP concentration reached 0.40 ± 0.09 μmol/g protein), and the concentrations of H2S3 (e) and H2S (f) were measured as monobromobimane adducts from cell lysates. ** and ##p < 0.01. **: the comparison with a value at 1 mM for (a,b). All data represent the mean ± standard error of the mean (SEM) of at least three experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4594004&req=5

f1: H2S3 and H2S are produced by 3MST from 3 MP.(a,b) The production of H2S3 (a) and H2S (b) from 3 MP with lysates of COS cells expressing 3MST (●), rhodanese (○), or empty vector (○) as a source of the enzymes incubated for 15 min. (c,d) Determination of the 3MST genotype by polymerase chain reaction (PCR) (c). Western blot analysis (d) of 3MST in the brains of wild-type (+/+) and 3MST-KO (−/−) mice. (+/−): heterozygote. GAPDH was used as a control. (e,f) Concentrations of H2S3 (e) and H2S (f) in whole cells prepared from wild-type (open bar) and 3MST-KO-mice (filled bar). Suspensions of brain cells were exposed to 500 μM 3 MP (distilled water for a control) for 15 min (the intracellular 3 MP concentration reached 0.40 ± 0.09 μmol/g protein), and the concentrations of H2S3 (e) and H2S (f) were measured as monobromobimane adducts from cell lysates. ** and ##p < 0.01. **: the comparison with a value at 1 mM for (a,b). All data represent the mean ± standard error of the mean (SEM) of at least three experiments.
Mentions: Because 3 MP, a substrate of 3MST, produces polysulfides, which bridge cysteine residues in the proteins1019, and cells expressing 3MST contain greater amounts of the form of polysulfides than do controls619, we hypothesized that 3MST may also produce diffusible polysulfides (H2Sn). To address this problem, we examined H2Sn production using lysates of COS cells expressing 3MST as a source of the enzyme. There are various methods to measure H2S including colorimetric methylene blue method, ion-selective or polarographic electrodes, gas chromatography, and monobromobimane assay20. The variations of endogenous levels of H2S in tissue and blood samples have been reported and are largely attributed to the contaminant of H2S released from acid-labile sulfur or bound sulfane sulfur mostly caused during the preparation of the samples rather than the methods21. The monobromobimane analysis with high performance liquid chromatography with fluorescence detection (LC-FL) was previously applied to the quantitative analysis of H2Sn13, and also to the analysis of H2S using both LC-FL and mass spectrometry22. In the present study LC-FL and LC-tandem mass spectrometry (LC-MS/MS) was used to analyze monobromobimane adducts of H2Sn and H2S. H2S3 and H2S were produced from 3 MP by 3MST in a concentration-dependent manner (Fig. 1a,b and Supplementary Fig. 1). Rhodanese, which is homologous to 3MST and may produce the bound form of polysulfides10, generated neither H2S3 nor H2S from 3 MP (Fig. 1a,b). Lysates of cells transfected with an empty vector did not produce either molecule (Fig. 1a,b). These observations suggest that 3MST produces H2S3 (as a major H2Sn) and H2S from 3 MP.

Bottom Line: We recently found H2Sn in the brain.Purified recombinant 3MST and lysates of COS cells expressing 3MST produced H2S3 from 3 MP, while those expressing defective 3MST mutants did not.The present study provides new insights into the physiology of H2S3 and H2S, as well as novel therapeutic targets for diseases in which these molecules are involved.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan.

ABSTRACT
Hydrogen polysulfides (H2Sn) have a higher number of sulfane sulfur atoms than hydrogen sulfide (H2S), which has various physiological roles. We recently found H2Sn in the brain. H2Sn induced some responses previously attributed to H2S but with much greater potency than H2S. However, the number of sulfur atoms in H2Sn and its producing enzyme were unknown. Here, we detected H2S3 and H2S, which were produced from 3-mercaptopyruvate (3 MP) by 3-mercaptopyruvate sulfurtransferase (3MST), in the brain. High performance liquid chromatography with fluorescence detection (LC-FL) and tandem mass spectrometry (LC-MS/MS) analyses showed that H2S3 and H2S were produced from 3 MP in the brain cells of wild-type mice but not 3MST knockout (3MST-KO) mice. Purified recombinant 3MST and lysates of COS cells expressing 3MST produced H2S3 from 3 MP, while those expressing defective 3MST mutants did not. H2S3 was localized in the cytosol of cells. H2S3 was also produced from H2S by 3MST and rhodanese. H2S2 was identified as a minor H2Sn, and 3 MP did not affect the H2S5 level. The present study provides new insights into the physiology of H2S3 and H2S, as well as novel therapeutic targets for diseases in which these molecules are involved.

No MeSH data available.


Related in: MedlinePlus