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Peroxynitrite is a reactive nitrogen species produced when nitric oxide and superoxide react. In vivo studies suggest that reactive oxygen species and, perhaps, peroxynitrite can influence Na-K-ATPase function. However, the direct effects of peroxynitrite on Na-K-ATPase function remain unknown. We show that a single bolus addition of peroxynitrite inhibited purified renal Na-K-ATPase activity, with [IC.sub.50] of 107 [+ or -] 9 [micro]M. To mimic cellular/physiological production of peroxynitrite, a syringe pump was used to slowly release (~0.85 [micro]M/s) peroxynitrite. The inhibition of Na-K-ATPase activity induced by this treatment was similar to that induced by a single bolus addition of equal cumulative concentration. Peroxynitrite produced 3-nitrotyrosine residues on the [alpha], [beta], and FXYD subunits of the Na pump. Interestingly, the flavonoid epicatechin, which prevented tyrosine nitration, was unable to blunt peroxynitrite-induced ATPase inhibition, suggesting that tyrosine nitration is not required for inhibition. Peroxynitrite led to a decrease in iodoacetamidofluorescein labeling, implying that cysteine modifications were induced. Glutathione was unable to reverse ATPase inhibition. The presence of [Na.sup.+] and low MgATP during peroxynitrite treatment increased the [IC.sub.50] to 145 [+ or -] 10 [micro]M, while the presence of [K.sup.+] and low MgATP increased the [IC.sub.50] to 255 [+ or -] 13 [micro]M. This result suggests that the EPNa conformation of the pump is slightly more sensitive to peroxynitrite than the E(K)_conformation. Taken together, these results show that peroxynitrite is a potent inhibitor of Na-K-ATPase activity and that peroxynitrite can induce amino acid modifications to the pump. P-type ATPase; reactive oxygen species; nitrotyrosine; nitric oxide

The impacts of three classes of organic cations on the inhibition of the plasma membrane Ca pump were determined and compared to inhibition of the Na pump. It was found that the monovalent cation quarternary amines are unable to form hydrogen bonds, and that the positive charge is localized at the center of the compound sterically hindered from direct contact with amino acid side chains.

Current models of the [Na.sup.+]-[K.sup.+]-ATPase reaction cycle have ATP binding with low affinity to the [K.sup.+]-occluded form and accelerating [K.sup.+] deocclusion, presumably by opening the inside gate. Implicit in this situation is that ATP binds after closing the extracellular gate and thus predicts that ATP binding and extracellular cation binding to be mutually exclusive. We tested this hypothesis. Accordingly, we needed a cation that binds outside and not inside, and we determined that tetrapropylammonium (TPA) behaves as such. TPA competed with [K.sup.+] (and not [Na.sup.+]) for ATPase, TPA was unable to prevent phosphoenzyme (EP) formation even at low [Na.sup.+], and TPA decreased the rate of EP hydrolysis in a [K.sup.+]-competitive manner. Having established that TPA binding is a measurement of extracellular access, we next determined that TPA and inorganic phosphate ([P.sub.i] were not mutually exclusive inhibitors of para-nitrophenylphosphatase (pNPPase) activity, implying that when [P.sub.i] is bound, the transport site has extracellular access. Surprisingly, we found that ATP and TPA also were not mutually exclusive inhibitors of pNPPase activity, implying that when the cation transport site has extracellular access, ATP can still bind. This is consistent with a model in which ATP speeds up the conformational changes that lead to intracellular or extracellular access, but that ATP binding is not, by itself, the trigger that causes opening of the cation site to the cytoplasm. quaternary ammonium; Dixon plot; P-type adenosine triphosphatase; inorganic phosphate

Chloro(2,2':6',2'-terpyridine) platinum, a bulky, hydrophilic reagent, inhibited the renal sodium pump with a single exponential time course. [K.sup.+] increased the rate constant of the reaction by about twofold; the [K.sup.+] concentration dependence was monotonic, with a half-maximal effect observed at 1 mM, consistent with [K.sup.+] acting at a transport site. [Na.sup.+], [Mg.sup.2+], eosin, and vanadate did not significantly alter the rate of reaction. The results of proteolysis and mass spectrometer analysis were consistent with terpyridine platinum labeling of Cys452, Cys456, or Cys457. Because phenylarsine oxide reacts with vicinal cysteines and did not prevent terpyridine platinum modification, terpyridine platinum most likely modifies Cys452. This modification prevents ADP binding; interestingly, the analogous residue in sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA) is on the exterior of the nucleotide-binding pocket. Thus it appears that the terpyridine platinum residue is more accessible in the presence of [K.sup.+] than in its absence and that terpyridine platinum modification prevents nucleotide binding. sodium pump; chemical modification; active transport

The effects of 0.3-10 nM extracellular protons (pH 9.5-8.0) on ouabain-sensitive rubidium influx were determined in 4,4'-diisocyanostilbene-2, 2'-disulfonate (DIDS)-treated human and rat erythrocytes. This treatment clamps the intracellular H. We found that rubidium binds much better to the protonated pump than the unprotonated pump; 13-fold better in rat and 34-fold better in human erythrocytes. This clearly shows that protons are not competing with rubidium in this proton concentration range. Bretylium and tetrapropylammonium also bind much better to the protonated pump than the unprotonated pump in human erythrocytes and in this sense they are potassium-like ions. In contrast, guanidinium and sodium bind about equally well to protonated and unprotonated pump in human red cells. In rat red cells, protons actually make sodium bind less well (about sevenfold). Thus, protons have substantially different effects on the binding of rubidium and sodium. The effect of protons on ouabain binding in rat red cells was intermediate between the effects of protons on rubidium binding and on sodium binding. Remarkably, all four cationic inhibitors (bretylium, guanidinium, sodium, and tetrapropylammonium) had similar apparent inhibitory constants for the unprotonated pump (~5-10 mM). The [K.sub.d] for proton binding to the human pump, with the empty transport site facing extracellularly is 13 nM, whereas the extracellular transport site loaded with sodium is 9.5 nM, and with rubidium is 0.38 nM. In rat red cells there is also a substantial difference in the [K.sub.d] for proton binding to the sodium-loaded pump (14.5 nM) and the rubidium-loaded pump (0.158 nM). These data suggest that important rearrangements occur at the extracellular pump surface as the pump moves between conformations in which the outward facing transport site has sodium bound, is empty, or has rubidium bound and that guanidinium is sodium-like and bretylium and tetrapropylammonium are rubidium-like. KEY WORDS: pH clamp * tetrapropylammonium * ion transport * Na(+)-K(+)-exchanging ATPase * red blood cells

Current models of the Na+-K+-ATPase reaction cycle have ATP binding with low affinity to the K+-occluded form and accelerating K+ deocclusion, presumably by opening the inside gate. Implicit in this situation is that ATP binds after closing the extracellular gate and thus predicts that ATP binding and extracellular cation binding to be mutually exclusive. We tested this hypothesis. Accordingly, we needed a cation that binds outside and not inside, and we determined that tetrapropylammonium (TPA) behaves as such. TPA competed with K+ (and not Nat) for ATPase, TPA was unable to prevent phosphoenzyme (EP) formation even at low Nat, and TPA decreased the rate of EP hydrolysis in a K+-competitive manner. Having established that TPA binding is a measurement of extracellular access, we next determined that TPA and inorganic phosphate (Pi) were not mutually exclusive inhibitors of para-nitrophenylphosphatase (pNPPase) activity, implying that when Pi is bound, the transport site has extracellular access. Surprisingly, we found that ATP and TPA also were not mutually exclusive inhibitors of pNPPase activity, implying that when the cation transport site has extracellular access, ATP can still bind. This is consistent with a model in which ATP speeds up the conformational changes that lead to intracellular or extracellular access, but that ATP binding is not, by itself, the trigger that causes opening of the cation site to the cytoplasm. [ABSTRACT FROM AUTHOR]

Chloro(2,2':6',2'-terpyridine) platinum, a bulky, hydrophilic reagent, inhibited the renal sodium pump with a single exponential time course. K[sup +] increased the rate constant of the reaction by about twofold; the K[sup +] concentration dependence was monotonic, with a half-maximal effect observed at I mM, consistent with K[sup +] acting at a transport site. Na[sup +], Mg[sup 2+], eosin, and vanadate did not significantly alter the rate of reaction. The results of proteolysis and mass spectrometer analysis were consistent with terpyridine platinum labeling of Cys452, Cys456, or Cys457. Because phenylarsine oxide reacts with vicinal cysteines and did not prevent terpyridine platinum modification, terpyridine platinum most likely modifies Cys452. This modification prevents ADP binding; interestingly, the analogous residue in sarco(endo)plasmic reticulum Ca[sup 2+]-ATPase (SERCA) is on the exterior of the nucleotide-binding pocket. Thus it appears that the terpyridine platinum residue is more accessible in the presence of K[sup +] than in its absence and that terpyridine platinum modification prevents nucleotide binding. [ABSTRACT FROM AUTHOR]