Modulation of gill Na+,K+-ATPase activity by ammonium ions: Putative coupling of nitrogen excretion and ion uptake in the freshwater shrimp Macrobrachium olfersii

The effect of NH4+ ions on (Na+,K+)-ATPase hydrolytic activity was examined in a gill microsomal fraction from M. olfersii. In the absence of NH4+ ions, K+ ions stimulated ATP hydrolysis, exhibiting cooperative kinetics (nH=0.8), to a maximal specific activity of V=556.1+/-22.2 nmol.min(-1).mg(-1) with K(0.5)=2.4+/-0.1 mmol.L(-1). No further stimulation by K+ ions was observed in the presence of 50 mmol.L(-1) NH4+ ions. ATP hydrolysis was also stimulated by NH4+ ions obeying Michaelian kinetics to a maximal specific activity of V=744.8+/-22.3 nmol.min(-1).mg(-1) and KM=8.4+/-0.2 mmol.L(-1). In the presence of 10 mmol.L(-1) K+ ions, ATP hydrolysis was synergistically stimulated by NH4+ ions to V=689.8+/-13.8 nmol.min(-1).mg(-1) and K(0.5)=6.6+/-0.1 mmol.L(-1), suggesting that NH4+ ions bind to different sites than K+ ions. PNPP hydrolysis was also stimulated cooperatively by K+ or NH4+ ions to maximal values of V= 235.5+/-11.8 nmol.min(-1).mg(-1) and V=234.8+/-7.0 nmol.min(-1).mg(-1), respectively. In contrast to ATP hydrolysis, K(+)-phosphatase activity was not synergistically stimulated by NH4+ and K+ ions. These data suggest that at high NH4+ ion concentrations, the (Na+, K+)-ATPase exposes a new site; the subsequent binding of NH4+ ions stimulates ATP hydrolysis to rates higher than those for K+ ions alone. This is the first demonstration that (Na+, K+)-ATPase activity in a freshwater shrimp gill is modulated by ammonium ions, independently of K+ ions, an effect that may constitute a fine-tuning mechanism of physiological relevance to osmoregulatory and excretory processes in palaemonid shrimps.     

Cation permeation through the voltage-dependent potassium channel in the squid axon. Characteristics and mechanisms

Characteristics of cation permeation through voltage-dependent delayed rectifier K channels in squid giant axons were examined. Axial wire voltage-clamp measurements and internal perfusion were used to determine conductance and permeability properties. These K channels exhibit conductance saturation and decline with increases in symmetrical K+ concentrations to 3 M. They also produce ion- and concentration-dependent current-voltage shapes. K channel permeability ratios obtained with substitutions of internal Rb+ or NH+4 for K+ are higher than for external substitution of these ions. Furthermore, conductance and permeability ratios of NH+4 or Rb+ to K+ are functions of ion concentration. Conductance measurements also reveal the presence of an anomalous mole fraction effect for NH+4, Rb+, or Tl+ to K+. Finally, internal Cs+ blocks these K channels in a voltage-dependent manner, with relief of block by elevations in external K+ but not external NH+4 or Cs+. Energy profiles for K+, NH+4, Rb+, Tl+, and Cs+ incorporating three barriers and two ion-binding sites are fitted to the data. The profiles are asymmetric with respect to the center of the electric field, have different binding energies and electrical positions for each ion, and (for K+) exhibit concentration-dependent barrier positions.     

Activation of calcium transport in skeletal muscle sarcoplasmic reticulum by monovalent cations.

The rates of calcium transport and Ca2+-dependent ATP hydrolysis by rabbit skeletal muscle sarcoplasmic reticulum were stimulated by monovalent cations. The rate of decomposition of phosphoprotein intermediate of the Ca2+-dependent ATPase of sarcoplasmic reticulum was also increased by these ions to an extent that is sufficient to account for the stimulation of calcium transport and Ca2+-dependent ATPase activity. The order of effectiveness of monovalent cations tested at saturating concentrations in increasing rate of phosphoprotein decomposition is: K+, Na+ greater than Rb+, NH4+ greater than Cs+ greater than Li+, choline+, Tris+.     

Membrane ionic currents in Rhodobacter capsulatus. Evidence for electrophoretic transport of K+, Rb+ and NH4+

1. The cytoplasmic membrane ionic current of cells of Rhodobacter capsulatus, washed to lower the endogenous K+ concentration, had a non-linear dependence on the membrane potential measured during photosynthetic illumination. Treatment of the cells with venturicidin, an inhibitor of the H(+)-ATP synthase, increased the membrane potential and decreased the membrane ionic current at values of membrane potential below a threshold. 2. The addition of K+ or Rb+, but not of Na+, led to an increase in the membrane ionic current and a decrease in the membrane potential in either the presence or absence of venturicidin. Approximately 0.4 mM K+ or 2.0 mM Rb+ led to a half-maximal response. At saturating concentrations of K+ and Rb+, the membrane ionic currents were similar. The membrane ionic currents due to K+ and Rb+ were not additive. The K(+)-dependent and Rb(+)-dependent ionic currents had a non-linear relationship with membrane potential: the alkali cations only increased the ionic current when the membrane potential lay above a threshold value. The presence of 1 mM Cs+ did not lead to an increase in the membrane ionic current but it had the effect of inhibiting the membrane ionic current due to either K+ or Rb+. 3. Photosynthetic illumination in the presence of either K+ or Rb+, and weak acids such as acetate, led to a decrease in light-scattering by the cells. This was attributed to the uptake of potassium or rubidium acetate and a corresponding increase in osmotic strength in the cytoplasm. 4. The addition of NH4+ also led to an increase in membrane ionic current and to a decrease in membrane potential (half-maximal at 2.0 mM NH4+). The relationship between the NH4(+)-dependent ionic currents and the membrane potential was similar to that for K+. The NH4(+)-dependent and K(+)-dependent ionic current were not additive. However, illumination in the presence of NH4+ and acetate did not lead to significant light-scattering changes. The NH4(+)-dependent membrane ionic current was inhibited by 1 mM Cs+ but not by 50 microM methylamine. 5. It is proposed that the K(+)-dependent membrane ionic current is catalysed by a low-affinity K(+)-transport system such as that described in Rb. capsulatus [Jasper, P. (1978) J. Bacteriol. 133, 1314-1322]. The possibility is considered that, as well as Rb+, this transport system can also operate with NH4+. However, in our experimental conditions NH4+ uptake is followed by NH3 efflux.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of ammonium (methylammonium)/potassium antiport in Escherichia coli

The energetics of ammonium ion transport by Escherichia coli have been studied using [14C]methylammonium as a substrate. Rapid assays for uptake allowed kinetic parameters (CH3NH3+ Km = 36 microM; Vmax = 4 nmol X s-1 X mg-1 to be determined in the absence of CH3NH3+ metabolism. Cells cultured in media containing 1 mM NH4+ failed to express CH3NH3+ transport activity. Methylammonium accumulated at levels which were 100-fold higher than those of the medium. This accumulation was dependent upon the addition of glucose or pyruvate. The entry of CH3NH3+ supported by glucose oxidation in an F1F0-ATPase-deficient mutant was blocked by uncoupler. Transport by wild-type cells under similar conditions was significantly inhibited by arsenate. Thus, CH3NH3+ uptake requires both ATP and an electrochemical H+ gradient. This transport activity was lost upon exposure of E. coli to osmotic shock, but could be recovered by incubation of shocked cells with boiled shock fluid or with glucose plus K+ in the presence of chloramphenicol. Similar reconstitution was observed in K+-depleted parental strains, but not in a mutant defective in K+ transport, demonstrating a requirement for internal K+. However, external K+ proved to be a noncompetitive inhibitor (Ki = 1 mM) of CH3NH3+ uptake by K+ -replete bacteria. External Na+ had no effect on transport. The addition of NH4+ or CH3NH3+ induced a rapid exodus of intracellular 86Rb+, an analog which was able to substitute for K+. The molar ratio of CH3NH3+ uptake to Rb+ exit was 1.12 +/- 0.11. These findings support a mechanism for CH3NH3+ (NH4+) accumulation which requires K+ antiport (exchange) and is driven by the electrochemical K+ gradient.     

Regulation of connexin hemichannels by monovalent cations

Opening of connexin hemichannels in the plasma membrane is highly regulated. Generally, depolarization and reduced extracellular Ca2+ promote hemichannel opening. Here we show that hemichannels formed of Cx50, a principal lens connexin, exhibit a novel form of regulation characterized by extraordinary sensitivity to extracellular monovalent cations. Replacement of extracellular Na+ with K+, while maintaining extracellular Ca2+ constant, resulted in >10-fold potentiation of Cx50 hemichannel currents, which reversed upon returning to Na+. External Cs+, Rb+, NH4+, but not Li+, choline, or TEA, exhibited a similar effect. The magnitude of potentiation of Cx50 hemichannel currents depended on the concentration of extracellular Ca2+, progressively decreasing as external Ca2+ was reduced. The primary effect of K+ appears to be a reduction in the ability of Ca2+, as well as other divalent cations, to close Cx50 hemichannels. Cx46 hemichannels exhibited a modest increase upon substituting Na+ with K+. Analyses of reciprocal chimeric hemichannels that swap NH2- and COOH-terminal halves of Cx46 and Cx50 demonstrate that the difference in regulation by monovalent ions in these connexins resides in the NH2-terminal half. Connexin hemichannels have been implicated in physiological roles, e.g., release of ATP and NAD+ and in pathological roles, e.g., cell death through loss or entry of ions and signaling molecules. Our results demonstrate a new, robust means of regulating hemichannels through a combination of extracellular monovalent and divalent cations, principally Na+, K+, and Ca2+.     

Potassium ion-activated hydrolysis of p-nitrophenyl phosphate in pancreatic islet-cell membranes.

Hydrolysis of p-nitrophenyl phosphate was measured in a fraction enriched in plasma membranes from pancreatic islets of non-inbred ob/ob mice. Hydrolysis was stimulated by K+ (10mM) in the pH range 5--10; a small peak of K+-induced activation was observed between pH7.5 and 8. Both the K+-induced activation and the hydrolysis in the absence of K+ were Mg2+-dependent; maximum activation was obtained with 10mM-K+ plus 5 mM-Mg2+. Rb+ was as effective an activator as K+. Ouabain was inhibitory, the effect being inversely related to the K+ concentration; 0.1--0.2mM-ouabain caused about 50% inhibition in the presence of 1 mM-K+, but had no demonstrable effect in the presence of 4--5mM-K+. The K+-stimulated activity was markedly inhibited by 0.1mM-ATP, 35--140 MM-Na+, or 0.01 mM-p-chloromercuribenzenesulphonic acid. Similarities to Rb+ accumulation suggest that catalysis of univalent cation flow in pancreatic beta-cells may be coupled to a phosphoryl-transfer reaction with ATP as natural substrate or regulator.     

Effects of K+ and other monovalent cations on yeast mitochondria.

In mitochondria from Saccharomyces cerevisiae and in the presence of ethanol or NADH, K+ or Na+ increased the rate of O2 uptake in states 3 and uncoupled as well as in sonicated mitochondria. The respiratory control, the ADP:O ratio and the synthesis of ATP also increased. ATP hydrolysis by sonicated mitochondria increased depending on the cation added as follows: K+ = NH4+ = Rb+ Na+ Li+. This correlated with the ionic radii of the cations. Monovalent cations increased the activity of: 1) F1F0ATPase which was sensitive to cation size and 2) complex I of the respiratory chain, which seemed to regulate the rate of oxidative phosphorylation, but did not discriminate between K+ or Na+.     

Monovalent Cation Activation of Plant Pyruvate Dehydrogenase Kinase

The pyruvate dehydrogenase kinase-catalyzed inactivation of the pyruvate dehydrogenase complex was studied using dialyzed, soluble proteins from mitochondria purified from green leaf tissue of Pisum sativum L. seedlings. At subsaturating ATP concentrations, K+ or NH4+, but not Na+, stimulated the pyruvate dehydrogenase kinase by lowering the Km(ATP). Micromolar concentrations of NH4+ were required to produce the same effect as millimolar concentrations of K+. This is apparent from the observations that the activation constant (Kact) for NH4+ was 0.1 mM, whereas the Kact(K+) was 0.7 mM. Maximal pyruvate dehydrogenase kinase velocities attained with NH4+ were higher than those with K+, and, therefore, NH4+ was able to stimulate PDH kinase further in the presence of saturating K+. This result supports our conclusion that photorespiratory NH4+ production in plant mitochondria may be involved in regulating the entry of carbon into the Krebs cycle by way of the pyruvate dehydrogenase complex.     

The interaction of “K+-like” cations with the apical K+ channel in frog skin

The apparent permeability of the apical K+ channel in the abdominal skin of the frog (Rana temporaria) for different monovalent cations was tested by comparing the short-circuit current (SCC) obtained after imposition of serosally directed ionic concentration gradients. Furthermore, the SCC was subjected to noise analysis. Of various cations tested, only the "K+-like" ions NH+4, Rb+ and Tl+, besides K+, were found to permeate the apical K+ channel, as reflected by SCC- and fluctuation analysis: (i) The SCC could be depressed by addition of the K+-channel blocker Ba2+ to the mucosal solution. (ii) With the K+-like ions (Ringer's concentration), a spontaneous Lorentzian noise was observed. Plateau values were similar for K+ and Tl+, and smaller for NH+4 and Rb+. The corner frequencies clearly increased in the order K+ less than NH+4 less than Tl+ much less than Rb+. The SCC dose-response relationships revealed a Michaelis-Menten-type current saturation only for pure K+- or Tl+-Ringer's solutions as mucosal medium, whereas a more complicated SCC behavior was seen with Rb+ and especially, NH+4. For K+-Tl+ mixtures an anomalous mole-fraction relationship was observed: At low [Tl+]/[K+] ratios, Tl+ ions appeared to inhibit competitively the K+ current while, at high [Tl+]/[K+] ratios, Tl+ seemed to be a permeant cation. This feature was also detected in the noise analysis of K+-Tl+ mixtures. Long-term exposure to mucosal Tl+ resulted in an irreversible deterioration of the tissue. The SCC depression by Ba2+ was of a simple saturation-type characteristic with, however, different half-maximal doses (NH+4 less than K+ less than Rb+). Ba2+ induced a "blocker noise" in presence of all permeant cations with corner frequencies that depended on the Ba2+ concentration. A linear increase of the corner frequencies of the Ba2+-induced noise with increasing Ba2+ concentration was seen for NH+4, Rb+ and K+. With the assumption of a pseudo two-state model for the Ba2+ blockade the on- and off-rate constants for the Ba2+ interaction with the NH+4/Rb+/K+ channel were calculated and showed marked differences, dependent on the nature of the permeant ion. The specific problems with Tl+ prevented such an analysis but SCC- and noise data indicated a comparably poor efficiency of Ba2+ as Tl+-current inhibitor. We attempted a qualitative analysis of our results in terms of a "two-sites, three-barriers" model of the apical K+ channel in frog skin.     

Multi-ion conduction and selectivity in the high-conductance Ca++-activated K+ channel from skeletal muscle.

Open-channel ion permeation properties were investigated for Ca++-activated K+ (CaK) channels in solutions of K+ and its analogues T1+, Rb+, and NH4+. Single CaK channels were inserted into planar lipid bilayers composed of neutral phospholipids, and open-channel current-voltage (I-V) relations were measured in symmetrical and asymmetrical solutions of each of these individual ions. For all concentrations studied, the zero-voltage conductance falls in the sequence K+ greater than T1+ greater than NH4+ greater than Rb+. The shape of the I-V curve in symmetrical solutions of a single permeant ion is non-ohmic and is species-dependent. The I-V shape is sublinear for K+ and T1+ and superlinear for Rb+ and NH4+. As judged by reversal potentials under bi-ionic conditions with K+ on one side of the bilayer and the test cation on the other, the permeability sequence is T1+ greater than K+ greater than Rb+ greater than NH4+ at 300 mM, which differs from the conductance sequence. Symmetrical mixtures of K+ or NH4+ with Rb+ show a striking anomalous mole fraction behavior, i.e., a minimum in single-channel conductance when the composition of a two-ion mixture is varied at constant total ion concentration. This result is incompatible with present models that consider the CaK channel a single-ion pore. In total, the results show that the CaK channel finely discriminates among K+-like ions, exhibiting different energy profiles among these species, and that several such ions can reside simultaneously within the conduction pathway.     

Effects of ATP and protons on the Na : K selectivity of the (Na+ + K+)-ATPase studied by ligand effects on intrinsic and extrinsic fluorescence

The effect of pH and of ATP on the Na : K selectivity of the (Na+ + K+)-ATPase has been tested under equilibrium conditions. The Na+ : K+-induced change in intrinsic tryptophan fluorescence and in fluorescence of eosin maleimide bound to the system has been used as a tool. 1 mol of eosin maleimide per mol of enzyme gives no loss in either ATPase or phosphatase activity and the fluorescence in the presence of Na+ is about 30% higher than in the presence of K+. Choline, protonated Tris, protonated histidine and Mg2+ have an 'Na+' effect on the extrinsic fluorescence, while Rb+, Cs+ and NH4+ have a 'K+' effect. Choline and protonated Tris have an Na+ effect on intrinsic fluorescence. A close correlation between the effect of Na+ compared to K+ on the fluorescence change and on Na+ activation of hydrolysis indicates that the observed changes in fluorescence are due to an effect of Na+ and of K+ on the internal sites of the system. The equilibrium between the two conformations, which are reflected by the difference in fluorescence with Na+ and K+, respectively, is highly influenced by the concentration of protons. At a given Na+ : K+ ratio, an increase in the proton concentration shifts the equilibrium towards the 'K+' fluorescence form while a decrease shifts the equilibrium towards the 'Na+' fluorescence form, i.e., protons increase the apparent affinity for K+ and vice versa, K+ increases pK values of importance for the Na+ : K+ selectivity. Conversely, a decrease in protons increases the apparent affinity for Na+ and vice versa, Na+ decreases the pK. ATP decreases the apparent pK for the protonation-deprotonation, i.e., ATP facilitates the deprotonation which accompanies Na+ binding. The results suggest two effects of ATP for the hydrolysis in the presence of Na+ and K+ : (i) at low ATP concentrations (K0.5 < 10 microM) on the K+-Na+ exchange on the internal sites and (ii) at higher, substrate, concentrations on the activation by K+ on the external sites.     

Passive transport of Rb+ by hog gastric (H+,K+)-ATPase

Gastric vesicles enriched in (H+,K+)-ATPase were prepared from hog fundic mucosa and studied for their ability to transport K+ using 86Rb+ as tracer. In the absence of ATP, the vesicles elicited a rapid uptake of 86Rb+ (t 1/2 = 45 +/- 9 s at 30 degrees C) which accounted for both transport and binding. Transport was osmotically sensitive and was the fastest phase. It was not limited by anion permeability (C1- was equivalent to SO2-4) but rather by availability of either H+ or K+ as intravesicular countercation suggesting a Rb+-K+ or a Rb+-H+ exchange. Selectivity was K+ greater than Rb+ greater than Cs+ much greater than Na+,Li+. The capacity of vesicles which catalyzed the fast transport of K+ was 83 +/- 4% of maximal vesicular capacity of the fraction. Addition of ATP decreased both rate and extent of 86Rb+ uptake (by 62 and 43%, respectively with 1 mM ATP) with an apparent Ki of 30 microM. Such an effect was not seen on 22Na+ transport. ATP inhibition of transport did not require the presence of Mg2+, and inhibition was also produced by ADP even in the presence of myokinase inhibitor. On the other hand, 86Rb+ uptake was as strongly inhibited by 200 microM vanadate in the presence of Mg2+. Efflux studies suggested that ATP inhibition was originally due to a decrease of vesicular influx with little or no modification of efflux. Since ATP, ADP, and vanadate are known modulators of the (H+,K+)-ATPase, we propose that, in the absence of ATP, (H+,K+)-ATPase passively exchanges K+ for K+ or H+ and that ATP, ADP, and vanadate regulate this exchange.     

Conduction properties of the cloned Shaker K+ channel.

The conduction properties of the cloned Shaker K+ channel were studied using electrophysiological techniques. Single channel conductance increases in a sublinear manner with symmetric increases in K+ activity, reaching saturation by 0.6 M K+. The Shaker K+ channel is highly selective among monovalent cations; under bi-ionic conditions, its selectivity sequence is K+ > Rb+ > NH+4 > Cs+ > Na+, whereas, by relative conductance in symmetric solutions, it is K+ > NH+4 > Rb+ > Cs+. In Cs+ solutions, single channel currents were too small to be measured directly, so nonstationary fluctuation analysis was used to determine the unitary Cs+ conductance. The single channel conductance displays an anomalous molefraction effect in symmetric mixtures of K+ and NH+4, suggesting that the conducting pore is occupied by multiple ions simultaneously.     

Ligand binding to (Na,K)-ATPase labeled with 5-iodoacetamidofluorescein

The equilibrium binding of sodium, potassium, and adenine nucleotides to dog kidney (Na,K)-ATPase was studied by measuring changes in the fluorescence of enzyme labeled with 5-iodoacetamidofluorescein (5-IAF). The intensity of the fluorescence emission at 520 nm of the bound fluorescein (excited at 490 nm) is increased by ATP, adenyl-5'-yl imidodiphosphate (AMP-PNP), ADP (but not AMP), and Na+, and decreased by K+, Rb+, NH+4, and LI+. Thus the fluorescence effects correlate with the ability of these groups of ligands to stabilize E1 and E2 conformations, respectively. The Na+-induced increase in fluorescence has two components: a slow, high-affinity increase of approximately 7% (K0.5 = 0.16 mM) with positive cooperativity; and a large (approximately 15%), rapid, low-affinity (K0.5 = 34 mM) increase that is not cooperative. The K0.5 for the high-affinity effect is decreased by oligomycin and increased by K+. ATP effects on the fluorescence follow Michaelis-Menten kinetics and are of high affinity (K0.5 = 0.12 microM); K+ increases the K0.5 for ATP, AMP-PNP, and ADP but does not induce cooperative behavior. K+ itself decreases the fluorescence signal by about 9%, with high affinity (K0.5 = 5 microM), showing Michaelis-menten behavior in the absence of other ligands, while with ATP, Na+, or Mg2+ present, K+ effects are cooperative and of lower affinity.     

The effects of ammonium, inorganic phosphate and potassium ions on the activity of phosphofructokinases from muscle and nervous tissues of vertebrates and invertebrates.

1. The effect of NH4+, Pi and K+ on phosphofructokinase from muscle and nervous tissues of a large number of animals was investigated. The activation of the enzyme from lobster abdominal muscle by NH4+ was increased synergistically by the presence of Pi or SO4(2-). In the absence of K+, NH4+ plus Pi markedly activated phosphofructokinase from all tissues studied. In the presence of 100 mM-K+, NH4+ plus Pi activated phosphofructokinase from nervous tissue and muscle of invertebrates and the enzyme from brain of vertebrates, but there was no effect of NH4+ plus Pi on the enzyme from the muscles of vertebrates. Nonetheless, NH4+ plus Pi increased the activity of vertebrate muscle phosphofructokinase in the presence of 50 mM-K+ at inhibitory concentrations of ATP, i.e. these ions de-inhibited the enzyme. In the absence of NH4+ plus Pi, K+ activated phosphofructokinase from vertebrate tissues at non-inhibitory ATP concentrations, but the effect was less marked with the enzyme from invertebrate tissues. Indeed, high concentrations of K+ (greater than 50 mM) caused inhibition of invertebrate tissue phosphofructokinase. Of the other alkali-metal ions tested, only Rb+ activated phosphofructokinase from lobster abdominal muscle and rat heart muscle. 2. The properties of lobster abdominal-muscle phosphofructokinase were studied in detail. This muscle was chosen as representative of invertebrate muscle because large quantities of tissue could be obtained from one animal and the enzyme was considerably more stable in tissue extracts than in extracts of insect flight muscle. In general, the properties of the enzyme from this tissue were similar to those of the enzyme from many other tissues: ATP concentrations above an optimum value inhibited the enzyme and this inhibition was decreased by raising the fructose 6-phosphate or the AMP concentration. In particular, NH4+ plus Pi activated the enzyme at noninhibitory concentrations of ATP and they also relieved ATP inhibition (see above). 3. It is suggested that increases in the concentration of NH4+ and Pi, under conditions of increased ATP utilization in certain muscles and/or nervous tissue, may play a part in the stimulation of glycolysis through the effects on phosphofructokinase (the effect may be a direct activation and/or a relief of ATP inhibition). Changes in the concentration of NH4+ and Pi are consistent with this theory in nervous tissue and the anaerobic type of muscles. The role of AMP deaminase in production of NH4+ from AMP in these tissues is discussed in relation to the control of glycolysis.     

Role of monovalent cations in reactions catalyzed by glycerol dehydrase from Aerobacter aerogenes]

Cobamide-dependent glyceroldehydrase (GDH) is shown to have an absolute requirement in monovalent cations: K+, NH4+, Tl+, Rb+ and Cs+. Dependencies of initial dehydratation rates of three substrates: glycerol, ethyleneglycol and 1,2-propandiol on the concentration of K+ are studied. Km values for K+, NH4+ and Tl+ are calculated to be 7-10-3, 4-10-3 and 1-10-3 M respectively. Effect of K+ on Km values for glycerol and coenzyme and on maximal reaction rate is investigated. It is shown that the apparent affinity of the substrate to the enzyme does not depend on monovalent cation; the apparent affinity of the coenzyme somewhat changes with the change of K+ concentration. Maximal reaction rate increases with the increase of K+ content. On the basis of kinetic data obtained possible mechanism of the activating effect of monovalent cations in reactions, catalyzed by GDH, is discussed.     

Properties of the conversion of an enzyme-ATP complex to a phosphorylated intermediate in the reaction of Na+-K+-dependent ATPase1.

Previously, we proposed the following reaction machanism for the transport ATPase (EC 3.6.1.3) reaction in the presence of high concentrations of Mg2+ and Na+:(see article). Some kinetic and thermodynamic properties of steps 3 and 4 were investigated, and the following results were obtained. 1. When the reaction was started by adding ATP to the enzyme in the presence of 50 mM Na+ and 0.5 mM K+ or in the presence of 50mM Na+ and 0.5mM Rb+, the amount of E ADP P increased with time and maintained a constant level after reaching a maximum. We could not observe the initial burst of EP formation, which was observed by Post er al. in the presence of 8 mM Na+ and 0.01 mM Rb+. 2. The existence of quasi-equilibrium between E2ATP and E ADP P in the presence of low concentrations of Na+ was suggested by the fact that the values of the reciprocal of the equilibrium constant, K3 of step 3 obtained by the following three methods were almost the same. a) The value of 1+K3 was estimated from the ratio of vo/[EP] to kd, where vo is the rate of ATP hydrolysis in the steady state, [EP] the concentration of EP, and kd the first-order rate constant of EP disappearance after stopping EP formation. b) This value was also calculated from the ratio of the amount of P1 liberated to that of decrease in EP after stopping EP formation. c) The value of K3 was also calculated from the initial rapid decrease in EP on adding K+ and EDTA, assuming that the rapid decrease was due to a shift of the equilibrium toward E2ATP on adding K+. For example, the value of K3 with 10mM NaCL and 0.5mM KCL was 7--11. Although ATP formation due to a shift of the equilibrium toward E2ATP by a K+ jump in the presence of a low concentration of Na+ was observed at 0 degrees, the amount of ATP formed by a K+ jump at 15 degrees was less than the value expected from the shift of the equilibrium. 3. The values of delta H degrees and delta S degrees of step 3 were estimated in the presence of a sufficient amount of Na+ and in the absence of K+. They were +4--+5 kcal mole minus 1 and +15--+16 entropy units mole minus1, respectively. On the basis of kinetic studies of the elementary steps and the overall reaction of Na+-K+-dependent ATPase [EC 3.6.1.3], we (1--4) showed that a phosphorylated intermediate, EP, is formed via two kinds of enzyme-substrate complex, E1ATP and E2ATP, that the EP is in K+-dependent quasi-equilibrium with E2ATP, and that in the presence of high concentration of Mg2+, EP is in a high-energy state and contains bound ADP, E ADP P.(see article).     

The multi-ion nature of the pore in Shaker K+ channels.

We have investigated some of the permeation properties of the pore in Shaker K channels. We determined the apparent permeability ratio of K+, Rb+, and NH4+ ions and block of the pore by external Cs+ ions. Shaker channels were expressed with the baculovirus/Sf9 expression system and the channel currents measured with the whole-cell variant of the patch clamp technique. The apparent permeability ratio, PRb/PK, determined in biionic conditions with internal K+, was a function of external Rb+ concentration. A large change in PRb/PK occurred with reversed ionic conditions (internal Rb+ and external K+). These changes in apparent permeability were not due to differences in membrane potential. With internal K+, PNH4/PK was not a function of external NH4+ concentration (at least over the range 50-120 mM). We also investigated block of the pore by external Cs+ ions. At a concentration of 20 mM, Cs+ block had a voltage dependence equivalent to that of an ion with a valence of 0.91; this increased to 1.3 at 40 mM Cs+. We show that a 4-barrier, 3-site permeation model can simulate these and many of the other known properties of ion permeation in Shaker channels.     

Caclium uptake and associated adenosine triphosphatase activity in fragmented sarcoplasmic reticulum. Requirement for potassium ions.

The effects of monovalent cations on calcium uptake by fragmented sarcoplasmic reticulum have been clarified. Homogenization of muscle tissue in salt-containing solutions leads to contamination of this subcellular fraction with actomyosin and mitochondrial membranes. When, in addition, inorganic cations are contributed by the microsomal suspension and in association with nucleotide triphosphate substrates there is an apparent inhibition of the calcium transport system by potassium and other cations. However, when purified preparations were obtained after homogenization in sucrose medium followed by centrifugation on a sucrose density gradient in a zonal rotor, calcium uptake and the associated adenosine triphosphatase activity were considerably activated by potassium and other univalent cations. When plotted against the log of the free calcium concentration there was only a slight increase in calcium uptake and ATPase activity in the absence of potassium ions but sigmoid-shaped curves were obtained in 100 mM K+ with half-maximal stimulation occurring at 2 muM Ca2+ for both calcium uptake and ATPase activity. The augmentation in calcium uptake was not due to an ionic strength effect as Tris cation at pH 6.6 was shown to be inactive in this respect. Other monovalent cations were effective in the order K+ greater than Na+ greater than NH4+=Rb+=Cs+ greater than Li+ with half-maximal stimulation in 11 mM K+, 16 mM Na+, 25 mM NH4+, Rb+, and Cs+ and in 50 mM Li+. There was nos synergistic action between K+ AND Na+ ions and both calcium uptak and associated ATPase were insensitive to ouabain. Thallous ions stimulate many K+-requiring enzymes and at one-tenth the concentration were nearly as effective as K+ ions in promoting calcium uptake. The ratio of Ca2+ ions transported to P1 released remained unchanged at 2 after addition of K+ ions indicating an effect on the rate of calcium uptake rather than an increased efficiency of uptake. In support of this it was found that during the stimulation of calcium uptake by Na+ ions there was a reduction in the steady state concentration of phosphorylated intermediate formed from [gamma-32P]ATP. It is considered that there is a physiological requirement for potassium ions in the relaxation process.