Activity-based metagenomic screening and biochemical characterization of bovine ruminal protozoan glycoside hydrolases.

The rumen, the foregut of herbivorous ruminant animals such as cattle, functions as a bioreactor to process complex plant material. Among the numerous and diverse microbes involved in ruminal digestion are the ruminal protozoans, which are single-celled, ciliated eukaryotic organisms. An activity-based screen was executed to identify genes encoding fibrolytic enzymes present in the metatranscriptome of a bovine ruminal protozoan-enriched cDNA expression library. Of the four novel genes identified, two were characterized in biochemical assays. Our results provide evidence for the effective use of functional metagenomics to retrieve novel enzymes from microbial populations that cannot be maintained in axenic cultures.

Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa AlgX.

AlgX is a periplasmic protein required for the production of the exopolysaccharide alginate in Pseudomonas sp. and Azotobacter vinelandii. AlgX has been overexpressed and purified and diffraction-quality crystals have been grown using iterative seeding and the hanging-drop vapor-diffusion method. The crystals grew as flat plates with unit-cell parameters a = 46.4, b = 120.6, c = 86.9 A, beta = 95.7 degrees . The crystals exhibited the symmetry of space group P2(1) and diffracted to a minimum d-spacing of 2.1 A. On the basis of the Matthews coefficient (V(M) = 2.25 A(3) Da(-1)), two molecules were estimated to be present in the asymmetric unit.

The reactive nitrogen species peroxynitrite is a potent inhibitor of renal Na-K-ATPase activity.

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 IC50 of 107+/-9 microM. To mimic cellular/physiological production of peroxynitrite, a syringe pump was used to slowly release (approximately 0.85 microM/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+ and low MgATP during peroxynitrite treatment increased the IC50 to 145+/-10 microM, while the presence of K+ and low MgATP increased the IC50 to 255+/-13 microM. 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.

Divalent cation interactions with Na,K-ATPase cytoplasmic cation sites: implications for the para-nitrophenyl phosphatase reaction mechanism.

The interactions of divalent cations with the adenosine triphosphatase (ATPase) and para-nitrophenyl phosphatase (pNPPase) activity of the purified dog kidney Na pump and the fluorescence of fluorescein isothiocyanate (FITC)-labeled pump were determined. Sr(2+) and Ba(2+) did not compete with K(+) for ATPase (an extracellular K(+) effect). Sr(2+) and Ba(2+) did compete with Na(+) for ATPase (an intracellular Na(+) effect) and with K(+) for pNPPase (an intracellular K(+) effect). These results suggest that Ba(2+) or Sr(2+) can bind to the intracellular transport site, yet neither Ba(2+) nor Sr(2+) was able to activate pNPPase activity; we confirmed that Ca(2+) and Mn(2+) did activate. As another measure of cation binding, we observed that Ca(2+) and Mn(2+), but not Ba(2+), decreased the fluorescence of the FITC-labeled pump; we confirmed that K(+) substantially decreased the fluorescence. Interestingly, Ba(2+) did shift the K(+) dose-response curve. Ethane diamine inhibited Mn(2+) stimulation of pNPPase (as well as K(+) and Mg(2+) stimulation) but did not shift the 50% inhibitory concentration (IC(50)) for the Mn(2+)-induced fluorescence change of FITC, though it did shift the IC(50) for the K(+)-induced change. These results suggest that the Mn(2+)-induced fluorescence change is not due to Mn(2+) binding at the transport site. The drawbacks of models in which Mn(2+) stimulates pNPPase by binding solely to the catalytic site vs. those in which Mn(2+) stimulates by binding to both the catalytic and transport sites are presented. Our results provide new insights into the pNPPase kinetic mechanism as well as how divalent cations interact with the Na pump.

Extracellular terbium and divalent cation effects on the red blood cell Na pump and chrysoidine effects on the renal Na pump.

We examined the effect of extracellular terbium (Tb(3+)) and divalent metal cations (Ca(2+), Sr(2+), and Ba(2+)) on (86)Rb(+) influx into rabbit and human red blood cells. We found that Tb(3+) at 15 and 25 microM was a non-competitive inhibitor of (86)Rb(+) influx suggesting that Tb(3+) is not binding to the transport site. This result reduces the usefulness of Tb(3+) as a potential probe for the E(out) conformation (the conformation with the transport site facing extracellularly). Ba(2+), Sr(2+) and Ca(2+), at concentrations >50 mM, had minimal effects on Rb(+) influx into red blood cells (1 mM Rb-out). This suggests that the outside transport site is very specific for monovalent cations over divalent cations, in contrast to the inside transport site. We also found that chrysoidine (4-phenylazo-m-phenylenediamine) competes with Na(+) for ATPase activity and K(+) for pNPPase activity suggesting it is binding to the E(in) conformation. Chrysoidine and similar compounds may be useful as optical probes of the E(in) conformation.

Similarities and differences between organic cation inhibition of the Na,K-ATPase and PMCA.

The effects of three classes of organic cations on the inhibition of the plasma membrane Ca pump (PMCA) were determined and compared to inhibition of the Na pump. Quaternary amines (tetramethylammonium, tetraethylammonium, and tetrapropylammonium, TMA, TEA, and TPA, respectively) did not inhibit PMCA. This is not to imply that PMCA is inherently selective against monovalent cations because guanidine and tetramethylguanidine inhibited PMCA by competing with Ca(2+). The divalent organic cation, ethyl diamine, inhibited PMCA but was not competitive with Ca(2+). In contrast, propyl diamine did compete with Ca(2+) and was about 10-fold more potent than butyl diamine in inhibiting PMCA. For the Na pump, both TEA and TPA inhibited, but TMA did not. TEA, guanidine, and tetramethylguanidine inhibition was competitive with Na(+) for ATPase activation and with K(+) for pNPPase activation, both of which are cytoplasmic substrate cation effects. Thus, these findings are consistent with TEA, guanidine, and tetramethylguanidine inhibiting from the cytoplasmic side of the Na pump; in contrast, we have previously shown that TPA did not inhibit from the cytoplasmic side. The divalent alkane diamines ethyl, propyl, and butyl diamine all inhibited the Na pump and all competed at the intracellular surface. The order of potency was ED > PD > BD consistent with an optimal size for binding; similarly, for the quaternary amines TMA is apparently too small to make appropriate contacts, and TPA is too large. Homology models based upon the high-resolution SERCA structure are included to contextualize the kinetic observations.

Kinetic characterization of tetrapropylammonium inhibition reveals how ATP and Pi alter access to the Na+-K+-ATPase transport site.

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 Na(+)) for ATPase, TPA was unable to prevent phosphoenzyme (EP) formation even at low Na(+), 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 (P(i)) were not mutually exclusive inhibitors of para-nitrophenylphosphatase (pNPPase) activity, implying that when P(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.

Bretylium, an organic quaternary amine, inhibits the Na,K-ATPase by binding to the extracellular K-site.

The quaternary amine, bretylium, is a class III antiarrhythmic drug used to treat ventricular tachycardia and fibrillation. The primary mode of action for bretylium is thought to be inhibition of voltage-gated K(+) channels. While the Na,K-ATPase has been the pharmacological target of cardiac glycosides for over a century, recent evidence has shown that bretylium may also inhibit the Na pump. Our experimental findings support and extend these previous reports and provide definitive evidence supporting the previous suggestion that bretylium and K compete for the Na pump. We find that bretylium inhibits the Na pump in a dose-dependent manner in both Na,K-ATPase (IC(50) 4.5 mM) and Rb flux experiments (IC(50) 3.5 mM). Furthermore, we show that bretylium and Rb(+) competes for an extracellular site by measuring ouabain-sensitive (86)Rb flux in intact human red blood cells; that is, there is an apparent increase in K(m) for Rb(+) in the presence of 5 mM bretylium, while V(max) remains unchanged. We also determined that unlike K(+), bretylium does not facilitate the hydrolysis of E2-P. However, it stabilizes this conformation by reducing the ability of K(+) to facilitate dephosphorylation. Finally, we show that bretylium, like K(+), reduces [(3)H]ouabain binding to the Na pump. Taken together, these data are consistent with bretylium binding to the extracellular facing cation site within the E2-P state of the enzyme. Moreover, these findings suggest that bretylium may serve as an effective tool for freezing the pump in an extracellularly cation-bound phosphorylated intermediate, which will aid in future structural analyses.

Chloro(2,2':6',2"-terpyridine) platinum inhibition of the renal Na+,K+-ATPase.

Chloro(2,2':6',2"-terpyridine) platinum, a bulky, hydrophilic reagent, inhibited the renal sodium pump with a single exponential time course. K(+) increased the rate constant of the reaction by about twofold; the K(+) concentration dependence was monotonic, with a half-maximal effect observed at 1 mM, consistent with K(+) acting at a transport site. Na(+), Mg(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(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(+) than in its absence and that terpyridine platinum modification prevents nucleotide binding.

Extracellular protons regulate the extracellular cation selectivity of the sodium pump.

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 ( approximately 5-10 mM). The K(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(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.