Fig. 3.

Sulfhydration of GAPDH occurs at Cys¹⁵⁰. (A) HPLC followed by LC-MS/MS on endogenous full-length GAPDH protein immunoprecipitated from mouse liver shows sulfhydration of Cys¹⁵⁰ with an additional mass of ~32.058 daltons. m/z is the mass-to-charge ratio; the amino acid sequence surrounding Cys¹⁵⁰ is shown at the bottom. (B) LC-MS/MS on purified full-length human GAPDH protein treated with 100 μM NaHS for 30 min at 37°C shows a mass shift consistent with Cys¹⁵⁰ (152 in human) sulfhydration. Sulfhydration or sulfination could not be detected after treatment with 100 μM NaHS and 1 mM DTT or 500 μM H₂O₂, respectively. (C) Modified biotin switch assay in HEK293 cells treated with 100 μM NaHS for 30 min at 37°C. C150S mutant is not sulfhydrated. (D) Sulfhydration of GAPDH with radiolabeled [³⁵S]cysteine and CSE at 37°C. GAPDH radiolabeling is reversed by the addition of 1 mM DTT or boiling of the CSE protein for 5 min. Data were quantified by Cherenkov scintillation counting. All results are the mean ± SEM. **P < 0.01. (E) Radiolabeling wild-type and C150S GAPDH with [³⁵S]cysteine and CSE. C150S mutant is not labeled. All results are the mean ± SEM. ** P < 0.01.