The Titrimetric Assay of H+ Excreted by Ricinus communis Cultivated on NH4+-N Nutrient Media: The Effect of Ionic Strength and Nutrient Phosphate Concentration

Allen, C. R. and Allen, S. 1987. The titrimetric assay of H⁺excreted by Ricinus communis cultivated on -N nutrient media; the effect of ionic strengthand nutrient phosphate concentration.— J. exp. Bot. 38:597–606. The simple titrimetric assay of H⁺ excreted by -N plants into spent nutrient solution was criticallyassessed on the basis of equations for charge balance, conservationof matter and chemical equilibrium (including activity coefficients).For Ricinus communis, a simple volumetric calculation from back-titrationof the spent medium to 6–5 underestimated proton excretionby 3%, compared with the corresponding error of up to 50% fortitrimetric assay of base excreted into spent -N media. Key words: Ricinus, ammonium-N nutrient medium, proton excretion, ionic activity coefficients     

Hepatic urea synthesis and pH regulation. Role of CO2, HCO3-, pH and the activity of carbonic anhydrase

In isolated perfused rat liver, urea synthesis from ammonium ions was dependent on extracellular HCO3- and CO2 concentrations when the HCO3-/CO2 ratio in the influent perfusate was constant (pH 7.4). Urea synthesis was half-maximal at HCO3- = 4 mM, CO2 = 0.19 mM and was maximal at HCO3- and CO2 concentrations above 20 mM and 0.96 mM, respectively. At physiological HCO3- (25 mM) and CO2 (1.2 mM) concentrations in the influent perfusate, acetazolamide, the inhibitor of carbonic anhydrase, inhibited urea synthesis from ammonium ions (1 mM) by 50-60% and led to a 70% decrease in citrulline tissue levels. Acetazolamide concentrations required for maximal inhibition of urea synthesis were 0.01-0.1 mM. At subphysiological HCO3- and CO2 concentrations, inhibition of urea synthesis by acetazolamide was increased up to 90%. Inhibition of urea synthesis by acetazolamide was fully overcome in the presence of unphysiologically high HCO3- and CO2 concentrations, indicating that the inhibitory effect of acetazolamide is due to an inhibition of carbonic-anhydrase-catalyzed HCO3- supply for carbamoyl-phosphate synthetase, which can be bypassed when the uncatalyzed intramitochondrial HCO3- formation from portal CO2 is stimulated in the presence of high portal CO2 concentrations. With respect to HCO3- supply of mitochondrial carbamoyl-phosphate synthetase, urea synthesis can be separated into a carbonic-anhydrase-dependent (sensitive to acetazolamide at 0.5 mM) and a carbonic-anhydrase-independent (insensitive to acetazolamide) portion. Carbonic-anhydrase-independent urea synthesis linearly increased with the portal 'total CO2 addition' (which was experimentally determined to be CO2 addition plus 0.036 HCO3- addition) and was independent of the perfusate pH. At a constant 'total CO2 addition', carbonic-anhydrase-dependent urea synthesis was strongly affected by perfusate pH and increased about threefold when the perfusate pH was raised from 6.9 to 7.8. It is concluded that the pH dependent regulation of urea synthesis is predominantly due to mitochondrial carbonic anhydrase-catalyzed HCO3- supply for carbamoyl phosphate synthesis, whereas there is no control of urea synthesis by pH at the level of the five enzymes of the urea cycle. Because HCO3- provision for carbamoyl phosphate synthetase increases with increasing portal CO2 concentrations even in the absence of carbonic anhydrase activity, susceptibility of ureogenesis to pH decreases with increasing portal CO2 concentrations. This may explain the different response of urea synthesis to chronic metabolic and chronic respiratory acidosis in vivo.     

Effect of radiation quality on growth, nitrogen uptake, and HCO3-, CO2 and pH interactions in Ulva pertusa

A floating sterile mutant of Ulva pertusa, which grows faster than the wild type and also during summer periods, was grown in the laboratory under "white light" (WR as reference), broad band isoquantic red (600 - 700 nm, RR) and blue (400 - 500 nm, BR) radiation. The observed specific growth rates at WR, BR, and RR were 8.6, 2.15, and 1.2 %, respectively. A stimulatory effect of BR on the growth at low irradiance (60 μmol m-2 s-1) was observed during the 15 d of culture. Around 42.1 % of total nitrogen was used by Ulva mutant in WR, but under BR and RR it was around 27.0 and 16.7 %, respectively. However, the utilization of nitrogen under BR was significantly higher than under RR. Considerable CO2 fluctuation in the light and dark periods was observed in all the cultures, and it was higher under WR, followed by BR and RR. The possible growth promoting effect of BR includes nitrogen uptake and the consumption of HCO3- which in turn leads to reduced air CO2 concentration in the medium. This revised version was published online in August 2006 with corrections to the Cover Date.     

Heterotrophic nitrification by Alcaligenes faecalis: NO2-, NO3-, N2O, and NO production in exponentially growing cultures

Heterotrophic nitrification by Alcaligenes faecalis DSM 30030 was not restricted to media containing organic forms of nitrogen. In both peptone-meat extract and defined media with ammonium and citrate as the sole nitrogen and carbon sources, respectively, NO2-, NO3-, NO, and N2O were produced under aerobic growth conditions. Heterotrophic nitrification was not attributable to old or dying cell populations. Production of NO2-, NO3-, NO, and N2O was detectable shortly after cultures started growth and proceeded exponentially during the logarithmic growth phase. NO2- and NO3- production rates were higher for cultures inoculated in media with pH values below 7 than for those in media at alkaline pH. Neither assimilatory nor dissimilatory nitrate or nitrite reductase activities were detectable in aerobic cultures.     

A mathematical model of tumour and blood pHe regulation: The HCO3-/CO2 buffering system

Malignant tumours are characterised by a low, acidic extracellular pH (pHe) which facilitates invasion and metastasis. Previous research has proposed the potential benefits of manipulating systemic pHe, and recent experiments have highlighted the potential for buffer therapy to raise tumour pHe, prevent metastases, and prolong survival in laboratory mice. To examine the physiological regulation of tumour buffering and investigate how perturbations of the buffering system (via metabolic/respiratory disorders or changes in parameters) can alter tumour and blood pHe, we develop a simple compartmentalised ordinary differential equation model of pHe regulation by the HCO3-/CO2 buffering system. An approximate analytical solution is constructed and used to carry out a sensitivity analysis, where we identify key parameters that regulate tumour pHe in both humans and mice. From this analysis, we suggest promising alternative and combination therapies, and identify specific patient groups which may show an enhanced response to buffer therapy. In addition, numerical simulations are performed, validating the model against well-known metabolic/respiratory disorders and predicting how these disorders could change tumour pHe.     

Heterotrophic nitrification by Alcaligenes faecalis: NO2-, NO3-, N2O, and NO production in exponentially growing cultures.

Heterotrophic nitrification by Alcaligenes faecalis DSM 30030 was not restricted to media containing organic forms of nitrogen. In both peptone-meat extract and defined media with ammonium and citrate as the sole nitrogen and carbon sources, respectively, NO2-, NO3-, NO, and N2O were produced under aerobic growth conditions. Heterotrophic nitrification was not attributable to old or dying cell populations. Production of NO2-, NO3-, NO, and N2O was detectable shortly after cultures started growth and proceeded exponentially during the logarithmic growth phase. NO2- and NO3- production rates were higher for cultures inoculated in media with pH values below 7 than for those in media at alkaline pH. Neither assimilatory nor dissimilatory nitrate or nitrite reductase activities were detectable in aerobic cultures.     

Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors

The intracellular pH (pHi) of squid giant axons has been measured using glass pH microelectrodes. Resting pHi in artificial seawater (ASW) (pH 7.6-7.8) at 23 degrees C was 7.32 +/- 0.02 (7.28 if corrected for liquid junction potential). Exposure of the axon to 5% CO2 at constant external pH caused a sharp decrease in pHi, while the subsequent removal of the gas caused pHi to overshoot its initial value. If the exposure to CO2 was prolonged, two additional effects were noted: (a) during the exposure, the rapid initial fall in pHi was followed by a slow rise, and (b) after the exposure, the overshoot was greatly exaggerated. Application of external NH4Cl caused pHi to rise sharply; return to normal ASW caused pHi to return to a value below its initial one. If the exposure to NH4Cl was prolonged, two additional effects were noted: (a) during the exposure, the rapid initial rise in pHi was followed by a slow fall, and (b) after the exposure, the undershoot was greatly exaggerated. Exposure to several weak acid metabolic inhibitors caused a fall in pHi whose reversibility depended upon length of exposure. Inverting the electrochemical gradient for H+ with 100 mM K- ASW had no effect on pHi changes resulting from short-term exposure to azide. A mathematical model explains the pHi changes caused by NH4Cl on the basis of passive movements of both NH3 and NH4+. The simultaneous passive movements of CO2 and HCO3-cannot explain the results of the CO2 experiments; these data require the postulation of an active proton extrusion and/or sequestration mechanism.     

Passive entry of CO2 and its energy-dependent intracellular conversion to HCO3- in cyanobacteria are driven by a photosystem I-generated deltamuH+

CO(2) entry into Synechococcus sp. PCC7942 cells was drastically inhibited by the water channel blocker p-chloromercuriphenylsulfonic acid suggesting that CO(2) uptake is, for the most part, passive via aquaporins with subsequent energy-dependent conversion to HCO3(-). Dependence of CO(2) uptake on photosynthetic electron transport via photosystem I (PSI) was confirmed by experiments with electron transport inhibitors, electron donors and acceptors, and a mutant lacking PSI activity. CO(2) uptake was drastically inhibited by the uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and ammonia but substantially less so by the inhibitors of ATP formation arsenate and N, N,-dicyclohexylcarbodiimide (DCCD). Thus a DeltamuH(+) generated by photosynthetic PSI electron transport apparently serves as the direct source of energy for CO(2) uptake. Under low light intensity, the rate of CO(2) uptake by a high-CO(2)-requiring mutant of Synechococcus sp. PCC7942, at a CO(2) concentration below its threshold for CO(2) fixation, was higher than that of the wild type. At saturating light intensity, net CO(2) uptake was similar in the wild type and in the mutant IL-3 suggesting common limitation by the rate of conversion of CO(2) to HCO3(-). These findings are consistent with a model postulating that electron transport-dependent formation of alkaline domains on the thylakoid membrane energizes intracellular conversion of CO(2) to HCO3(-).     

Quantification of the Contribution of CO2, HCO3-, and External Carbonic Anhydrase to Photosynthesis at Low Dissolved Inorganic Carbon in Chlorella saccharophila

cDNAs encoding the large subunit and a possibly truncated small subunit of the potato tuber (Solanum tuberosum L.) adenosine 5'-diphosphate-glucose pyrophosphorylase have been expressed in Escherichia coli (A.A. Iglesias, G.F. Barry, C. Meyer, L. Bloksberg, P.A. Nakata, T. Greene, M.J. Laughlin, T.W. Okita, G.M. Kishore, J. Preiss, J Biol Chem [1993] 268: 1081-1086). However, some properties of the transgenic enzyme were different from those reported for the enzyme from potato tuber. In this work, extension of the cDNA was performed to elongate the N terminus of the truncated small subunit by 10 amino acids. This extension is based on the almost complete conservation seen at the N-terminal sequence for the potato tuber and the spinach leaf small subunits. Expressing the extended cDNA in E. coli along with the large subunit cDNA yielded a transgenic heterotetrameric enzyme with similar properties to the purified potato tuber enzyme. It was also found that the extended small subunit expressed by itself exhibited high enzyme activity, with lower affinity for activator 3-phosphoglycerate and higher sensitivity toward inorganic phosphate inhibition. It is proposed that a major function of the large subunit of adenosine 5'-diphosphate-glucose pyrophosphorylases from higher plants is to modulate the regulatory properties of the native heterotetrameric enzyme, and the small subunit's major function is catalysis.     

Influence of carbon availability on the production of NO, N2O, N2 and CO2 by soil cores during anaerobic incubation

Net productions of permanent soil atmosphere gases (N₂, CO₂, O₂) and temporary gases (N₂O, NO) were monitored in soil cores using a non-interfering, fully automated measuring technique allowing highly time resolved measurements over prolonged periods. The influence of changes in available organic carbon on CO₂, N₂O, NO and N₂ production was studied by changing the soil carbon content through aerobic preincubations of different length, up to 21 days.The aerobic preincubation caused an increase in NO₃ ^- concentration and a decrease in available carbon content. Available carbon content dominated both CO₂ and total N gas (N₂+N₂O+NO) production during anaerobiosis. Both CO₂ and total N gas production rates decreased with increasing length of the previous aerobic preincubation, this in spite of the higher initial NO₃ ^- concentration.Total denitrification rates were closely related to the anaerobic CO₂ production rates. No relation was found between water soluble carbon content and total denitrification. The N₂O/N₂ ratio could be explained by an interaction of carbon availability, NO₃ ^- concentration and enzyme status. Net N₂O consumption was monitored. The balance between cumulative total N gas production and NO₃ ^- consumption varied according to the different treatments. Cumulative N₂O production exceeded cumulative N₂ production for 0 up to 5 days.     

The Effect of pH, O(2), and Temperature on the CO(2) Compensation Point of Isolated Asparagus Mesophyll Cells

The effect of pH, O₂ concentration, and temperature on the CO₂ compensation point (Г[CO₂]) of isolated Asparagus sprengeri Regel mesophyll cells has been determined in a closed, aqueous environment by a sensitive gas-chromatographic technique. Measured values range between 10 and 100 microliters per liter CO₂ depending upon experimental conditions. The Г(CO₂) increases with increasing temperature. The rate of increase is dependent upon the O₂ concentration and is more rapid at high (250-300 micromolar), than at low (30-60 micromolar), O₂ concentrations. The differential effect of temperature on Г(CO₂) is more pronounced at pH 6.2 than at pH 8.0, but this pH-dependence is not attributable to a direct, differential effect of pH on the relative rates of photosynthesis and photorespiration, as the O₂-sensitive component of Г(CO₂) remains constant over this range. The Г(CO₂) of Asparagus cells at 25°C decreases by 50 microliters per liter when the pH is raised from 6.2 to 8.0, regardless of the prevailing O₂ concentration. It is suggested that the pH-dependence of Г(CO₂) is related to the ability of the cell to take up CO₂ from the aqueous environment. The correlation between high HCO₃⁻ concentrations and low Г(CO₂) at alkaline pH indicates that extracellular HCO₃⁻ facilitates the uptake of CO₂, possibly by increasing the flux of inorganic carbon from the bulk medium to the cell surface. The strong O₂− and temperature-dependence of Г(CO₂) indicates that isolated Asparagus mesophyll cells lack an efficient means for concentrating intracellular CO₂ to a level sufficient to reduce or suppress photorespiration.     

Carbon,Nitrogen, and Nutrient Interactions in Beta vulgaris L. as Influenced by Nitrogen Source,NO3- versus NH4+

Sugar beets (Beta vulgaris L. cv F58-554H1) were grown hydroponically in a 16-h light, 8-h dark period (photosynthetic photon flux density of 0.5 mmol m-2 s-1) for 4 weeks from sowing in half-strength Hoagland nutrient solution containing 7.5 mM nitrate. Half of the plants were then transferred to 7.5 mM ammonium N; the rest remained in solution with 7.5 mM nitrate N. Upon transfer from nitrate to ammonium, the total N concentration decreased sharply in the fibrous roots and petiole/midribs and increased substantially in the leaf blades. This was because of the decreased nitrate concentrations in fibrous roots and petioles and a concomitant increase in amino acid/amide-N and protein N in leaf blades. Sugar beets acclimated to ammonium partly by a 2.5-fold increase in glutamine synthase activity in fibrous roots and a 1.7-fold increase in leaf blades. Rapid ammonium assimilation into glutamine consumed carbon skeletons, leading to a depletion of foliar starch, sucrose, and maltose. Ammonium treatment stimulated activities of some glycolytic/Krebs cycle enzymes, e.g. pyruvate dehydrogenase. Nitrate-fed leaf blades contained substantially larger concentrations of osmolytes (i.e. nitrate, cations, and sucrose), which may have contributed to the faster rates of leaf expansion in nitrate-fed compared to ammonium-fed plants.     

Influence of CO2-HCO3− Levels and pH on Growth, Succinate Production, and Enzyme Activities of Anaerobiospirillum succiniciproducens

Growth and succinate versus lactate production from glucose by Anaerobiospirillum succiniciproducens was regulated by the level of available carbon dioxide and culture pH. At pH 7.2, the generation time was almost doubled and extensive amounts of lactate were formed in comparison with growth at pH 6.2. The succinate yield and the yield of ATP per mole of glucose were significantly enhanced under excess-CO₂-HCO₃⁻ growth conditions and suggest that there exists a threshold level of CO₂ for enhanced succinate production in A. succiniciproducens. Glucose was metabolized via the Embden-Meyerhof-Parnas route, and phosphoenolpyruvate carboxykinase levels increased while lactate dehydrogenase and alcohol dehydrogenase levels decreased under excess-CO₂-HCO₃⁻ growth conditions. Kinetic analysis of succinate and lactate formation in continuous culture indicated that the growth rate-linked production rate coefficient (K) cells was much higher for succinate (7.2 versus 1.0 g/g of cells per h) while the non-growth-rate-related formation rate coefficient (K′) was higher for lactate (1.1 versus 0.3 g/g of cells per h). The data indicate that A. succiniciproducens, unlike other succinate-producing anaerobes which also form propionate, can grow rapidly and form high final yields of succinate at pH 6.2 and with excess CO₂-HCO₃⁻ as a consequence of regulating electron sink metabolism.     

The regulation of intracellular pH in monkey kidney epithelial cells (BSC-1). Roles of Na+/H+ antiport, Na+-HCO3(-)-(NaCO3-) symport, and Cl-/HCO3- exchange

Using the pH-sensitive absorbance of 5 (and 6)-carboxy-4',5'- dimethylfluorescein, we investigated the regulation of cytoplasmic pH (pHi) in monkey kidney epithelial cells (BSC-1). In the absence of HCO3-, pHi is 7.15 +/- 0.1, which is not significantly different from pHi in 28 mM HCO3-, 5% CO2 (7.21 +/- 0.07). After an acid load, the cells regulate pHi in the absence of HCO3- by a Na+ (or Li+)-dependent, amiloride-inhibitable mechanism (indicative of Na+/H+ antiport). In 28 mM HCO3-, while still dependent on Na+, this regulation is only blocked in part by 1 mM amiloride. A partial block is also observed with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (1 mM). With cells pretreated with DIDS, 1 mM amiloride nearly totally inhibits this regulation. Cl- had no effect on pHi regulation in the acidic range. In HCO3(-)-free saline, Na+ removal leads to an amiloride-insensitive acidification, which is dependent on Ca2+. In 28 mM HCO3-, Na+ (and Ca2+) removal led to a pronounced reversible and DIDS-sensitive acidification. When HCO3- was lowered from 46 to 10 mM at constant pCO2 (5%), pHi dropped by a DIDS-sensitive mechanism. Identical changes in pHo (7.6 to 6.9) in the nominal absence of HCO3- led to smaller changes of pHi. In the presence but not in the absence of HCO3-, removal of Cl- led to a DIDS-sensitive alkalinization. This was also observed in the nominal absence of Na+, which leads to a sustained acidification. It is concluded that in nominally bicarbonate-free saline, the amiloride-sensitive Na+/H+ antiport is the predominant mechanism of pHi regulation at acidic pHi, while being relatively inactive at physiological values of pHi. In bicarbonate saline, two other mechanisms effect pHi regulation: a DIDS-sensitive Na+-HCO3- symport, which contributes to cytoplasmic alkalinization, and a DIDS-sensitive Cl-/HCO3- exchange, which is apparently independent of Na+.     

Short-term studies of 15NO3 - and 15NH4 + uptake by micropropagated strawberry shoots cultured with or without CO2 enrichment

The uptake of ¹⁵NO₃ ^- and ¹⁵NH₄ ⁺ has been examined in 5-,10- and 28-day-old micropropagated strawberry (Fragaria x ananassa Duch. cv. Kent) shoots rooted in one-half strength Murashige and Skoog (MS) liquid medium on cellulose plugs (Sorbarods). The results indicated that the plantlets absorbed both NO₃ ^- and NH₄ ⁺ during the culture with a greater uptake of NH₄ ⁺ at 5 days of culture. Furthermore, a pronounced reduction in NO₃ ^- and NH₄ ⁺ uptake at 10 and 28 days of culture was observed within 6 h of the short-term uptake study. This reduction could be explained by the low CO₂ concentration in test tubes during the photoperiod, since no reduction in nitrogen uptake occurred in the CO₂ enriched condition. The results are interpreted as an indication of the important role for photosynthetic CO₂ fixation in the process of nitrogen uptake by the plantlets during the rooting stage.Contribution No. CRH 82, Centre de Recherche en Horticulture, F.S.A.A., Université Laval, Québec.     

Photosynthetic Assimilation of NO(3) by Intact Cells of the Cyanobacterium Anacystis nidulans: Influence of NO(3) and NH(4) Assimilation on CO(2) Fixation

Illuminated suspensions of Anacystis nidulans, supplied with saturating concentrations of CO₂ evolved O₂ at a greater rate when nitrate was simultaneously present. The extent of the stimulation of noncyclic electron flow induced by nitrate was dependent on light intensity, being maximal under light saturating conditions. Accordingly, nitrate depressed the rate of CO₂ fixation at limiting but not at saturating light, this depression reflecting the competition between both processes for assimilatory power. In contrast, ammonium stimulated CO₂ fixation at any light intensity assayed, the stimulation being dependent on the incorporation of ammonium to carbon skeletons. The positive effect of ammonium on CO₂ fixation also appeared to occur when nitrate was the nitrogen source, since with either nitrogen source an increase in the incorporation of newly fixed carbon into acid-soluble metabolites took place. From these results, the in vivo partitioning of assimilatory power between photosynthetic nitrogen and carbon assimilation and the quantitative and qualitative effects of inorganic nitrogen assimilation on CO₂ fixation are discussed.