Effects of oxidative and nitrosative stress on Tetrahymena pyriformis glyceraldehyde-3-phosphate dehydrogenase.
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.
Laboratoire de Physiologie et Génétique moléculaire, Département de Biologie, Faculté des Sciences Aïn-Chock, Université Hassan-II, Km 8 route d'El Jadida, B.P. 5366 Mâarif, Casablanca, Morocco.
Latifa
L
Fourrat
Abdelghani
A
Iddar
Federico
F
Valverde
Aurelio
A
Serrano
Abdelaziz
A
Soukri
D000818:Animals; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000187:drug effects / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology*; D009599:Nitroprusside / Q000494:pharmacology; D018384:Oxidative Stress; D013769:Tetrahymena pyriformis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics
D009599:Nitroprusside; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases