Vanadate selectively inhibits the K0+-activated Na+ efflux in squid axons

The effects of internally applied 1 mM vanadate on the Na⁺ efflux in dialysed squid axons were found to depend on the presence of external K⁺. In K⁺-free artificial sea water, vanadate did not produce any change in the rate of Na⁺ efflux, whereas in the presence of 10 mM K⁺ the Na⁺ efflux was reduced to values even lower than those observed in the absence of K⁺ (inversion of the K⁺-free effect). In vanadate-poisoned axons, K⁺ and NH₄⁺ at low concentrations activated Na⁺ efflux, but at high concentrations both cations were inhibitory. However, NH₄⁺ was always a better activator and a poorer inhibitor than K⁺.     

Monovalent cation requirement for ADP inhibition of pyruvate dehydrogenase kinase

ADP is a competitive inhibitor with respect to ATP for pyruvate dehydrogenase kinase. Evidence is presented that K⁺ or NH₄⁺ ions are required for inhibition of the kinase by ADP. K⁺ at 30–90 mM and NH₄⁺ at 1–5 mM decrease markedly the apparent K_i of bovine kidney pyruvate dehydrogenase kinase for ADP and also decrease, to a lesser extent, the apparent K_m for ATP. Na⁺ is less effective and, in addition, inhibits kinase activity. Since K⁺ and NH₄⁺ are not required for kinase activity, their effect appears to be primarily of regulatory significance. K⁺ and NH₄⁺ have little effect, if any, on pyruvate dehydrogenase phosphatase activity. When both the kinase and the phosphatase are present and functional, the near steady state activity of the pyruvate dehydrogenase complex is affected significantly by varying the concentration of K⁺ or NH₄⁺ at a fixed ADP/ATP concentration ratio and by varying the $\text{ADP}\text{ATP}$ ratio at a fixed concentration of monovalent cation.     

Regulation of ligand binding to cardiac muscarinic receptors by ammonium ion and guanine nucleotides

Guanine nucleotides and Na⁺ are known to regulate ligand binding to cardiac muscarinic receptors, which are netagively couple to the adenylate cyclase system. In the present study, we found that NH₄⁺ was more potent than Na⁺ or other monovalent cations in regulating the affinity of the muscarinic receptor for agonists and antagonists. The effect of NH₄⁺ (or Na⁺) on the binding of the antagonist [³H]quinuclidinyl benzilate (QNB) to muscarinic receptors in homogenates of embryonic chick hearts depended on the assay buffer used. NH₄⁺ increased K_d in phosphate buffer or histidine and increased B_max in Tris. NH_f4⁺ (0.1 M) increased the IC₅₀ value for actylcholine inhibition of [³H]QNB binding 20-fold compared to 3–4-fold with 0.1 M Na⁺ or K⁺. Furthermore, NH₄⁺ could substitute for and was more potent than Na⁺ in producing synergistic effects with Gpp[NH]p to reduce the affinity of the receptor of acetylcholine. Tris depressed these effects. Gpp[NH]p plus 0.4 M NH₄Cl totally converted the receptor population to a low affinity agonist state and increased the IC₅₀ for acetylcholine by more than 2000-fold. Two conclusions can be made from the present results. First, NH₄⁺ appears to be the most potent effector yet studied of the monovalent cation site of the muscarinic receptor system. Second, the use of Tris in muscarinic receptor ligand binding assays will produce anomalous results concerning the properties of both agonist antagonist binding to the receptor.     

Kinetic analysis of gill (Na⁺,K⁺)-ATPase activity in selected ontogenetic stages of the Amazon River shrimp, Macrobrachium amazonicum (Decapoda, Palaemonidae): interactions at ATP- and cation-binding sites

We investigated modulation by ATP, Mg²⁺, Na⁺, K⁺ and NH₄ ⁺ and inhibition by ouabain of (Na⁺,K⁺)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, Macrobrachium amazonicum. (Na⁺,K⁺)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP (K _M = 0.09 ± 0.01 mmol L⁻¹) of the decapodid III (Na⁺,K⁺)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na⁺,K⁺)-ATPase activity by K⁺ also revealed a threefold greater affinity for K⁺ (K _0.5 = 0.91 ± 0.04 mmol L⁻¹) in decapodid III than in other stages; NH₄ ⁺ had no modulatory effect. The affinity for Na⁺ (K _0.5 = 13.2 ± 0.6 mmol L⁻¹) of zoea I (Na⁺,K⁺)-ATPase was fourfold less than other stages. Modulation by Na⁺, Mg²⁺ and NH₄ ⁺ obeyed cooperative kinetics, while K⁺ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg²⁺ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg²⁺-stimulated ATPases other than (Na⁺,K⁺)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na⁺-ATPase may be involved in the ontogeny of osmoregulation in larval M. amazonicum. The NH₄ ⁺-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.     

Regulation of Na,K-ATPase Transport Activity by Protein Kinase C

Considerable evidence indicates that the renal Na⁺,K⁺-ATPase is regulated through phosphorylation/dephosphorylation reactions by kinases and phosphatases stimulated by hormones and second messengers. Recently, it has been reported that amino acids close to the NH₂-terminal end of the Na⁺,K⁺-ATPase α-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na⁺,K⁺-ATPase activity. To determine whether the α-subunit NH₂-terminus is involved in the regulation of Na⁺,K⁺-ATPase activity by PKC, we have expressed the wild-type rodent Na⁺,K⁺-ATPase α-subunit and a mutant of this protein that lacks the first thirty-one amino acids at the NH₂-terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant phenotype characteristic of rodent kidney cells. The presence of the α-subunit NH₂-terminal segment was not necessary to express the maximal Na⁺,K⁺-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb⁺-transport in intact cells were the same in cells transfected with the wild-type rodent α1 and the NH₂-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na⁺,K⁺-ATPase mediated Rb⁺-uptake and reduced the intracellular Na⁺ concentration of cells transfected with wild-type α1 cDNA. In contrast, these effects were not observed in cells expressing the NH₂-deletion mutant of the α-subunit. Treatment with phorbol ester appears to affect specifically the Na⁺,K⁺-ATPase activity and no evidence was observed that other proteins involved in Na⁺-transport were affected. These results indicate that amino acid(s) located at the α-subunit NH₂-terminus participate in the regulation of the Na⁺,K⁺-ATPase activity by PKC. Received: 10 July 1996/Revised: 19 September 1996     

Involvement of cation channels and NH4+-sensitive K+ transporters in Na+ uptake by cowpea roots under salinity

Na⁺ accumulation was investigated in the roots of 11-d-old cowpea [Vigna unguiculata (L.) Walp.] plants. The relative contribution of different membrane transporters on Na⁺ uptake was estimated by applying Ca²⁺, K⁺, NH₄ ⁺, and pharmacological inhibitors. Na⁺ accumulation into the root symplast was decreased by half in the presence of 1 mM Ca²⁺ and it was almost abolished by 100 mM K⁺. The inhibitory effect of external NH⁴⁺ on Na⁺ accumulation was more pronounced in the roots of NH₄ ⁺-free growing plants. Na⁺ accumulation was reduced about 73 % by 0.1 mM flufenamate and it was almost blocked by 2 mM quinine. In addition, 20 mM tetraethylammonium and 1.0 mM Cs⁺ decreased Na⁺ accumulation by 28 and 30 %, respectively. These results evidenced that low-affinity Na⁺ uptake by cowpea roots depends on Ca²⁺-sensitive and Ca²⁺-insensitive pathways. The Ca²⁺-sensitive pathway is probably mediated by nonselective cation channels and the Ca²⁺-insensitive one may involve K⁺ channels and to a lesser extent NH₄ ⁺-sensitive K⁺ transporters.     

Influence of inorganic nitrogen sources on K+/Na+ homeostasis and salt tolerance in sorghum plants

This work aimed to study the regulation of K⁺/Na⁺ homeostasis and the physiological responses of salt-treated sorghum plants [Sorghum bicolor (L.) Moench] grown with different inorganic nitrogen (N) sources. Four days after sowing (DAS), the plants were transferred to complete nutrient solutions containing 0.75 mM K⁺ and 5 mM N, supplied as either NO₃ ^? or NH₄ ⁺. Twelve DAS, the plants were subjected to salt stress with 75 mM NaCl, which was applied in two doses of 37.5 mM. The plants were harvested on the third and seventh days after the exposure to NaCl. Under the salt stress conditions, the reduction of K⁺ concentrations in the shoot and roots was higher in the culture with NO₃ ^? than with NH₄ ⁺. However, the more conspicuous effect of N was on the Na⁺ accumulation, which was severely limited in the presence of NH₄ ⁺. This ionic regulation had a positive influence on the K⁺/Na⁺ ratio and the selective absorption and transport of K⁺ in the plants grown with NH₄ ⁺. Under control and salt stress conditions, higher accumulation of free amino acids and soluble proteins was promoted in NH₄ ⁺ grown roots than NO₃ ^? grown roots at both harvesting time, whereas higher accumulation of soluble sugars was observed only at 7 days of salt stress exposure. Unlike the NH₄ ⁺ grown plants, the gas exchanges of the NO₃ ^? grown plants were reduced after 7 days of salt stress. These results suggest that external NH₄ ⁺ may limit Na⁺ accumulation in sorghum, which could contribute to improving its physiological and metabolic responses to salt stress.     

Selectivity and sensitivity to hydrogen ion blocking of batrachotoxin-modified sodium channels in nerve fiber membrane

Currents through batrachotoxin-modified sodium channels were measured by the voltage clamp method on the Ranvier node membrane. In experiments with replacement of Na⁺ in the external solution by K⁺ or NH ₄ ⁺ the following series of permeabilities, determined as reversal potentials according to the equation of a static field, was obtained — P_Na: \(P_{NH_4 }\) :P_K=1:0.47:0.19. The relative permeability for H⁺ was determined by measuring currents after replacement of Na⁺ in the external solution by nonpenetrating choline ions and lowering pH to 3.7–3.8. The ratio p_H/p_Na for sodium channels modified by batrachotoxin averaged 528±46. Modified channels were less sensitive to the blocking action of H⁺ than normal sodium channels. The difference in the effective values of pK of the acid group of normal and modified channels was 0.40–0.45.     

Effect of potassium and ammonium ions upon glycolysis catalyzed by an extract of rat brain

The stimulatory effect of ammonium and potassium ions upon glycolysis, as catalyzed by an extract prepared from an acetone powder of rat brain, has been investigated. The effect is most pronounced when small amounts of adenosine triphosphate (ATP) are employed and it becomes progressively less apparent as the ATP concentration is increased.Unlike the extracts and homogenates obtained from fresh brain tissue (1,2), glycolysis by the acetone extracts is not inhibited significantly by sodium ion. This is apparently due to the low apyrase content of these extracts. In addition the experiments indicate that the NH₄⁺ and K⁺ have direct stimulatory effects which are not due to antagonism between Na⁺ and NH₄⁺ or K⁺. This suggestion gains credence by the observation that the same concentration of NH₄⁺ is required for stimulation whether or not the concentration of Na⁺ is reduced through substitution of trishydroxymethylaminomethane buffer for the NaHCO₃ buffer. Apparently NH₄⁺ and K⁺ serve to maintain ATP in these systems by initiating or accelerating phosphorylation reactions. In so doing, they prevent the formation of adenylic acid which is fairly rapidly dephosphorylated in those systems by a 5-nucleotidase.     

Ion complexation in nonactin,monactin, and dinactin: A Raman spectroscopic study

The ability of the macrotetrolide nactins to complex selectivity with a wide variety of cations makes these ionophorous antibiotics important model systems for the study of biologic ionic transport. We report a Raman spectroscopic investigation of the Na⁺, K⁺, Rb⁺, Cs⁺, Tl⁺, NH₄⁺, NH₃OH⁺, C(NH₂)₃⁺, and Ba⁺⁺ complexes of nonactin, monactin, and dinactin in 4:1 (v/v) CH₃OH/CHCl₃ and in the solid state. The nactins display characteristic spectral changes upon complexation, some of which are specific for a given cation. In the K⁺, Rb⁺, Cs⁺, NH₃OH⁺, and C(NH₂)₃⁺ complexes, which are apparently isosteric, the ester carbonyl stretch frequency is found to be linearly proportional to the cation–carbonyl electrostatic interaction energy, as calculated from a simplified model. Deviations for the Na⁺, NH₄⁺, Tl⁺, and Ba⁺⁺ complexes are interpreted as arising from additional nonelectrostatic interactions. Additional information is obtained from other spectral regions and from measurements of depolarization ratios. Spectra of the nactin complexes differ from each other more in the solid state than in solution, reflecting the effects of crystalline contact forces.     

Effect of four heavy metals on the biology of Nostoc muscorum

Summary This study presents the effects of Cr, Pb, Ni and Ag on growth, pigments, protein, DNA, RNA, heterocyst frequency, uptake of NH₄ ⁺ and N0₃ ^–, loss of electrolytes (Na⁺ and K⁺), nitrate reductase and glutamine synthetase activities ofNostoc muscorum. The statistical tests revealed a direct positive correlation between the metal concentration and inhibition of different processes. Ni was found to be more toxic against growth, pigments and heterocyst differentiation compared to the other metals. Inhibition of pigment showed the following trend: chlorophyll > phycocyanin > carotenoid. No generalized trend for inhibition of macromolecules was observed. The loss of K⁺ and Na⁺ as affected by Cr, Ni and Pb was similar but more pronounced for K⁺ than Na⁺. The inhibition of physiological variables depicted the following trend: Na⁺ loss > K⁺ loss > glutamine synthetase > NH₄ uptake > growth > N0₃ ^– uptake > nitrate reductase > heterocyst frequency. This study therefore suggests that loss of electrolytes can be used as a first signal of metal toxicity in cyanobacteria. However, further study is needed to confirm whether the abnormality induced by nickel (branch formation) is a physiological or genetic phenomenon.     

Ammonia excretion in the seawater blue crab (Callinectes sapidus) occurs by diffusion, and not Na+/NH4+ exchange

Summary A series of experiments was conducted to investigate whether ammonia is excreted across the seawater-acclimated blue crab's gills as ionized NH ₄ ⁺ or as the free base, NH₃. The net excretion rate of ammonia was not changed by transfer of the crabs to reduced (150 mM) Na⁺ solutions, by transfer to Na⁺- and K⁺-free artificial sea water, or by the sodium transport inhibitor amiloride. Ammonia excretion, therefore, does not appear to be linked to Na⁺ uptake in these animals, and appears to take place by passive diffusion. Since ammonia could diffuse either as NH ₄ ⁺ or NH₃, we examined two other kinds of evidence. The trans-epithelial potential was measured in sea water and the various artificial media. In spite of a 10 mV more negative potential in Na⁺-, K⁺-free medium, the ammonia excretion was not reduced. Also, in alkalinized seawater in which the partial pressure gradient of NH₃ was reduced, but the concentration gradient of NH ₄ ⁺ increased, ammonia excretion was reduced by about 70%. These results are consistent with the conclusion that ammonia excretion takes place by diffusion of the free base, NH₃.Abbreviations SW sea water - ASW artificial sea water - t.e.p. transepithelial potential The University of Texas Marine Science Institute Contribution No. 461Supported by NSF Grant PCM77-24358     

Comparison between drug-induced and K+-induced changes in molar acid extrusion fluxes (JH +) and in energy consumption rates in astrocytes

Neuronal excitation leads to an increase of the extracellular K⁺ concentration ([K⁺]_o) in brain. This increase has at least two energy-consuming consequences: (1) a depolarization-mediated change in intracellular pH (pH_i) in astrocytes due to depolarization-mediated increased activity of the acid-extruding Na⁺/bicarbonate transporter NBCe1 (driven by secondary active transport, supported by ion gradients established by the Na⁺, K⁺-ATPase); and (2) activation of cellular reuptake of K⁺ mediated by the Na⁺, K⁺-ATPase in both neurons and astrocytes. Astrocytic, but not neuronal increase in NBCe1 activity and pH_i is also seen after chronic treatment with either of the two anti-bipolar drugs carbamazepine or valproic acid. The third ‘classical’ anti-bipolar drug, ‘lithium’ increases astrocytic pH_i by a different mechanism (stimulation of the acid extruding Na⁺/H⁺ exchanger NHE1). The acid extruder fluxes, which depend upon the change in pH_i per time unit (ΔpH_i/Δt) and intracellular buffering power, have not been established in most of these situations. Therefore their stimulatory effects on energy metabolism has not been quantitated. This has been done in the present study in cultured mouse astrocytes. pH_i was determined using the fluorescent pH-sensitive indicator BCECF–AM and an Olympus IX71 live cell imaging fluorescence microscope. Molar acid extrusion fluxes (indicating transporter activity) were determined as pH_i changes/min during recovery after acid-loading with NH₃/NH₄ ⁺, NBCe1 mRNA and protein expression in the cultured cells by, respectively RT-PCR and Western blotting. Drug-induced up-regulation of acid extrusion flux was slow and less than physiologically seen after increase in K⁺ concentration. Energetically, K⁺ uptake is much costlier than NBCe1 activity.     

The human non-gastric H,K-ATPase has a different cation specificity than the rat enzyme

The primary sequence of non-gastric H,K-ATPase differs much more between species than that of Na,K-ATPase or gastric H,K-ATPase. To investigate whether this causes species-dependent differences in enzymatic properties, we co-expressed the catalytic subunit of human non-gastric H,K-ATPase in Sf9 cells with the β₁ subunit of rat Na,K-ATPase and compared its properties with those of the rat enzyme (Swarts et al., J. Biol. Chem. 280, 33115-33122, 2005). Maximal ATPase activity was obtained with NH₄⁺ as activating cation. The enzyme was also stimulated by Na⁺, but in contrast to the rat enzyme, hardly by K⁺. SCH 28080 inhibited the NH₄⁺-stimulated activity of the human enzyme much more potently than that of the rat enzyme. The steady-state phosphorylation level of the human enzyme decreased with increasing pH, [K⁺], and [Na⁺] and nearly doubled in the presence of oligomycin. Oligomycin increased the sensitivity of the phosphorylated intermediate to ADP, demonstrating that it inhibited the conversion of E₁P to E₂P. All three cations stimulated the dephosphorylation rate dose-dependently. Our studies support a role of the human enzyme in H⁺/Na⁺ and/or H⁺/NH₄⁺ transport but not in Na⁺/K⁺ transport.     

Properties and Expression of Na+/K+-ATPase α-Subunit Isoforms in the Brain of the Swamp Eel,Monopterus albus,Which Has Unusually High Brain Ammonia Tolerance

The swamp eel, Monopterus albus, can survive in high concentrations of ammonia (>75 mmol l⁻¹) and accumulate ammonia to high concentrations in its brain (∼4.5 µmol g⁻¹). Na⁺/K⁺-ATPase (Nka) is an essential transporter in brain cells, and since NH₄ ⁺ can substitute for K⁺ to activate Nka, we hypothesized that the brain of M. albus expressed multiple forms of Nka α-subunits, some of which might have high K⁺ specificity. Thus, this study aimed to clone and sequence the nka α-subunits from the brain of M. albus, and to determine the effects of ammonia exposure on their mRNA expression and overall protein abundance. The effectiveness of NH₄ ⁺ to activate brain Nka from M. albus and Mus musculus was also examined by comparing their Na⁺/K⁺-ATPase and Na⁺/NH₄ ⁺-ATPase activities over a range of K⁺/NH₄ ⁺ concentrations. The full length cDNA coding sequences of three nkaα (nkaα1, nkaα3a and nkaα3b) were identified in the brain of M. albus, but nkaα2 expression was undetectable. Exposure to 50 mmol l⁻¹ NH₄Cl for 1 day or 6 days resulted in significant decreases in the mRNA expression of nkaα1, nkaα3a and nkaα3b. The overall Nka protein abundance also decreased significantly after 6 days of ammonia exposure. For M. albus, brain Na⁺/NH₄ ⁺-ATPase activities were significantly lower than the Na⁺/K⁺-ATPase activities assayed at various NH₄ ⁺/K⁺ concentrations. Furthermore, the effectiveness of NH₄ ⁺ to activate Nka from the brain of M. albus was significantly lower than that from the brain of M. musculus, which is ammonia-sensitive. Hence, the (1) lack of nkaα2 expression, (2) high K⁺ specificity of K⁺ binding sites of Nkaα1, Nkaα3a and Nkaα3b, and (3) down-regulation of mRNA expression of all three nkaα isoforms and the overall Nka protein abundance in response to ammonia exposure might be some of the contributing factors to the high brain ammonia tolerance in M. albus.     

Altered Ionic Selectivity of the Sodium Channel Revealed by Cysteine Mutations within the Pore

To explore the role of pore-lining amino acids in Na⁺ channel ion-selectivity, pore residues were  replaced serially with cysteine in cloned rat skeletal muscle Na⁺ channels. Ionic selectivity was determined by measuring permeability and ionic current ratios of whole-cell currents in Xenopus oocytes. The rSkM1 channels displayed an ionic selectivity sequence Na⁺>Li⁺>NH₄ ⁺>>K⁺>>Cs⁺ and were impermeable to divalent cations.  Replacement of residues in domain IV showed significantly enhanced current and permeability ratios of NH₄ ⁺ and K⁺, and negative shifts in the reversal potentials recorded in the presence of external Na⁺ solutions when compared to cysteine mutants in domains I, II, and III (except K1237C). Mutants in domain IV showed altered selectivity sequences: W1531C (NH₄ ⁺>K⁺>Na⁺≥Li⁺≈Cs⁺), D1532C, and G1533C (Na⁺>Li⁺≥NH₄ ⁺>K⁺>Cs⁺). Conservative replacement of the aromatic residue in domain IV (W1531) with phenylalanine or tyrosine retained Na⁺ selectivity of the channel while the alanine mutant (W1531A) reduced ion selectivity. A single mutation within the third pore forming region (K1237C) dramatically altered the selectivity sequence of the rSkM1 channel (NH₄ ⁺>K⁺>Na⁺≥Li⁺≈Cs⁺) and was permeable to divalent cations having the selectivity sequence Ca²⁺≥Sr²⁺>Mg²⁺>Ba²⁺. Sulfhydryl modification of K1237C, W1531C or D1532C with methanethiosulfonate derivatives that introduce a positively charged ammonium group, large trimethylammonium moiety, or a negatively charged sulfonate group within the pore was ineffective in restoring Na⁺ selectivity to these channels. Selectivity of D1532C mutants could be largely restored by increasing extracellular pH suggesting altering the ionized state at this position influences selectivity. These data suggest that K1237 in domain III and W1531, D1532, and G1533 in domain IV play a critical role in determining the ionic selectivity of the Na⁺ channel.     

Subcellular Localization and Kinetic Characterization of a Gill (Na+, K+)-ATPase from the Giant Freshwater Prawn Macrobrachium rosenbergii

The stimulation by Mg²⁺, Na⁺, K⁺, NH₄ ⁺, and ATP of (Na⁺, K⁺)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na⁺, K⁺)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M _r. Under saturating Mg²⁺, Na⁺, and K⁺ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V _M = 115.0 ± 2.3 U mg^?1, K _0.5 = 0.10 ± 0.01 mmol L^?1. Stimulation by Na⁺ (V _M = 110.0 ± 3.3 U mg^?1, K _0.5 = 1.30 ± 0.03 mmol L^?1), Mg²⁺ (V _M = 115.0 ± 4.6 U mg^?1, K _0.5 = 0.96 ± 0.03 mmol L^?1), NH₄ ⁺ (V _M = 141.0 ± 5.6 U mg^?1, K _0.5 = 1.90 ± 0.04 mmol L^?1), and K⁺ (V _M = 120.0 ± 2.4 U mg^?1, K _M = 2.74 ± 0.08 mmol L^?1) followed single saturation curves and, except for K⁺, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K _I = 12.4 ± 1.3 mol L^?1. Complementary inhibition studies suggest the presence of F₀F₁–, Na⁺-, or K⁺-ATPases, but not V(H⁺)- or Ca²⁺-ATPases, in the gill microsomal preparation. K⁺ and NH₄ ⁺ synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH₄ ⁺, and K⁺ of the gill enzyme.     

Effect of NH+4 and K+ on the activity of the ribosomal subunits in the EF-G- and EF-T-dependent GTR hydrolysis

$\text{E. coli}$ 50S ribosomal subunits show in the absence of 30S subunits and at low NH₄⁺ or K⁺ high turnover activity in EF-G-dependent GTP hydrolysis which is inhibited by increasing concentrations of monovalent cations. At 80 mM NH₄⁺ or K⁺ this activity is already 70–80% inhibited. This effect is reversed by 30S which are stimulatory with an optimum at about 80 mM for NH₄⁺ and 20–40 mM for K⁺. At low NH₄⁺ or K⁺ (<5 mM) stimulation by 30S of maximal 50S activity depends on the [EF-G]/[50S]. Unlike EF-G, EF-T does not show any Phe-tRNA-dependent GTPase activity with 50S alone even at low concentrations of NH₄⁺ or K⁺.     

Ion conduction and selectivity in acid-sensing ion channel 1

The ability of acid-sensing ion channels (ASICs) to discriminate among cations was assessed based on changes in conductance and reversal potential with ion substitution. Human ASIC1a was expressed in Xenopus laevis oocytes, and acid-induced currents were measured using two-electrode voltage clamp. Replacement of extracellular Na⁺ with Li⁺, K⁺, Rb⁺, or Cs⁺ altered inward conductance and shifted the reversal potentials consistent with a selectivity sequence of Li ∼ Na > K > Rb > Cs. Permeability decreased more rapidly than conductance as a function of atomic size, with P_K/P_Na = 0.1 and G_K/G_Na = 0.7 and P_Rb/P_Na = 0.03 and G_Rb/G_Na = 0.3. Stimulation of Cl⁻ currents when Na⁺ was replaced with Ca²⁺, Sr²⁺, or Ba²⁺ indicated a finite permeability to divalent cations. Inward conductance increased with extracellular Na⁺ in a hyperbolic manner, consistent with an apparent affinity (K_m) for Na⁺ conduction of 25 mM. Nitrogen-containing cations, including NH₄⁺, NH₃OH⁺, and guanidinium, were also permeant. In addition to passing through the channels, guanidinium blocked Na⁺ currents, implying competition for a site within the pore. The role of negative charges in an external vestibule of the pore was evaluated using the point mutation D434N. The mutant channel had a decreased single-channel conductance, measured in excised outside-out patches, and a macroscopic slope conductance that increased with hyperpolarization. It had a weakened interaction with Na⁺ (K_m = 72 mM) and a selectivity that was shifted toward larger atomic sizes. We conclude that the selectivity of ASIC1 is based at least in part on interactions with binding sites both within and internal to the outer vestibule.     

Kinetic Studies of a (Na++ K++ Mg2+) ATPase in Sugar Beet Roots

Kinetic studies of a dithiothreitol treated membrane ATPase fraction from sugar beet roots led to the following conclusions: 1) In the presence of MgATP, Na⁺ and K⁺ stimulate the ATPase activity in different ways following simple Michaelis-Menten kinetics. Thus separate sites for Na⁺ and K⁺ are suggested. 2) In the absence of K⁺, Na⁺ acts as an uncompetitive modifier raising the apparent K_m and V_max for MgATP. 3) In the absence of Na⁺, K⁺ activates non-competitively with respect to MgATP. Thus K⁺ increases V_max but does not affect the apparent affinity constant. 4) K⁺ and Na⁺ double the rate constants. 5) In the presence of Na⁺ or K⁺, Mg²⁺ in excess acts as a weak inhibitor to Na⁺ and/or K⁺ activity. 6) The temperature-activity dependence in the 5–40°C interval shows biphasic Arrhenius plots with the transition point between 15–18°C. The activation energy is lowered at temperatures > 18°C.