Analysis of Plant L-Cysteine Desulfhydrase (LCD) Isozymes by Non-denaturing Polyacrylamide Gel Electrophoresis

H2S-Generating Cytosolic L-Cysteine Desulfhydrase and Mitochondrial D-Cysteine Desulfhydrase from Sweet Pepper (Capsicum annuum L.) Are Regulated During Fruit Ripening and by Nitric Oxide

“…Then, the gels were incubated in the following staining solution prepared in 100 m M Tris-HCl, pH 8.0 containing 20 m M L-Cys or D-Cys, 0.4 m M lead acetate, 50 μ M PLP hydrate, and 5 m M dithiothreitol. The generated H 2 S reacts with lead acetate to produce lead sulfide (PbS) forming dark brown precipitates corresponding to the enzymatic activity (Muñoz-Vargas et al, 2023 ). To corroborate the specificity of the isozymes, several internal controls were performed, one in the absence of the L-Cys or D-Cys substrate and another in the absence of the cofactor PLP/Vitamin B6 (Mooney and Hellmann, 2010 ).…”

Isoenzymatic Pattern of Hydrogen Sulfide (H2S)-Generating L-Cysteine Desulfhydrase (LCD) in Arabidopsis thaliana Seedlings: Effect of Nitric Oxide (NO) and H2S

“…Arabidopsis samples were separated non-denaturing polyacrylamide gel electrophoresis (PAGE) on 8% acrylamide gels. After the electrophoresis, to visualize the LCD (EC 4.4.1.28) activity bands, the gels were incubated in the dark in a staining buffer containing Tris-HCl 100 mM, pH 7.5, L-cysteine 20 mM, lead acetate 0.4 mM, pyridoxal 50-phosphate hydrate 50 µM and dithiothreitol 5 mM, until the appearance of brown bands over a colorless background [10,11]. Two internal controls were accomplished to check the specificity of the LCD activity; one was to incubate the gel in the absence of the substrate L-cysteine or the cofactor pyridoxal 5 -phosphate.…”

“…Among the twenty-six identified enzymes involved in the metabolism of H 2 S, five enzymes directly generate H 2 S in plants including the cytosolic L-cysteine desulfhydrase (LCD), bifunctional cystathionine γlyase/cysteine synthase (DES1), the chloroplastic sulfite reductase (SiR), the mitochondrial bifunctional D-cysteine desulfhydrase/1-aminocyclopropane-1-carboxylate deaminase (DCDES1), and D-cysteine desulfhydrase 2 (DCDES2) [5][6][7]. The cytosolic LCD is considered one of the primary sources of H 2 S, which catalyzes the following reaction: L-cysteine + H 2 O → pyruvate + NH 4 + + H 2 S + H + , using pyridoxal 5-phosphate (PLP) as a necessary cofactor [8][9][10][11].…”

“…Based on previous work, where an optimized method in polyacrylamide gels under non-denaturing conditions allowed the identification of different LCD isoenzymes in several species of the genus Allium and pepper fruit [8,11], the present study aims to analyze the isozymatic pattern of LCD in Arabidopsis seedlings and evaluate if any of these LCD isozymes could be modulated via NO and/or H 2 S.…”