Krista L. Arnett

Activity-Based Metagenomic Screening and Biochemical Characterization of Bovine Ruminal Protozoan Glycoside Hydrolases

ABSTRACT 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.

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.

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 (∼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.

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. Sr2+ and Ba2+ did not compete with K+ for ATPase (an extracellular K+ effect). Sr2+ and Ba2+ did compete with Na+ for ATPase (an intracellular Na+ effect) and with K+ for pNPPase (an intracellular K+ effect). These results suggest that Ba2+ or Sr2+ can bind to the intracellular transport site, yet neither Ba2+ nor Sr2+ was able to activate pNPPase activity; we confirmed that Ca2+ and Mn2+ did activate. As another measure of cation binding, we observed that Ca2+ and Mn2+, but not Ba2+, decreased the fluorescence of the FITC-labeled pump; we confirmed that K+ substantially decreased the fluorescence. Interestingly, Ba2+ did shift the K+ dose-response curve. Ethane diamine inhibited Mn2+ stimulation of pNPPase (as well as K+ and Mg2+ stimulation) but did not shift the 50% inhibitory concentration (IC50) for the Mn2+-induced fluorescence change of FITC, though it did shift the IC50 for the K+-induced change. These results suggest that the Mn2+-induced fluorescence change is not due to Mn2+ binding at the transport site. The drawbacks of models in which Mn2+ stimulates pNPPase by binding solely to the catalytic site vs. those in which Mn2+ 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.