Modulation of the protein environment in the hydrophilic pore of the ammonia transporter protein AmtB upon GlnK protein binding

The conduction of ammonia/ammonium (NH3/NH4(+)) through the channel protein AmtB is inhibited by the binding of the signal transduction protein GlnK. In the AmtB-GlnK binding interface, there exists an NH3/NH4(+) binding site--Am6. The calculated pK(a) values at the Am6 sites in both the AmtB-GlnK complex and isolated AmtB implies the dominance of an uncharged NH3 state. The GlnK protein binding causes a significant downshift in the Am6 pK(a) value of the AmtB. However, this downshift is perfectly compensated by the reorientation of the protein backbone (carbonyl group of Cys312 from the AmtB part) upon AmtB-GlnK complex formation.     

Modulation of electroneutral Na transport in sheep rumen epithelium by luminal ammonia

Ammonia is an abundant fermentation product in the forestomachs of ruminants and the intestine of other species. Uptake as NH3 or NH4+ should modulate cytosolic pH and sodium-proton exchange via Na+/H+ exchanger (NHE). Transport rates of Na+, NH4+, and NH3 across the isolated rumen epithelium were studied at various luminal ammonia concentrations and pH values using the Ussing chamber method. The patch-clamp technique was used to identify an uptake route for NH4+. The data show that luminal ammonia inhibits electroneutral Na transport at pH 7.4 and abolishes it at 30 mM (P < 0.05). In contrast, at pH 6.4, ammonia stimulates Na transport (P < 0.05). Flux data reveal that at pH 6.4, approximately 70% of ammonia is absorbed in the form of NH4+, whereas at pH 7.4, uptake of NH3 exceeds that of NH4+ by a factor of approximately four. The patch-clamp data show a quinidine-sensitive permeability for NH4+ and K+ but not Na+. Conductance was 135 +/- 12 pS in symmetrical NH(4)Cl solution (130 mM). Permeability was modulated by the concentration of permeant ions, with P(K) > P(NH4) at high and P(NH4) > P(K) at lower external concentrations. Joint application of both ions led to anomalous mole fraction effects. In conclusion, the luminal pH determines the predominant form of ammonia absorption from the rumen and the effect of ammonia on electroneutral Na transport. Protons that enter the cytosol through potassium channels in the form of NH4+ stimulate and nonionic diffusion of NH3 blocks NHE, thus contributing to sodium transport and regulation of pH.     

Permeability of the urinary bladder of the turtle, Pseudemys scripta elegans, to ammonia

1. The permeability of the isolated turtle urinary bladder to ammonia was investigated by varying the ratio of NH3 to NH4+ concentration in the serosal bath and measuring the flux of ammonia into the mucosal bath. 2. The permeability of the turtle bladder to NH3 was in the range of 5.4-6.1 x 10(-3) cm/sec and the permeability of the unstirred layer to NH4+ was in the range of 1.1-2.1 x 10(-5) cm/sec. 3. The flux of ammonia calculated from the permeability of NH3 can account for the decrease in mucosal H+ secretion observed with addition of NH4Cl to the serosal bath.     

Ammonium Uptake by Rice Roots (II. Kinetics of 13NH4+ Influx across the Plasmalemma)

Short-term influxes of 13NH4+ were measured in intact roots of 3-week-old rice (Oryza sativa L. cv M202) seedlings that were hydroponically grown at 2, 100, or 1000 [mu]M NH4+. Below 1 mM external concentration ([NH4+]0), influx was saturable and due to a high-affinity transport system (HATS). For the HATS, Vmax values were negatively correlated and Km values were positively correlated with NH4+ provision during growth and root [NH4+]. Between 1 and 40 mM [NH4+]0, 13NH4+ influx showed a linear response due to a low-affinity transport system (LATS). The 13NH4+ influxes by the HATS, and to a lesser extent the LATS, are energy-dependent processes. Selected metabolic inhibitors reduced influx of the HATS by 50 to 80%, but of the LATS by only 31 to 51%. Estimated values for Q10 (the ratio of rates at temperatures differing by 10[deg]C) for HATS were greater than 2.4 at root temperatures from 5 to 10[deg]C and were constant at approximately 1.5 between 5 and 30[deg]C for the LATS. Influx of 13NH4+ by the HATS was insensitive to external pH in the range from 4.5 to 9.0, but influx by the LATS declined significantly beyond pH 6.0. The data presented are discussed in the context of the kinetics, energy dependence, and the regulation of ammonium influx.     

Ammonia uptake and its effects on ionoregulation in the freshwater crayfish Pacifastacus leniusculus (Dana)

Exposure of adult crayfish Pacifastacus leniusculus to Artificial Freshwater (AFW) media containing 1.5 m and 0.15 mmol x l(-1) total ammonia [Tamm; 0.1 x acute lethal concentration (24 h LC50) and 0.01 x 24 h LC50] and adjusted to pH 6.5, pH 8.2 and pH 10.5 resulted in significant increases in haemolymph ammonia over a 24-h period. Ammonia accumulated most rapidly at pH 10.5. These media were chosen to expose animals to a range of different un-ionised ammonia (UIA) [NH3] and ionised ammonia [NH4+] concentrations. From comparisons of measured transepithelial potential differences (PDte) with calculated Nernst potentials (PDNH4+) for the known haemolymph-to-medium gradients of [NH4+], it was deduced that, in pH 8.2 and pH 6.5 AFW, NH4+ was not in thermodynamic equilibrium across the integument (presumably gill epithelium). In pH 10.5 AFW with 1.5 mmol x l(-1) Tamm (predominantly NH3), the accumulation of ammonia in the haemolymph was in the NH4+ form due to haemolymph pH regulation by the crayfish in this alkaline external medium. Measured net fluxes of ammonia (Jamm(net)) were inwardly directed and maximal when [NH3] was the main component externally, but were also significant at pH 8.2 with high [NH4+] ([NH4+]:[NH3] approximately 20:1). Haemolymph Na+ depletion was significant and, over the 24-h exposure period, most rapid in high [NH3] medium but [Cl-] was unaffected. However, paradoxically, sodium uptake (measured JNa(in) on immediate transfer to high Tamm medium) was not significantly inhibited when [NH3] was the predominant ammonia species. In 1.5 mmol x l(-1) Tamm (mainly [NH4+), VNa(in) (the active component of JNa(in)) was significantly inhibited, particularly at low external [Na+]. This inhibition could not be demonstrated as one of competition at an Na+/NH4+ apical gill exchange site. The resultant net efflux of sodium from the animal showed that the ability of the animals to balance sodium losses at low external [Na+] was severely affected. Longer exposure to pH 10.5 AFW with high [NH3] (12 h) resulted in significantly increased JNa(out), while not significantly affecting JNa(in). Analysis of urinary Na+ losses showed that, while urinary flow rate and water reabsorption was most likely unaffected by ammonia exposure, final urine [Na+] was significantly elevated. The resulting urinary Na+ loss accounted for 63% of the increased JNa(out) in high [NH3] medium.     

Ammonia Flux between Oilseed Rape Plants and the Atmosphere in Response to Changes in Leaf Temperature,Light Intensity,and Air Humidity (Interactions with Leaf Conductance and Apoplastic NH4+ and H+ Concentrations)

NH3 exchange between oilseed rape (Brassica napus) plants and the atmosphere was examined at realistic ambient NH3 levels under controlled environmental conditions. Different leaf conductances to NH3 diffusion were obtained by changing leaf temperature (10 to 40[deg]C), light intensity (0 to 600 [mu]mol m-2 s-1), and air humidity (20 to 80%), respectively. NH3 adsorption to the cuticle with subsequent NH3 transport through the epidermis had no significant effect on the uptake of atmospheric NH3, even at 80% relative air humidity. NH3 fluxes increased linearly with leaf conductance when light intensities were increased from 0 to 600 [mu]mol m-2 s-1. Increasing leaf temperatures from 10 to 35[deg]C caused an exponential increase in NH3 emission from plants exposed to low ambient NH3 concentrations, indicating that leaf conductance was not the only factor responding to the temperature increase. The exponential relationship between NH3 emission and temperature was closely matched by the temperature dependence of the mole fraction of gaseous NH3 above the leaf apoplast (NH3 compensation point), as calculated on the basis of NH4+ and H+ concentrations in the leaf apoplast at the different leaf temperatures. NH3 fumigation experiments showed that an increase in leaf temperature may cause a plant to switch from being a strong sink for atmospheric NH3 to being a significant NH3 source. In addition to leaf temperature, the size of the NH3 compensation point depended on plant N status and was related to plant ontogeny.     

Planktonic rotifers and temperature

The influence of temperature (t) upon rotifer embryonic development rate (D_e) has been analysed using data from the literature, and the author's own results from experimental and natural populations. For Keratella cochlearis (Gosse), within the temperature range of 1–28°C, this relationship is best expressed by the equation: 1/D_e = 0.002 + 0.00025t + 0.000065t².For Brachionus calyciflorus Pallas, between 8°C and 35°C, the best relationship is given by the equation: 1/D_e = 0.005 + 0.00013t + 0.00013t².Increasing the incubation temperature to 37–40°C resulted in a decrease in development rate and a sharp reduction in life length.Analysis of the relationship between respiration rate and temperature in experimental and natural populations of Brachionus calyciflorus and Hexarthra mira (Hudson) showed that the maximum rate of oxygen consumption occurred at 32–33°C.The effects of temperature upon the ingestion rates of rotifers is greatly influenced by food concentration. Consequently, this factor also influences the secondary production of experimental populations at different temperatures.     

Interaction of external H+ with the Na+-H+ exchanger in renal microvillus membrane vesicles

We examined the effects of external H+ on the kinetics of Na+-H+ exchange in microvillus membrane vesicles isolated from the rabbit renal cortex. The initial rate of Na+ influx into vesicles with internal pH 6.0 was optimal at external pH 8.5 and was progressively inhibited as external pH was reduced to 6.0. A plot of 1/V versus [H+]o was linear and yielded apparent KH = 35 nM (apparent pK 7.5). In vesicles with internal pH 6.0 studied at external pH 7.5 or 6.6, apparent KNa was 13 or 54 mM, Ki for inhibition of Na+ influx by external Li+ was 1.2 or 5.2 mM, Ki for inhibition by external NH4+ was 11 or 50 mM, and Ki for inhibition by external amiloride was 7 or 25 microM, respectively. These findings were consistent with competition between each cation and H+ at a site with apparent pK 7.3-7.5. Lastly, stimulation of 22Na efflux by external Na+ (i.e. Na+-Na+ exchange) was inhibited as external pH was reduced from 7.5 to 6.0, also consistent with competition between external H+ and external Na+. Thus, in contrast with internal H+, which interacts at both transport and activator sites, external H+ interacts with the renal microvillus membrane Na+-H+ exchanger at a single site, namely the external transport site, where H+, Na+, Li+, NH4+, and amiloride all compete for binding.     

K+ and NH4(+) modulate gill (Na+, K+)-ATPase activity in the blue crab, Callinectes ornatus: fine tuning of ammonia excretion

To better comprehend the mechanisms of ionic regulation, we investigate the modulation by Na+, K+, NH4(+) and ATP of the (Na+, K+)-ATPase in a microsomal fraction from Callinectes ornatus gills. ATP hydrolysis obeyed Michaelis-Menten kinetics with KM=0.61+/-0.03 mmol L(-1) and maximal rate of V=116.3+/-5.4 U mg(-1). Stimulation by Na+ (V=110.6+/-6.1 U mg(-1); K0.5=6.3+/-0.2 mmol L(-1)), Mg2+ (V=111.0+/-4.7 U mg(-1); K0.5=0.53+/-0.03 mmol L(-1)), NH4(+) (V=173.3+/-6.9 U mg(-1); K0.5=5.4+/-0.2 mmol L(-1)) and K+ (V=116.0+/-4.9 U mg(-1); K0.5=1.5+/-0.1 mmol L(-1)) followed a single saturation curve, although revealing site-site interactions. In the absence of NH4(+), ouabain (K(I)=74.5+/-1.2 micromol L(-1)) and orthovanadate inhibited ATPase activity by up to 87%; the inhibition patterns suggest the presence of F0F1 and K+-ATPases but not Na+-, V- or Ca2+-ATPase as contaminants. (Na+, K+)-ATPase activity was synergistically modulated by K+ and NH4(+). At 10 mmol L(-1) K+, increasing NH4(+) concentrations stimulated maximum activity to V=185.9+/-7.4 U mg(-1). However, at saturating NH4(+) (50 mmol L(-1)), increasing K+ concentrations did not stimulate activity further. Our findings provide evidence that the C. ornatus gill (Na+, K+)-ATPase may be particularly well suited for extremely efficient active NH4(+) excretion. At elevated NH4(+) concentrations, the enzyme is fully active, regardless of hemolymph K+ concentration, and K+ cannot displace NH4(+) from its exclusive binding sites. Further, the binding of NH4(+) to its specific sites induces an increase in enzyme apparent affinity for K+, which may contribute to maintaining K+ transport, assuring that exposure to elevated ammonia concentrations does not lead to a decrease in intracellular potassium levels. This is the first report of modulation by ammonium ions of C. ornatus gill (Na+, K+)-ATPase, and should further our understanding of NH4(+) excretion in benthic crabs.     

Rat liver alcohol dehydrogenase. I. Purification and characterization

Alcohol dehydrogenase was purified in 14 h from male Fischer-344 rat livers by differential centrifugation, (NH4)2SO4 precipitation, and chromatography over DEAE-Affi-Gel Blue, Affi-Gel Blue, and AMP-agarose. Following HPLC more than 240-fold purification was obtained. Under denaturing conditions, the enzyme migrated as a single protein band (Mr congruent to 40,000) on 10% sodium dodecyl sulfate-polyacrylamide gels. Under nondenaturing conditions, the protein eluted from an HPLC I-125 column as a symmetrical peak with a constant enzyme specific activity. When examined by analytical isoelectric focusing, two protein and two enzyme activity bands comigrated closely together (broad band) between pH 8.8 and 8.9. The pure enzyme showed pH optima for activity between 8.3 and 8.8 in buffers of 0.5 M Tris-HCl, 50 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), and 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), and above pH 9.0 in 50 mM glycyl-glycine. Kinetic studies with the pure enzyme, in 0.5 M Tris-HCl under varying pH conditions, revealed three characteristic ionization constants for activity: 7.4 (pK1); 8.0-8.1 (pK2), and 9.1 (pK3). The latter two probably represent functional groups in the free enzyme; pK1 may represent a functional group in the enzyme-NAD+ complex. Pure enzyme also was used to determine kinetic constants at 37 degrees C in 0.5 M Tris-HCl buffer, pH 7.4 (I = 0.2). The values obtained were Vmax = 2.21 microM/min/mg enzyme, Km for ethanol = 0.156 mM, Km for NAD+ = 0.176 mM, and a dissociation constant for NAD+ = 0.306 mM. These values were used to extrapolate the forward rate of ethanol oxidation by alcohol dehydrogenase in vivo. At pH 7.4 and 10 mM ethanol, the rate was calculated to be 2.4 microM/min/g liver.     

Untersuchung der Beziehung zwischen extracellulärer Glykolsäure-Ausscheidung und der photosynthetischen CO2-Aufnahme bei der Blaualge Anacystis nidulans

Summary The formations of transients in CO₂ exchange in the blue-green alga Anacystic nidulans is dependent on the temperature used during the measurements. The algae were grown in a low light intensity (4000 lux) under normal air conditions and measured in the same low CO₂ concentration (0.03 vol. %) but under a higher light intensity (10 000 lux). At a temperature of +20°C the stationary rate of CO₂ uptake was reached directly. At a temperature of +35°C, on the other hand, a maximum of CO₂ uptake could be observed at the beginning of the light period followed by a steady rate of photosynthesis, which was higher than at +20°C. In the beginning of the dark period a CO₂ outburst appeared at 35°C.Only at a low temperature (+20°C) did we find a light induced glycollate excretion; after a maximum at 7 1/2 minutes illumination the release of glycollate ceases and the level decreases to a lower value. A similar time course exists during illumination in red light (621 nm, 1.5·10^-8 einsteins) and a temperature of +20°C. In blue light (432 nm, 1,5·10^-8 einsteins, +20°C) and in white light at a high temperature (+35°C) we could not find any light induced glycollate excretion. Our results are discussed in reference to the photorespiration. We explain the formation of transients in CO₂ uptake of Anacystis at a high temperature (+35°C) and in blue light (+20°C) on the basis of the influence of photorespiration.     

Role of NH3 and NH4+ transporters in renal acid-base transport

Renal ammonia excretion is the predominant component of renal net acid excretion. The majority of ammonia excretion is produced in the kidney and then undergoes regulated transport in a number of renal epithelial segments. Recent findings have substantially altered our understanding of renal ammonia transport. In particular, the classic model of passive, diffusive NH3 movement coupled with NH4+ "trapping" is being replaced by a model in which specific proteins mediate regulated transport of NH3 and NH4+ across plasma membranes. In the proximal tubule, the apical Na+/H+ exchanger, NHE-3, is a major mechanism of preferential NH4+ secretion. In the thick ascending limb of Henle's loop, the apical Na+-K+-2Cl- cotransporter, NKCC2, is a major contributor to ammonia reabsorption and the basolateral Na+/H+ exchanger, NHE-4, appears to be important for basolateral NH4+ exit. The collecting duct is a major site for renal ammonia secretion, involving parallel H+ secretion and NH3 secretion. The Rhesus glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), are recently recognized ammonia transporters in the distal tubule and collecting duct. Rhcg is present in both the apical and basolateral plasma membrane, is expressed in parallel with renal ammonia excretion, and mediates a critical role in renal ammonia excretion and collecting duct ammonia transport. Rhbg is expressed specifically in the basolateral plasma membrane, and its role in renal acid-base homeostasis is controversial. In the inner medullary collecting duct (IMCD), basolateral Na+-K+-ATPase enables active basolateral NH4+ uptake. In addition to these proteins, several other proteins also contribute to renal NH3/NH4+ transport. The role and mechanisms of these proteins are discussed in depth in this review.     

Responses to reversed NH3 and NH4+ gradients in a teleost (Ictalurus punctatus), an elasmobranch (Raja erinacea), and a crustacean (Callinectes sapidus):evidence for NH4+/H+ exchange in the teleost and the elasmobranch

Ammonia excretion rates of channel catfish, Ictalurus punctatus, little skate (Raja erinacea), and blue crab (Callinectes sapidus) were measured in experimental regimes which permitted simultaneous assessment of the partial pressure gradients for nonionized NH3 and the chemical concentration gradients of NH4+. Under conditions of low external ammonia, the average ammonia excretion was +295 microM kg-1 h-1 for catfish, +149 microM kg-1 h-1 for blue crabs, and +59 microM kg-1 h1 for skates with partial pressure gradients of +72.5 mu Torr, +413 mu Torr, and +24.4 mu Torr, respectively; and [NH4+] gradients of +189 microM l-1, +643 microM l-1, and +107 microM l-1 (positive indicating greater from inside to medium). When the external ammonia was increased to 1.15 mM l-1, both gradients were reversed, and the net ammonia movement was initially from the external water into all three species. In the catfish the inward movement ceased, however, and ammonia excretion eventually resumed in the face of reversed gradients of both NH3 partial pressure and [NH4+]. Unidirectional Na+ influx, indicative of a Na+/NH4+ exchange, did not increase. The ammonia data, changes in titratable acidity, and net apparent H+ efflux were all consistent with a linked extrusion of internal NH4+ for external H+. Incorporation of such an exchange into a computer simulation model of the ammonia equilibrium and exchange system duplicated the experimental data. Other hypotheses failed to match experimental data, or failed to predict internal ammonia levels lower than outside. In the crab, internal ammonia levels rose rapidly to concentrations and partial pressures above the external medium until excretion was reestablished, with no evidence of maintenance of a reversed gradient. In the skate, internal concentrations rose appreciably in the first hour and continued to rise for 6-8 h, with no resumption of ammonia excretion. The interspecies differences appear to be due at least partly to differences in ammonia permeability of the gills.     

Mass production of poly-β-hydroxybutyric acid by fully automatic fed-batch culture of methylotroph

Summary Fifty-one methylotrophs were checked with respect to their ability of poly--hydroxybutyric acid (PHB) production from methanol. One of them, Pseudomonas sp. K, was chosen from its good growth on a minimum synthetic medium. Optimal temperature and pH for its growth were 30° C and 7.0, respectively. Concentrations of PO ₄ ^3- and NH ₄ ⁺ in the medium should be kept at low levels. PHB formation was stimulated by deficiency of nutrient such as NH ₄ ⁺ , SO ₄ ^2- , Mg²⁺, Fe²⁺ or Mn²⁺. Among them, nitrogen deficiency was chosen from its effectiveness and easiness for PHB accumulation.The microorganism was cultivated to produce a large amount of poly--hydroxybutyric acid (PHB) from methanol by means of microcomputer-aided fully automatic fed-batch culture technique. During the cultivation, temperature, dissolved oxygen concentration (DO), and methanol concentration in the culture broth were maintained at 30° C 2.5±0.5 ppm and 0.5±0.2 g/l, respectively. Other nutrients, nitrogen source and mineral ions, were also controlled to maintain their initial concentrations in the medium during cell growth phase. When the high cell concentration was achieved (160 g/l), feedings of ammonia and minerals were stopped and only methanol was supplied successively to accumulate PHB. At 175 h, high concentration of PHB (136 g/l) was obtained and total cell concentration became 206 g/l. DO must be maintained above the critical level during the PHB formation phase, too. PHB yield from methanol (g PHB/g methanol) was 0.18 and the maximum PHB content reached 66% of dry weight. Solid PHB produced by the strain had the melting point of 176° C and the average molecular weight of 3.0x10⁵.     

Use of electrodialysis and reverse osmosis for the recovery and concentration of ammonia from swine manure

This project aimed at producing a concentrated nitrogen fertilizer from liquid swine manure using electrodialysis (ED) and reverse osmosis (RO), as a mean to help resolve the excess nutrient problem faced by many swine producers, and offer an alternative to chemical nitrogen fertilizer production. Different types of ED membranes were evaluated based on the NH4+ transfer rate, current efficiency and membrane stability. A combination of CMB/AMX membranes was retained due to its high NH4+ transfer rate and chemical stability. The maximum total ammonia concentration (NH3-N) achievable by ED was limited by water transport from the manure to the concentrate compartment, and ammonia volatilization (17%) from the open concentrate compartment. Results suggested that, under the conditions of this experiment, a maximum total NH3-N concentration of about 16g/L could be reached with the ED system. An ED concentrate (8.7g/L of total NH3-N) was also fed to TFC-HF reverse osmosis membranes. A mass balance analysis revealed that the RO permeate, which represented 49.6% of the initial volume, contained 8.6% of the ammonia. However, the RO concentrate contained only 66.6% of the initial total NH3-N, suggesting that 21.2% of the ammonia was volatilized during the concentration test with RO membranes. Ammonia concentration in the RO concentrate reached approximately 13g/L, which is similar to the maximum concentration that could be achieved by ED. These results suggest that the use of ED and RO membranes to recover and concentrate ammonia is potentially interesting but the process must include an approach to minimize ammonia volatilization or trap volatilized ammonia.     

Computational study of the binding affinity and selectivity of the bacterial ammonium transporter AmtB

We report results from microscopic molecular dynamics and free energy perturbation simulations of substrate binding and selectivity for the Escherichia coli high-affinity ammonium transporter AmtB. The simulation system consists of the protein embedded in a model membrane/water surrounding. The calculated absolute binding free energies for the external NH(4)(+) ions are between -5.8 and -7.3 kcal/mol and are in close agreement with experimental data. The apparent pK(a) of the bound NH(4)(+) increases by more than 4 units, indicating a preference for binding ammonium ion and not neutral ammonia. The external binding site is also selective for NH(4)(+) toward monovalent metal cations by 2.4-4.4 kcal/mol. The externally bound NH(4)(+) shows strong electrostatic interactions with the proximal buried Asp160, stabilized in the anionic form, whereas the interactions with the aromatic rings of Phe107 and Trp148, lining the binding cavity, are less pronounced. Simulated mutation of the highly conserved Asp160 to Asn reduces the pK(a) of the bound ammonium ion by approximately 7 units and causes loss of its binding. The calculations further predict that the substrate affinity of E. coli AmtB depends on the ionization state of external histidines. The computed free energies of hypothetical intermediate states related to transfer of NH(3), NH(4)(+), or H(2)O from the external binding site to the first position inside the internal channel pore favor permeation of the neutral species through the channel interior. However, the predicted change in the apparent pK(a) of NH(4)(+) upon translocation from the external site, Am1, to the first internal site, Am2, indicates that ammonium ion becomes deprotonated only when it enters the channel interior.     

Comparative growth of the thermal alga Cyanidium caldarium on nitrate and ammonia at different temperatures

Summary The growth of Cyanidium caldarium on nitrate and ammonia as nitrogen sources was studied at different temperatures from 21 to 54°C.Algal growth occurred at temperatures of 24° C or above when ammonia was the nitrogen source, whereas with nitrate, growth occurred at 30° C or above. The optimum and the maximum growth temperatures were 45 and 54° C respectively on both substrates.Arrhenius plots show that the logarithm of the growth rate is not linear with the reciprocal of absolute temperature, but exhibit sharply defined breaks at 30° C on ammonia and at 40° C on nitrate.It is assumed that below 40° C, when Cyanidium grows on nitrate, the utilization of this substrate represents the master reaction which controls the growth rate of the alga.     

Investigation of substrate specificity of creatine kinase using chromium (III) and cobalt(III) complexes of adenosine 5'-diphosphate

The specificity for substrate binding to creatine kinase for metal-nucleotide complexes of the type Cr-(H2O)4-n(NH3)nADP (where n = 0, 3, or 4) and Co-(H2O)4-m(NH3)mADP (for m = 3 or 4) has been investigated over the pH range 5.5-7.8 with the delta-alpha, beta-bidentate diastereoisomers. These inert nucleotide complexes acted as competitive inhibitors vs. MgADP over this range. In addition, the pH dependence of the V, V/K, and Km values for MgADP has been determined. Metal-nucleotide binding to the enzyme is strongest below an approximate pK of 6.45 but again becomes pH independent above pH 7. This pK is not associated with the metal-nucleotide complex. Instead, we conclude that the pK of the acid-base catalyst (thought to be histidine) is about 6.45 in the absence of nucleotide but is raised to 7.2 in its presence. This perturbation of the pK may result from a protein conformational change that allows a hydrogen bond to form between the phosphorylated nitrogen of phosphocreatine and the acid-base catalyst. The pK of the water in Cr(H2O)(NH3)3ADP has been determined to be 6.6, and by comparison of the binding affinity of this complex with that of Cr(NH3)4ADP or Cr(H2O)4ADP, it can be deduced that the hydroxo species binds more strongly than the aquo complex. In general, chromium nucleotides are bound more strongly than cobalt complexes, and binding affinity increases as water replaces ammonia in the first coordination sphere of the metal. Both trends are a result of stronger hydrogen-bond interactions between the metal complex and protein.     

Ammonia assimilation pathways in nitrogen-fixing Clostridium kluyverii and Clostridium butyricum.

Pathways of ammonia assimilation into glutamic acid were investigated in ammonia-grown and N2-fixing Clostridium kluyverii and Clostridium butyricum by measuring the specific activities of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. C. kluyverii had NADPH-glutamate dehydrogenase with a Km of 12.0 mM for NH4+. The glutamate dehydrogenase pathway played an important role in ammonia assimilation in ammonia-grown cells but was found to play a minor role relative to that of the glutamine synthetase/NADPH-glutamate synthase pathway in nitrogen-fixing cells when the intracellular NH4+ concentration and the low affinity of the enzyme for NH4+ were taken into account. In C. butyricum grown on glucose-salt medium with ammonia or N2 as the nitrogen source, glutamate dehydrogenase activity was undetectable, and the glutamine synthetase/NADH-glutamate synthase pathway was the predominant pathway of ammonia assimilation. Under these growth conditions, C. butyricum also lacked the activity of glucose-6-phosphate dehydrogenase, which catalyzes the regeneration of NADPH from NADP+. However, high activities of glucose-6-phosphate dehydrogenase as well as of NADPH-glutamate dehydrogenase with a Km of 2.8 mM for NH4+ were present in C. butyricum after growth on complex nitrogen and carbon sources. The ammonia-assimilating pathway of N2-fixing C. butyricum, which differs from that of the previously studied Bacillus polymyxa and Bacillus macerans, is discussed in relation to possible effects of the availability of ATP and of NADPH on ammonia-assimilating pathways.     

Basis for the equilibrium constant in the interconversion of l-lysine and l-beta-lysine by lysine 2,3-aminomutase

l-beta-lysine and beta-glutamate are produced by the actions of lysine 2,3-aminomutase and glutamate 2,3-aminomutase, respectively. The pK(a) values have been titrimetrically measured and are for l-beta-lysine: pK(1)=3.25 (carboxyl), pK(2)=9.30 (beta-aminium), and pK(3)=10.5 (epsilon-aminium). For beta-glutamate the values are pK(1)=3.13 (carboxyl), pK(2)=3.73 (carboxyl), and pK(3)=10.1 (beta-aminium). The equilibrium constants for reactions of 2,3-aminomutases favor the beta-isomers. The pH and temperature dependencies of K(eq) have been measured for the reaction of lysine 2,3-aminomutase to determine the basis for preferential formation of beta-lysine. The value of K(eq) (8.5 at 37 degrees C) is independent of pH between pH 6 and pH 11; ruling out differences in pK-values as the basis for the equilibrium constant. The K(eq)-value is temperature-dependent and ranges from 10.9 at 4 degrees C to 6.8 at 65 degrees C. The linear van't Hoff plot shows the reaction to be enthalpy-driven, with DeltaH degrees =-1.4 kcal mol(-1) and DeltaS degrees =-0.25 cal deg(-1) mol(-1). Exothermicity is attributed to the greater strength of the bond C(beta)-N(beta) in l-beta-lysine than C(alpha)-N(alpha) in l-lysine, and this should hold for other amino acids.