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The reactive nitrogen species peroxynitrite is a potent inhibitor of Na,K ‐ ATPase activity
Author(s) -
Reifenberger Matthew Stanton,
Milanick Mark
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1157.2
Subject(s) - peroxynitrite , chemistry , reactive nitrogen species , peroxynitrous acid , nitric oxide , nitrotyrosine , biochemistry , tyrosine , superoxide , glutathione , reactive oxygen species , atpase , biophysics , nitric oxide synthase , enzyme , organic chemistry , biology
Peroxynitrite (ONOO − ) is a potent oxidant formed by nitric oxide (NO) reacting with superoxide (O 2 − ). Peroxynitrite modifies tyrosine, cysteine and other residues and affects protein function which might contribute to pathophysiological responses. Peroxynitrite reacts with tyrosine to form 3‐nitrotyrosine which is elevated in a number of diseases. We investigated the effects of peroxynitrite on the Na‐pump and show that a single bolus addition of peroxynitrite inhibited Na, K – ATPase activity with an IC 50 of 107 ± 9 uM. To mimic cellular/physiological production of peroxynitrite (chronic, low levels) a syringe pump was used to slowly release (≈0.1 uM / second) peroxynitrite. This treatment was equally effective at inhibiting Na,K ATPase activity as a single bolus addition of equal cumulative concentration. Peroxynitrite led to Na pump nitrotyrosine formation and preliminary data showed modification to cysteines as detected by 5‐(iodoacetamido)fluorescein. Glutathione (GSH) and dithioreitol (DTT) were unable to reverse inhibition. The flavonoid epicatechin which prevented tyrosine nitration enhanced ATPase inhibition. The presence of Na and Mg ATP during peroxynitrite treatment increased the IC 50 to 145 ± 10 uM while the presence of K and Mg ATP increased the IC 50 to 255 ± 13 uM. This result suggests that the E‐PNa conformation of the pump is more sensitive to peroxynitrite than the E (K) conformation.