Effects of Oxidative and Nitrosative Stress on Tetrahymena pyriformis Glyceraldehyde-3-Phosphate Dehydrogenase

• Published in: The Journal of eukaryotic microbiology 2007-07, Vol.54 (4), p.338-346
• Main Authors: FOURRAT, LATIFA, IDDAR, ABDELGHANI, VALVERDE, FEDERICO, SERRANO, AURELIO, SOUKRI, ABDELAZIZ
• Format: Article
• Language: English
• Subjects: Animals; Biochemistry. Physiology. Immunology. Molecular biology; Biological and medical sciences; Central carbon metabolism; Fundamental and applied biological sciences. Psychology; Glyceraldehyde-3-Phosphate Dehydrogenases - drug effects; Glyceraldehyde-3-Phosphate Dehydrogenases - metabolism; glycolysis; Hydrogen Peroxide - pharmacology; Nitric Oxide - pharmacology; Nitroprusside - pharmacology; oxidative reagents; Oxidative Stress; protein modification; Protozoa; regulatory network; Tetrahymena pyriformis; Tetrahymena pyriformis - drug effects; Tetrahymena pyriformis - enzymology; Tetrahymena pyriformis - genetics
• ISSN: 1066-5234; 1550-7408
• DOI: 10.1111/j.1550-7408.2007.00275.x

Previous reports showed that hydrogen peroxide and the NO-generating reagent sodium nitroprusside (SNP)-modulated enzymatic activity of animal glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12). These modifications are suggested to have a physiological regulatory role. To gain further insight into this regulatory process the model ciliated protozoan Tetrahymena pyriformis was chosen. Both reagents inhibited growth of T. pyriformis cultures and produced a specific increase of GAPDH protein but only NO seemed to reduce GAPDH activity in cell-free extracts. Both specific activity and pI were found to be altered in the in vivo NO-treated purified enzyme, but no effect was detected by the in vivo H₂O₂ treatment. Analytical chromatofocusing showed a single basic isoform (pI 8.8) in enzyme preparations from control and H₂O₂-treated cells. In contrast to this, three more acidic isoforms (pIs, 8.6, 8.0 and 7.3) were resolved in purified fractions from SNP-treated cells, suggesting post-translational modification of the enzyme by NO. Nevertheless, a decrease of GAPDH activity by H₂O₂ and NO, mainly due to a decrease in its Vmax without apparent change in substrate affinity, was observed in vitro in the whole enzyme population. The increase of GAPDH protein level found in vivo suggests a cell response in order to compensate for the inhibitory effect on activity observed in the purified enzyme. This is the first report of NO- and H₂O₂-dependent effects on GAPDH of T. pyriformis, and identifies this key protein of central carbon metabolism as a physiological target of oxidative and nitrosative stress in this ciliated protozoan.