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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

Production of H2S3 from H2S by 3MST and rhodanese.(a) Production of H2S3 from H2S by 3MST or rhodanese. Lysates of COS cells expressing 3MST (●), rhodanese (○), or empty vector (○) as a source of the enzymes were incubated with Na2S for 15 min. (b) The production of H2S3 from H2S by 3MST mutants. The production of H2S3 from 100 μM Na2S in lysates of COS cells expressing various defective 3MST mutants was compared with that of wild-type 3MST. Control: lysates of cells transfected with an empty vector. (c) Production of H2S3 from H2S in whole cells. Suspensions of brain cells prepared from wild-type and 3MST-KO mice were exposed to 500 μM Na2S for 15 min (the intracellular H2S concentration reached 0.11 ± 0.08 μmol/g protein), and H2S3 levels in the cells were analyzed. Distilled water was applied as a control. (d) The production of H2S3 from H2S by rhodanese mutants. The production of H2S3 from 100 μM Na2S by lysates of COS cells expressing various defective rhodanese mutants as a source of the enzymes was compared with that of wild-type rhodanese. Control: lysates of cells transfected with an empty vector. The amounts of H2Sn species produced by the oxidation of Na2S in the absence of cells or cell lysates were subtracted. * and #p < 0.05, ** and ##p < 0.01. * and **: the comparison with a value at 1 μM Na2S for (a), and with that of an empty vector for (b,d). All data represent the mean ± SEM of at least three experiments.
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f5: Production of H2S3 from H2S by 3MST and rhodanese.(a) Production of H2S3 from H2S by 3MST or rhodanese. Lysates of COS cells expressing 3MST (●), rhodanese (○), or empty vector (○) as a source of the enzymes were incubated with Na2S for 15 min. (b) The production of H2S3 from H2S by 3MST mutants. The production of H2S3 from 100 μM Na2S in lysates of COS cells expressing various defective 3MST mutants was compared with that of wild-type 3MST. Control: lysates of cells transfected with an empty vector. (c) Production of H2S3 from H2S in whole cells. Suspensions of brain cells prepared from wild-type and 3MST-KO mice were exposed to 500 μM Na2S for 15 min (the intracellular H2S concentration reached 0.11 ± 0.08 μmol/g protein), and H2S3 levels in the cells were analyzed. Distilled water was applied as a control. (d) The production of H2S3 from H2S by rhodanese mutants. The production of H2S3 from 100 μM Na2S by lysates of COS cells expressing various defective rhodanese mutants as a source of the enzymes was compared with that of wild-type rhodanese. Control: lysates of cells transfected with an empty vector. The amounts of H2Sn species produced by the oxidation of Na2S in the absence of cells or cell lysates were subtracted. * and #p < 0.05, ** and ##p < 0.01. * and **: the comparison with a value at 1 μM Na2S for (a), and with that of an empty vector for (b,d). All data represent the mean ± SEM of at least three experiments.

Mentions: Because H2Sn can also be generated from H2S26, it is possible that 3MST is involved in its generation. We examined this problem using lysates of COS cells expressing 3MST. Although H2S was oxidized to generate H2S3, even in the absence of 3MST, 3MST significantly accelerated the production of H2S3 from Na2S, a sodium salt of H2S; control lysates did not generate H2S3 (Fig. 5a).


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)

Production of H2S3 from H2S by 3MST and rhodanese.(a) Production of H2S3 from H2S by 3MST or rhodanese. Lysates of COS cells expressing 3MST (●), rhodanese (○), or empty vector (○) as a source of the enzymes were incubated with Na2S for 15 min. (b) The production of H2S3 from H2S by 3MST mutants. The production of H2S3 from 100 μM Na2S in lysates of COS cells expressing various defective 3MST mutants was compared with that of wild-type 3MST. Control: lysates of cells transfected with an empty vector. (c) Production of H2S3 from H2S in whole cells. Suspensions of brain cells prepared from wild-type and 3MST-KO mice were exposed to 500 μM Na2S for 15 min (the intracellular H2S concentration reached 0.11 ± 0.08 μmol/g protein), and H2S3 levels in the cells were analyzed. Distilled water was applied as a control. (d) The production of H2S3 from H2S by rhodanese mutants. The production of H2S3 from 100 μM Na2S by lysates of COS cells expressing various defective rhodanese mutants as a source of the enzymes was compared with that of wild-type rhodanese. Control: lysates of cells transfected with an empty vector. The amounts of H2Sn species produced by the oxidation of Na2S in the absence of cells or cell lysates were subtracted. * and #p < 0.05, ** and ##p < 0.01. * and **: the comparison with a value at 1 μM Na2S for (a), and with that of an empty vector for (b,d). All data represent the mean ± SEM of at least three experiments.
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Related In: Results  -  Collection

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f5: Production of H2S3 from H2S by 3MST and rhodanese.(a) Production of H2S3 from H2S by 3MST or rhodanese. Lysates of COS cells expressing 3MST (●), rhodanese (○), or empty vector (○) as a source of the enzymes were incubated with Na2S for 15 min. (b) The production of H2S3 from H2S by 3MST mutants. The production of H2S3 from 100 μM Na2S in lysates of COS cells expressing various defective 3MST mutants was compared with that of wild-type 3MST. Control: lysates of cells transfected with an empty vector. (c) Production of H2S3 from H2S in whole cells. Suspensions of brain cells prepared from wild-type and 3MST-KO mice were exposed to 500 μM Na2S for 15 min (the intracellular H2S concentration reached 0.11 ± 0.08 μmol/g protein), and H2S3 levels in the cells were analyzed. Distilled water was applied as a control. (d) The production of H2S3 from H2S by rhodanese mutants. The production of H2S3 from 100 μM Na2S by lysates of COS cells expressing various defective rhodanese mutants as a source of the enzymes was compared with that of wild-type rhodanese. Control: lysates of cells transfected with an empty vector. The amounts of H2Sn species produced by the oxidation of Na2S in the absence of cells or cell lysates were subtracted. * and #p < 0.05, ** and ##p < 0.01. * and **: the comparison with a value at 1 μM Na2S for (a), and with that of an empty vector for (b,d). All data represent the mean ± SEM of at least three experiments.
Mentions: Because H2Sn can also be generated from H2S26, it is possible that 3MST is involved in its generation. We examined this problem using lysates of COS cells expressing 3MST. Although H2S was oxidized to generate H2S3, even in the absence of 3MST, 3MST significantly accelerated the production of H2S3 from Na2S, a sodium salt of H2S; control lysates did not generate H2S3 (Fig. 5a).

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