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

Abstract

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 H(2)O(2) treatment. Analytical chromatofocusing showed a single basic isoform (pI 8.8) in enzyme preparations from control and H(2)O(2)-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 H(2)O(2) and NO, mainly due to a decrease in its V(max) 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 H(2)O(2)-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.

Purification and partial characterization of glyceraldehyde-3-phosphate dehydrogenase from the ciliate <italic>Tetrahymena thermophila</italic>

“…5(B)]. These observations are in accordance with those reported by Fourrat et al [20] for the same enzyme from T. pyriformis, and suggest that GAPDH could be used to characterize the physiological effect of oxidative and nitrosative stress. Study in U937 cells (human promonocytes derived from a histiocytic lymphoma) has found that GAPDH is inactivated by the hydrogen peroxide, as a part of a cell defense pathway against oxidative stress induced apoptosis [6].…”

“…Effect of H 2 O 2 and SNP on the purified T. thermophila GAPDH In order to investigate the physiological effect of oxidative and nitrosative stress on T. thermophila, we selected GAPDH as H 2 O 2 and NO-sensitive enzyme, based on previous studies in animal cells, Tetrahymena pyriformis [20] and Saccharomyces cerevisiae [21]. The purified T. thermophila GAPDH was incubated at various concentrations of two agents of stress; H 2 O 2 and SNP a NO-donating compound.…”

Protective and antioxidant potential of the argan oil on induced oxidative stress in Tetrahymena pyriformis

“…In this study, the experimental perturbations in redox status were caused by the H 2 O 2 , it belongs to the group of reactive oxygen species (ROS). The action of this oxidant agent on T. pyriformis showed an increase in MDA level; this result is also observed by Fourat et al (2007) in T. pyriformis after oxidative and nitrosative stress. This increase of lipid peroxidation might be explained by the attack of radicals, such as ROS and reactive nitrogen species (RNS) to polyunsaturated fatty acid residues of phospholipids generating many aldehydes including the MDA (Nair et al, 2007).…”

Nuclear Accumulation of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Cadmium-Stressed Arabidopsis Roots

“…sulfonation, nitrosylation, or glutathionylation; Bedhomme et al, 2012) were not the cause of the apparent inactivation. Interestingly, Fourrat et al (2007) showed that treatments of the protozoan Tetrahymena pyriformis with either sodium nitroprusside (NO donor) or H 2 O 2 led to a dramatic increase of GAPDH protein level without affecting total enzymatic activity.…”