Response of rat liver glutaminase to pH, ammonium, and citrate. Possible regulatory role of glutaminase in ureagenesis

Liver glutaminase is stimulated by an increase in NH4+ concentration and NH4+ is an absolute requirement for activity at approximate physiological concentrations of phosphate and glutamine. Increases in the concentration of NH4+ cannot, however, overcome the inhibitory effect of a decrease in pH. In addition, the concentration of NH4+ required for half-maximal rate decreases as pH increases. This decrease is the result of two factors: a direct effect of pH on the apparent affinity of the enzyme for NH4+, and an indirect effect of pH brought about by an increase in the apparent affinity of the enzyme for phosphate which results in a further decrease in the M0.5 for NH4+. In addition, liver glutaminase responds strongly to the concentration of citrate over a physiologically relevant range at approximate physiological concentrations of NH4+, phosphate, and glutamine. An increase in citrate concentration stimulates glutaminase by increasing the affinity of the enzyme for glutamine. The apparent affinity of the enzyme for citrate increases as pH increases. The strong response of liver glutaminase to pH, NH4+, and citrate and the fact that the hydrolysis of glutamine can supply metabolites and effectors for urea synthesis suggest a possible regulatory role of glutaminase in ureagenesis.     

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.     

Regulation of K+ transport in tomato roots by the TSS1 locus. Implications in salt tolerance

The tss1 tomato (Lycopersicon esculentum) mutant exhibited reduced growth in low K+ and hypersensitivity to Na+ and Li+. Increased Ca2+ in the culture medium suppressed the Na+ hypersensitivity and the growth defect on low K+ medium of tss1 seedlings. Interestingly, removing NH4+ from the growth medium suppressed all growth defects of tss1, suggesting a defective NH4(+)-insensitive component of K+ transport. We performed electrophysiological studies to understand the contribution of the NH4(+)-sensitive and -insensitive components of K+ transport in wild-type and tss1 roots. Although at 1 mm Ca2+ we found no differences in affinity for K+ uptake between wild type and tss1 in the absence of NH4+, the maximum depolarization value was about one-half in tss1, suggesting that a set of K+ transporters is inactive in the mutant. However, these transporters became active by raising the external Ca2+ concentration. In the presence of NH4+, a reduced affinity for K+ was observed in both types of seedlings, but tss1 at 1 mm Ca2+ exhibited a 2-fold higher Km than wild type did. This defect was again corrected by raising the external concentration of Ca2+. Therefore, membrane potential measurements in root cells indicated that tss1 is affected in both NH4(+)-sensitive and -insensitive components of K+ transport at low Ca2+ concentrations and that this defective transport is rescued by increasing the concentration of Ca2+. Our results suggest that the TSS1 gene product is part of a crucial pathway mediating the beneficial effects of Ca2+ involved in K+ nutrition and salt tolerance.     

Features of the effect of ammonium ion on the enzymatic activity of myosin and subfragment-1]

The kinetic isotope effect of hydrolysis of ATP by myosin subfragment-I in the presence of K+, NH4+, Rb+ was measured. VH/VD was found to be 1.8; 1.3; 2.0, respectively. According to the thermodynamic isotope effect induced by hydration, the kinetic isotope effect must increase with the increase of cation size from K+ to Rb+. The size of ammonium ions is the intermediate between K+ and Rb+, but the observed isotope effect in the presence of ammonium is much lower than in the presence of K+ and Rb+. The results suggest that monovalent cations occur near the active center of the enzyme and contribute to some extent to the hydrolysis reaction but the specificity of ammonium ions seems not to be due to its ideal steric accordance. The obtained results support the viewpoint that NH4+ ions as donor of protons participate in the chemical stage of ATP hydrolysis by subfragment-I.     

Ammonium effects on colonic Cl- secretion: anomalous mole fraction behavior

A significant amount of ammonium (NH4+) is absorbed by the colon. The nature of NH4+ effects on transport and NH4+ transport itself in colonic epithelium is poorly understood. The goal of this study was to elucidate the effects of NH4+ on cAMP-stimulated Cl- secretion in the colonic cell line T84. In HEPES-buffered solutions, application of basolateral NH4+ resulted in a reduced level of Cl- secretory current. The effect of NH4+ appears to occur by at least three mechanisms: 1) basolateral membrane depolarization, 2) a competitive effect with K+, and 3) a long-term (>20 min) increase in transepithelial resistance (TER). The competitive effect with K+ exhibits anomalous mole fraction behavior. Transepithelial current relative to that in 10 mM basolateral K+ was inhibited 15% by 10 mM NH4+ alone and by 30% with a mixture of 2 mM K+ and 8 mM NH4+. A mole fraction mix of 2 mM K+:8 mM NH4+ produced a greater inhibition of basolateral membrane K+ current than pure K+ or NH4+ alone. Similar anomalous behavior was also observed for inhibition of bumetanide-sensitive 36Cl- uptake, e.g., Na+-K+-2Cl- -cotransporter (NKCC-1). No anomalous effect was observed on Na+-K+-ATPase current. Both NKCC-1 and Na+-K+-ATPase activity were elevated in 10 mM NH4+ with respect to 10 mM K+. The effect on TER did not exhibit anomalous mole fraction behavior. The overall effect of basolateral NH4+ on cAMP-stimulated transport is dependent on the [K+]o /[NH4+]o ratio at the basolateral membrane, where o is outside of the cell.     

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.     

Anomalous mole fraction effect induced by mutation of the H5 pore region in the Shaker K+ channel.

Mutagenesis of the H5 region of the Shaker K+ channel has provided strong evidence that these amino acids form a major portion of the ionic pore. We have previously observed that a single-site mutation (T441S) in this region increased the apparent relative permeability of the channel to NH4+. We now report that this increased relative permeability to NH4+ is sensitive to small changes in external K+ in a pattern consistent with an anomalous mole fraction effect. The effect is not apparent in the wild-type channel. These findings, in combination with other studies showing effects of this particular mutation on the binding of tetraethylammonium and hydroxylamine, support the hypothesis that T441S alters the affinity of a putative ion binding site for NH4+ and ammonium derivatives. The mutation T441S alters ionic selectivity and reveals the multi-ion nature of the mutant Shaker K+ channel.     

The regulatory properties of yeast pyruvate kinase. Effects of NH4+ and K+ concentrations.

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied at 25 degrees C and pH 6.2 as a function of the concentrations of ADP, phosphoenolpyruvate, Mg2+ and either NH4+ or K+. The data were analysed by the exponential model for four substrates, obtained by extension of the model described by Ainsworth, Kinderlerer & Gregory [(1983) Biochem. J. 209, 401-411]. On that basis, it was concluded that NH4+ binding is almost non-interactive but leads to the appearance of positive interaction in the velocity response to increase in its concentration because of positive interactions with phosphoenolpyruvate and Mg2+. The data obtained with K+ lead to the same conclusions and differ only in suggesting that NH4+ is bound more strongly to the enzyme than is K+. Both data sets are used as the basis for a discussion of the substrate interactions of pyruvate kinase and it appears therefrom that the heterotropic interactions accord with what is known of the events that take place at the active site during catalysis. The paper also reports a determination of the dissociation constants for the NH4+ complexes with ADP and phosphoenolpyruvate and an examination of the simultaneous activation of pyruvate kinase by K+ and NH4+ ions.     

Interactions between K+ and ATP binding to the (Na+ + K+)-dependent ATPase.

K+ appears to decrease the affinity of the (Na+ + K+)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) for its substrate, Mg2+ - ATP, and Mg2+ - ATP, in turn, appears to decrease the affinity of the enzyme for K+. These antagonisms have been investigated in terms of a quantitative model defining the magnitude of the effects as well as identifying the class of K+ sites on the enzyme involved. K+ increased the apparent Km for Mg2+ - ATP, an effect that was antagonized competitively by Na+. The data can be fitted to a model in which Mg2+ - ATP binding is prevented by occupancy of alpha-sites on the enzyme by K+ (i.e. sites of moderate affinity for K+ accessible on the "free" non-phosphorylated enzyme, in situ on the external membrane surface). By contrast, occupancy of these alpha-sites by Na+ has no effect on Mg2+ - ATP binding to the enzyme. On the other hand, Mg2+ - ATP decreased the apparent affinity of the enzyme for K+ at the alpha-sites, in terms of (i) the KD for K+ measured by K+-accelerated inactivation of the enzyme by F-, and (ii) the concentration of K+ for half-maximal activation of the K+-dependent phosphatase reaction (which reflects the terminal hydrolytic steps of the overall ATPase reaction). These data fit the same quantitative model. Although this formulation does not support schemes in which ATP binding effects the release of transported K+ from discharge sites, it is consistent with observations that K+ can inhibit the enzyme at low substrate concentrations, and that Li+, which has poor efficacy when occupying these alpha-sites, can stimulate enzymatic activity at high K+ concentrations by displacing the inhibitory K+.     

The effect of cytoplasmic K+ on the activity of the Na+/K(+)-ATPase.

Experiments with the reconstituted (Na+ + K+)-ATPase show that besides the ATP-dependent cytoplasmic Na(+)-K+ competition for Na+ activation there is a high affinity inhibitory effect of cytoplasmic K+. In contrast to the high affinity K+ inhibition seen with the unsided preparation at a low ATP especially at a low temperature, the high affinity inhibition by cytoplasmic K+ does not disappear when the ATP concentration an-or the temperature is increased. The high affinity inhibition by cytoplasmic K+ is also observed with Cs+, Li+ or K+ as the extracellular cation, but the fractional inhibition is much less pronounced than with Na+ as the extracellular cation. The results suggest that either there are two populations of enzyme, one with the normal ATP dependent cytoplasmic Na(+)-K+ competition, and another which due to the preparative procedure has lost this ATP sensitivity. Or that the normal enzyme has two pathways for the transition from E2-P to E1ATP. One on which the enzyme with the translocated ion binds cytoplasmic K+ with a high affinity but not ATP, and another on which ATP is bound but not K+. A kinetic model which can accommodate this is suggested.     

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.     

High-affinity K+ uptake in pepper plants

High-affinity K+ uptake is an essential process for plant nutrition under K+-limiting conditions. The results presented here demonstrate that pepper (Capsicum annuum) plants grown in the absence of NH4+ and starved of K+ show an NH4+-sensitive high-affinity K+ uptake that allows plant roots to deplete external K+ to values below 1 microM. When plants are grown in the presence of NH4+, high-affinity K+ uptake is not inhibited by NH4+. Although NH4+-grown plants deplete external K+ below 1 microM in the absence of NH4+, when 1 mM NH4+ is present they do not deplete external K+ below 10 microM. A K+ transporter of the HAK family, CaHAK1, is very likely mediating the NH4+-sensitive component of the high-affinity K+ uptake in pepper roots. CaHAK1 is strongly induced in the roots that show the NH4+-sensitive high-affinity K+ uptake and its induction is reduced in K+-starved plants grown in the presence of NH4+. The NH4+-insensitive K+ uptake may be mediated by an AKT1-like K+ channel.     

Effects of sterol manipulation on microsomal desaturase activities in Tetrahymena: with regard to thermal acclimation

The role of NH+4 ion and AMP deaminase reaction in the activation of phosphofructokinase with respect to its response to the adenylate energy charge was investigated using permeabilized yeast cells. (a) Phosphofructokinase and AMP deaminase were activated by the decrease in the adenylate energy charge. The addition of NH+4 further stimulated the phosphofructokinase activity in the presence of intracellular level of K+, and the optimal energy charge value giving the maximal response of the enzyme was shifted from 0.3 to the value above 0.5. (b) The increase in NH+4 ion produced through the activation of AMP deaminase by spermine which shows no direct action on the phosphofructokinase activity can activate phosphofructokinase with shift of the optimal energy charge value of the enzyme to 0.5 in the presence of K+, whereas the optimal energy charge value for AMP deaminase reaction was not affected by the addition of spermine. Phosphofructokinase can be activated most effectively by the physiological decrease in the energy charge under the condition of increased NH+4 in the presence of K+. The possibility that the interaction of phosphofructokinase with AMP deaminase under hypoxic condition might be a contributing factor to the Pasteur effect is discussed.     

The non-gastric H,K-ATPase is oligomycin-sensitive and can function as an H+,NH4(+)-ATPase

We used the baculovirus/Sf9 expression system to gain new information on the mechanistic properties of the rat non-gastric H,K-ATPase, an enzyme that is implicated in potassium homeostasis. The alpha2-subunit of this enzyme (HKalpha2) required a beta-subunit for ATPase activity thereby showing a clear preference for NaKbeta1 over NaKbeta3 and gastric HKbeta. NH4(+), K+, and Na+ maximally increased the activity of HKalpha2-NaKbeta1 to 24.0, 14.2, and 5.0 micromol P(i) x mg(-1) protein x h(-1), respectively. The enzyme was inhibited by relatively high concentrations of ouabain and SCH 28080, whereas it was potently inhibited by oligomycin. From the phosphorylation level in the presence of oligomycin and the maximal NH4(+)-stimulated ATPase activity, a turnover number of 20,000 min(-1) was determined. All three cations decreased the steady-state phosphorylation level and enhanced the dephosphorylation rate, disfavoring the hypothesis that Na+ can replace H+ as the activating cation. The potency with which vanadate inhibited the cation-activated enzyme decreased in the order K+ > NH4(+) > Na+, indicating that K+ is a stronger E2 promoter than NH4(+), whereas in the presence of Na+ the enzyme is in the E1 form. For K+ and NH4(+), the E2 to E1 conformational equilibrium correlated with their efficacy in the ATPase reaction, indicating that here the transition from E2 to E1 is rate-limiting. Conversely, the low maximal ATPase activity with Na+ is explained by a poor stimulatory effect on the dephosphorylation rate. These data show that NH4(+) can replace K+ with similar affinity but higher efficacy as an extracellular activating cation in rat nongastric H,K-ATPase.     

Conformational changes required for pyruvate kinase activity as modulated by monovalent cations.

The interaction of a series of alkylamines with muscle pyruvate kinase was investigated by kinetic and physical studies in order to understand the mechanisms by which certain monovalent cations can activate the enzyme and to define several of the important conformational changes necessary for catalytic activity. Monomethylammonium ion interacts with pyruvate kinase to activate the enzyme. Dimethyland trimethylammonium ions do not activate, but are competitive inhibitors against activating cations. Tetramethylammonium ion neither activates nor inhibits pyruvate kinase activity. When the enzyme is in the presence of monomethylammonium ion or dimethylammonium ion, a conformational change is observed by ultraviolet difference spectroscopy. This conformational change is similar to that observed with other activating cations and appears to be a necessary but no sufficient conformational change in the formation of an active complex. The interaction of the substrate phosphoenolpyruvate with the pyruvate kinase-Mn2+ complex in the presence of these cations was studied by water proton relaxation rate measurements. The affinity of the enzyme-Mn2+ complex for phosphoenolpyruvate is decreased by a factor of 5 in the presence of any of the alkylamines compared to the affinity measured in the presence of K+ or NH4+. No change in the Km of phosphoenolpyruvate is observed however when it is measured in the presence of monomethylammonium ion, suggesting that the decrease in affinity for the substrate is not the reason for lack of enzymic activity. The conformation of the ternary enzyme-Mn2+-phosphoenolpyruvate complex about the bound Mn2+, as reflected by the enhancement values (epsilont) measured, differs depending upon the nature of the monovalent cation. The epsilon t values measured in the presence of the alkylamines are larger (epsilont - 5.7 +/- 0.2) than those measured in the presence of K+ or NH4+ (epsilont = 1.9 +/- 0.1).     

Brain (Na+,K+)-ATPase. Opposite effects of ethanol and dimethyl sulfoxide on temperature dependence of enzyme conformation and univalent cation binding

We examined effects of ethanol and dimethyl sulfoxide on the regulation and apparent thermodynamic properties of moderate affinity Na+ and K+ binding that regulates the K+-dependent phosphatase activity of (Na+,K+)-ATPase. Ethanol and other alcohols reduced the apparent affinity for Na+ and K+ at their moderate affinity sites and increased the negative delta H and delta S of cation binding. Dimethyl sulfoxide had the opposite effects. Inhibition by ethanol was favored by high temperature or low K+. Ethanol potentiated inhibition of K+ binding by ATP or Mg2+. Ethanol also shifted the equilibrium between K+-sensitive and -insensitive forms of (Na+,K+)-ATPase toward the K+-sensitive form; in this case, it reduced the negative delta H and delta S for the transition to K+-sensitive enzyme. Again, dimethyl sulfoxide had the opposite effects. These data indicate that ethanol and other agents considered to affect membrane fluidity act by a combination of membrane (on cation binding) and solvent (on conformation) effects. The most important effect of ethanol and similar agents on the enzyme is to prevent the formation of K+-sensitive enzyme by cation binding and to destabilize K+-sensitive enzyme in the presence of ATP. These results also add further evidence that the sites by which Na+ and K+ produce K+-sensitive enzyme are similar or identical.     

Low-affinity Na+ sites on (Na+ +K+)-ATPase modulate inhibition of Na+-ATPase activity by vanadate

Na+-ATPase activity is extremely sensitive to inhibition by vanadate at low Na+ concentrations where Na+ occupies only high-affinity activation sites. Na+ occupies low-affinity activation sites to reverse inhibition of Na+-ATPase and (Na+, K+)-ATPase activities by vanadate. This effect of Na+ is competitive with respect to both vanadate and Mg2+. The apparent affinity of the enzyme for vanadate is markedly increased by K+. The principal effect of K+ may be to displace Na+ from the low-affinity sites at which it activates Na+-ATPase activity.     

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.     

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.     

Preliminary characterization of adenosine deaminase from Bacillus cereus

Adenosine deaminase from Bacillus cereus is quite unstable, similarly to other bacterial deaminases, but it shows a peculiar stabilizing effect by some monovalent cations. These include K+, Li+, NH4+ and to a lesser extent Cs+. Maximal stabilization of the deaminase is exerted by K+ at concentrations higher than 20 mM. The enzyme can be rapidly inactivated by sulphydryl reagents such as p-hydroxymercuribenzoate. Since adenosine deaminase from B. cereus, in addition to monovalent cations, is stabilized also by dithiothreitol, a possible influence of monovalent cations on the reactivity of some sulphydryl groups on the enzyme has been suggested.