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. 2011 Jul 15;15(2):363-72.
doi: 10.1089/ars.2010.3781. Epub 2011 May 5.

The quantitative significance of the transsulfuration enzymes for H2S production in murine tissues

Omer Kabil  1 , Victor Vitvitsky, Peter Xie, Ruma Banerjee
Affiliations

Affiliation

  • 1 Biological Chemistry Department, University of Michigan Medical Center, Ann Arbor, Michigan, 48109-0600, USA.

The quantitative significance of the transsulfuration enzymes for H2S production in murine tissues

Omer Kabil et al. Antioxid Redox Signal. .
. 2011 Jul 15;15(2):363-72.
doi: 10.1089/ars.2010.3781. Epub 2011 May 5.

Authors

Omer Kabil  1 , Victor Vitvitsky, Peter Xie, Ruma Banerjee

Affiliation

  • 1 Biological Chemistry Department, University of Michigan Medical Center, Ann Arbor, Michigan, 48109-0600, USA.

Abstract

The enzymes of the transsulfuration pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), are important for the endogenous production of hydrogen sulfide (H(2)S), a gaseous signaling molecule. The relative contributions of CBS and CSE to H(2)S generation in different tissues are not known. In this study, we report quantification of CBS and CSE in murine liver and kidney and their contribution to H(2)S generation in these tissues and in brain at saturating substrate concentrations. We show that CBS protein levels are significantly lower than those of CSE; 60-fold and 20-fold in liver and kidney, respectively. Each enzyme is more abundant in liver compared with kidney, twofold and sixfold for CBS and CSE, respectively. At high substrate concentrations (20 mM each cysteine and homocysteine), the capacity for liver H(2)S production is approximately equal for CBS and CSE, whereas in kidney and brain, CBS constitutes the major source of H(2)S, accounting for ∼80% and ∼95%, respectively, of the total output. At physiologically relevant concentrations of substrate, and adjusting for the differences in CBS versus CSE levels, we estimate that CBS accounts for only 3% of H(2)S production by the transsulfuration pathway enzymes in liver.

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Figures

FIG. 1.

FIG. 1.

Schematic of H 2 S-generation…

FIG. 1.

Schematic of H 2 S-generation (A) and H 2 S-generating reactions catalyzed by…
FIG. 1.
Schematic of H2S-generation (A) and H2S-generating reactions catalyzed by CBS and CSE (B) in the transsulfuration pathway (black arrows). GSH, glutathione; CAT, cysteine aminotransferase; MST, 3-mercaptopyruvate sulfurtransferase.
FIG. 2.

FIG. 2.

Quantitative Western blot analysis of…

FIG. 2.

Quantitative Western blot analysis of transsulfuration pathway enzymes in liver. Representative Western blot…
FIG. 2.
Quantitative Western blot analysis of transsulfuration pathway enzymes in liver. Representative Western blot in which the indicated amounts of liver extract were separated in parallel with known amounts of CBS (A) or CSE (B). Quantitation of CBS and CSE protein levels in liver (C).
FIG. 3.

FIG. 3.

Quantitative Western blot analysis of…

FIG. 3.

Quantitative Western blot analysis of the transsulfuration pathway enzymes in kidney. Representative Western…
FIG. 3.
Quantitative Western blot analysis of the transsulfuration pathway enzymes in kidney. Representative Western blot in which the indicated amounts of kidney extract were separated in parallel with known amounts of CBS (A) or CSE (B). Quantitation of CBS and CSE protein levels in kidney (C).
FIG. 4.

FIG. 4.

Western blot analysis of CBS…

FIG. 4.

Western blot analysis of CBS (A) and CSE (B) in brain. Representative Western…
FIG. 4.
Western blot analysis of CBS (A) and CSE (B) in brain. Representative Western blot for CBS contains, from left to right, 0.1, 0.25, 0.5, and 1 ng of purified CBS per lane and 10, 20, and 40 μg of total brain protein per lane. Representative Western blot for CSE contains, from left to right, 2.5, 5, and 10 ng of purified CSE per lane and 25, 50, and 100 μg of total brain protein per lane.
FIG. 5.

FIG. 5.

Gas chromatogram showing separation of…

FIG. 5.

Gas chromatogram showing separation of sulfur compounds (A) and standard curve for H …
FIG. 5.
Gas chromatogram showing separation of sulfur compounds (A) and standard curve for H2S generated by injecting different amounts of H2S into the GC, as described under Methods (B). (A) The following standards were injected in a total volume of 200 μl: 1-H2S (2.7 ppm), 2-COS (5 ppm), 3-CH3SH (5 ppm), 4-CH3CH2SH (5 ppm), and 5-CH3SCH3 (5 ppm), respectively, in nitrogen. The standards were obtained from Cryogenic Gases (Detroit, MI).
FIG. 6.

FIG. 6.

H 2 S generation by…

FIG. 6.

H 2 S generation by murine tissues in the presence of different substrates. …
FIG. 6.
H2S generation by murine tissues in the presence of different substrates. H2S production rates were measured in liver (A, B), kidney (C), and brain (D) extract±3 mM propargylglycine, as described under Methods in the presence of either 20 mM cysteine+20 mM homocysteine, 20 mM cysteine, or 20 mM homocysteine. When the effect of AdoMet (500 μM) was examined, all reaction mixtures contained 3 mM propargylglycine to inhibit CSE. The control reaction mixture lacked tissue. The data represent the mean±SD of three independent experiments.
FIG. 7.

FIG. 7.

Simulated contributions of fully activated…

FIG. 7.

Simulated contributions of fully activated CBS and CSE to liver H 2 S…
FIG. 7.
Simulated contributions of fully activated CBS and CSE to liver H2S production at physiologically relevant substrate concentrations and varying grades of hyperhomocysteinemia. The contributions of the transsulfuration enzymes were estimated based on previously reported simulations (33) and by taking into account the 1:60 ratio for CBS to CSE in mouse liver, as determined in this study (Fig. 1). The substrate concentrations used for simulation were 560 μM serine, 100 μM cysteine, and varying homocysteine [10 μM (normal), 40 μM (mild hyperhomocysteinemia), and 200 μM (severe hyperhomocysteinemia)].
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References

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