Phlorizin inhibits hexose transport across the plasmalemma of Riccia fluitans

The effect of phlorizin has been tested on hexose transport and hexose-induced changes of electrical potential (_m) and conductance (g _m) across the plasmalemma of rhizoid and thallus cells of the aquatic liverwort Riccia fluitans. The decrease of _m (depolarization) and g _m induced by 1 mM 3-oxymethyl-D-glucose (3-OMG) is substantially inhibited by simultaneous addition of 2 mM phlorizin, whereas no significant response was observed when phlorizin was added alone or several minutes after the sugar. Current-voltage data show that the 3-OMG-generated electrical inward current of 0.016 A m^-2 drops to 0.010 A m^-2 when phlorizin is present. Uptake as well as efflux of [¹⁴C]-3-OMG is strongly and reversibly inhibited by phlorizin between 0.2 and 5 mM. The results are consistent with our hypothesis that the hexose carrier has one binding site with competitive inhibition of glucose uptake by phlorizin (k _i=0.08 mM). The electrical data indicate that phlorizin affects an _m step of the carrier transport cycle.Abbreviation 3-OMG 3-oxymethyl-D-glucose     

Stereospecificity and electrogenicity of amino acid transport in Riccia fluitans

In the aquatic liverwort Riccia fluitans, membrane depolarization (_m), change in membrane conductance (g_m), and current-voltage (I-V) characteristics in the presence of different amino acids as well as the uptake of ¹⁴C-labeled amino acids were measured. L-isomers of the tested amino acids generate larger electrical effects (_m, g_m) than D-isomers, and the I-V characteristics show that the positive electrical inward-current of 20 mA m^-2 generated by 0.5 mM D-serine is only about 50% of the current generated by adding 0.5 mM L-serine. Whereas - and -amino acids rapidly depolarize the membrane to the same extend, with -aminobutyric acid (-AB) and dipeptides no significant electrical effects have been measured. The uptake kinetics of ¹⁴C-labeled amino acids display three components: (I) A saturable high-affinity component with K_s-values of 48 M D-alanine, 12 M -aminoisobutyric acid (AIB), 9 M L-alanine, 8 M L-proline, and 6 M L-serine, respectively; (2) an apparently linear low-affinity component, and (3) an also linear but unspecific component at concentrations >20 times the given K_s-value. Uptake of ¹⁴C-labeled AIB can be inhibited competitively by all tested neutral amino acids, the L-isomers being more effective than the D-isomers, as well as by ammonium or methylamine. Vice versa, AIB competitively inhibits uptake of L-serine and L-alanine. It is concluded that an uncharged stereospecific carrier for the investigated amino acids exists in the plasmalemma of Riccia fluitans. Accumulation ratios of about 50 suggest secondary active transport driven by a transmembrane electro-chemical gradient (mainly _m) which is generated by the electrogenic proton pump. It is suggested that this carrier binds to the amino group forming either a charged binary complex with positively charged amines (Felle 1980), or an uncharged complex with -AB or dipeptides, whereas electrogenic transport of - and -amino acids is mediated by a ternary carrier complex, probably charged by a proton.Symbols and Abbreviations _m membrane potential (mV) - E_co equilibrium potential (mV) of the transport system - g_m membrane (slope) conductance (Sm^-2) - g_m change in g_m - I-V curve current-voltage curve - AIB -aminoisobutytric acid - -AB -aminobutyric acid     

Hexose transport and membrane depolarization in Riccia fluitans

In the aquatic liverwort Riccia fluitans, the uptake of ¹⁴C-labeled 3-O-methyl glucose (3-OMG) and membrane depolarization ( _m ) caused by different hexoses has been studied as a function of time and concentration of hexose, K⁺ and H⁺, respectively. The rate of uptake of the non-metabolized 3-OMG shows two components: (A)A pH-dependent saturable uptake with a k_m value around 0.1 mM which saturates at 2.1 and 7.2 mol G _DW ^-1 h^-1 at pH 6.8 and 5.0, respectively; and (B) a pH-insensitive uptake component which increases linearly with the external 3-OMG concentration and does not saturate 4 mM. Hexoses rapidly depolarize the plasmalemma of the thallus cell and increase its electrical conductance. The maximal _m was 60±2 mV, the concentrations (mM) for half-maximal _m were 0.24 glucose, 0.32 galactose, 0.37 2-deoxy glucose, 0.38 3-OMG, 0.57 mannose, and 34 fructose. In terms of a hexose carrier model and an equivalent circuit for the hexose-induced depolarized state of the membrane, it is proposed that a hexose carrier operates either electrogenically in its protonated, pH-and voltage-sensitive state, or by transmembrane diffusion of its uncharged state.Symbols and Abbreviations _m membrane potential (mV) - g _m membrane (slope) conductance (Sm^-2) - 3-OMG 3-O-methyl glucose     

Transport of basic amino acids in Riccia fluitans: Evidence for a second binding site

The transport of several amino acids with different side-chain characteristics has been investigated in the aquatic liverwort Riccia fluitans. i) The saturation of system I (neutral amino acids) by addition of excess -aminoisobutyric acid to the external medium completely eliminated the electrical effects which are usually set off by neutral amino acids. Under these conditions arginine and lysine significantly depolarized the plasmalemma. ii) L- and D-lysine/arginine were discriminated against in favour of the L-isomers. iii) Increasing the external proton concentration in the interval pH 9 to 4.5 stimulated plasmalemma depolarization, electrical net current, and uptake of [¹⁴C]-basic amino acids. iv) Uptake of [¹⁴C]-glutamic acid took place only at acidic pHs. v) [¹⁴C]-histidine uptake had an optimum between pH 6 and 5.5. vi) Overlapping of the transport of basic, neutral, and acidic amino acids was common. It is suggested that besides system I, a second system (II), specific for basic amino acids, exists in the plasmalemma of Riccia fluitans. It is concluded that the amino-acid molecule with an uncharged side chain is the substrate for system I, which also binds and transports the neutral species of acidic amino acids, whereas system II is specific for amino acids with a positively charged side chain. The possibility of system II being a proton cotransport is discussed.Abbreviation AiB -aminoisobutyric acid     

Electron transport across the plasmalemma of Lemna gibba G1

Lemna gibba L., grown in the presence or absence of Fe, reduced extracellular ferricyanide with a V _max of 3.09 mol · g^-1 fresh weight · h^-1 and a K _m of 115 M. However, Fe³⁺-ethylenediaminetetraacetic acid (EDTA) was reduced only after Fe-starvation. External electron acceptors such as ferricyanide, Fe³⁺-EDTA, 2,6-dichlorophenol indophenol or methylene blue induced a membrane depolarization of up to 100 mV, but electron donors such as ferrocyanide or NADH had no effect. Light or glucose enhanced ferricyanide reduction while the concomitant membrane depolarization was much smaller. Under anaerobic conditions, ferricyanide had no effect on electrical membrane potential difference (E_m). Ferricyanide reduction induced H⁺ and K⁺ release in a ratio of 1.16 H⁺+1 K⁺/2 e^- (in +Fe plants) and 1.28 H⁺+0.8 K⁺/2 e^- (in -Fe plants). Anion uptake was inhibited by ferricyanide reduction. It is concluded that the steady-state transfer of electrons and protons proceeds by separate mechanisms, by a redox system and by a H⁺-ATPase.Abbreviations E _m electrical membrane potential difference - EDTA ethylenediaminetetraacetic acid - DCPIP dichlorophenol indophenol - +Fe control plant - -Fe iron-deficient plant - FW fresh weight - H⁺ electrochemical proton gradient     

Characterization of amino-acid transport in Ricinus communis roots using isolated membrane vesicles

The mechanism and specificity of amino-acid transport at the plasma membrane of Ricinus communis L. roots was investigated using membrane vesicles isolated by phase partitioning. The transport of glutamine, isoleucine, glutamic acid and aspartic acid was driven by both a pH gradient and a membrane potential (internally alkaline and negative), created artificially across the plasma membrane. This is consistent with transport via a proton symport. In contrast, the transport of the basic amino acids, lysine and arginine, was driven by a negative internal membrane potential but not by a pH gradient, suggesting that these amino acids may be taken up via a voltage-driven uniport. The energized uptake of all of the amino acids tested showed a saturable phase, consistent with carrier-mediated transport. In addition, the membrane-potential-driven transport of all the amino acids was greater at pH 5.5 than at pH 7.5, which suggests that there could be a direct pH effect on the carrier. Several amino-acid carriers could be resolved, based on competition studies: a carrier with a high affinity for a range of neutral amino acids (apart from asparagine) but with a low affinity for basic and acidic amino acids; a carrier which has a high affinity for a range of neutral amino acids except isoleucine and valine, but with a low affinity for basic and acidic amino acids; and a carrier which has a higher affinity for basic and some neutral amino acids but has a lower affinity for acidic amino acids. The existence of a separate carrier for acidic amino acids is discussed.Abbreviations PM plasma membrane - TPP⁺ tetraphenylphosphonium ion - pH pH gradient - membrane potential This work was supported by the Agricultural and Food Research Council and The Royal Society. We would like to thank Mrs. Sue Nelson for help with some of the membrane preparations.     

Effects of external pH,fusicoccin and butyrate on the cytoplasmic pH in barley root tips measured by 31P-nuclear magnetic resonance spectroscopy

³¹P-Nuclear magnetic resonance spectroscopy was used to measure the cytoplasmic pH (pH_c) in barley (Hordeum vulgare L.) root tips. As the external pH was raised from 4–10, pH_c was found to increase from 7.44 to 7.75. The sensitivity of pH_c to changes in external pH decreased with increasing external pH. Metabolic inhibition by sodium azide caused pH_c to fall by 0.3 units. Addition of 10 mM butyrate resulted in a gradual decline in pH_c, by approx. 0.3 units over 90 min. At a concentration of 1 mM, butyrate had no effect on pH_c even after 2 h. Fusicoccin caused pH_c to rise by 0.1–0.2 units. In maize (Zea mays L.) root tips, pH_c was shown to have a similar sensitivity to fusicoccin. The results are discussed in relation to the regulation of pH_c and the possible role of pH_c in determining transmembrane electrical potential differences.Abbreviations and symbols FC Fusicoccin - NMR nuclear magnetic resonance - p.d. membrane electrical potential difference - pH_c cytoplasmic pH - P1 inorganic phosphate - chemical shift     

Protonmotive force driven 6-deoxyglucose uptake by the oral pathogen,Streptococcus mutans Ingbritt

Streptococcus mutans Ingbritt was grown in glucose-excess continuous culture to repress the glucose phosphoenolpyruvate phosphotransferase system (PTS) and allow investigation of the alternative glucose process using the non-PTS substrate, (³H) 6-deoxyglucose. After correcting for non-specific adsorption to inactivated cells, the radiolabelled glucose analogue was found to be concentrated approximately 4.3-fold intracellularly by bacteria incubated in 100 mM Tris-citrate buffer, pH 7.0. Mercaptoethanol or KCl enhanced 6-deoxyglucose uptake, enabling it to be concentrated internally by at least 8-fold, but NaCl was inhibitory to its transport. Initial uptake was antagonised by glucose but not 2-deoxyglucose. Evidence that 6-deoxyglucose transport was driven by protonmotive force (p) was obtained by inhibiting its uptake with the protonophores, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazine, gramicidin and nigericin, and the electrical potential difference () dissipator, KSCN. The membrane ATPase inhibitor, N,N¹-dicyclohexyl carbodiimide, also reduced 6-deoxyglucose uptake as did 100 mM lactate. In combination, these two inhibitors completely abolished 6-deoxyglucose transport. This suggests that the driving force for 6-deoxyglucose uptake is electrogenic, involving both the transmembrane pH gradient (pH) and . ATP hydrolysis, catalysed by the ATPase, and lactate excretion might be important contributors to pH.Abbreviations DNP 2,4-dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N¹-dicyclohyxyl carbodiimide - p protonmotive force - pH transmembrane pH gradient - transmembrane electrical potential difference     

Measurement of motive force for cytoplasmic streaming in characean internodes

Summary With an attempt to measure the motive force responsible for cytoplasmic streaming in characean internodal cells, the difference between densities of cytoplasm and vacuolar sap was heightened by about 10 times (density of vacuolar sap was made larger than that of cytoplasm) by replacing the natural vacuolar sap ofChara corallina with an artificial one of higher density. Endoplasmic flow contiguous to the peripheral actin cables (peripheral flow of endoplasm) in the centrifugal direction was not influenced at all by the application of centrifugal acceleration up to 1400 g. We thus concluded that the motive force for the peripheral flow should be much larger than 12dyn/cm², a figure more than 10 times larger than that for bulk endop lasmic flow so far reported.Dedicated to Emeritus Professor Noburo Kamiya on the occasion of his 80th birthday     

The role of pH gradients across the plasmalemma of Catharanthus roseus and its involvement in the release of alkaloids

During growth, Catharanthus roseus cells exhibit an acidification of the culture medium that may be controlled by Ca²⁺. With a view to enhance the productivity of alkaloids by plant cells, the effect of extracellular pH modifications on the excretion processes has been investigated. Ca²⁺ dependent proton pumping leads to the release of various lipophilic amine-like compounds (benzylamine, methylamine, nicotine) initially accumulated by the cells, but also facilitates the excretion of endogenous ajmalicine. Once released in the medium, these compounds are however taken up again by the cells, probably as the charged form. For the alkaloid contained in C. roseus some evidence suggests that the diffusible form comes from the cytosolic compartment and not from the storage vacuoles. This appears to be a major production limitation to the use of pH gradients in order to favour alkaloid excretion.     

Abscisic acid causes changes in gene expression involved in the induction of the landform of the liverwortRiccia fluitans L.

The conversion of the submerged form ofRiccia fluitans to the landform either by transfer to a moist solid surface or by treatment with abscisic acid (ABA), is accompanied by the formation of a set of new polypeptides and concomitant down-regulation of other polypeptides. Changes in gene expression were analyzed by twodimensional separations of proteins and differential screening of a cDNA library. One of the landform-specific proteins might depend on the expression of the newly discoveredRic 1 gene. The deduced amino acid sequence of the isolatedRic 1 cDNA clone codes for a protein with a molecular mass of 30.1 kDa. This polypeptide possesses two amino acid sequences which are repeated five times each and it is largely hydrophilic with the exception of a hydrophobic carboxyl-terminal region. Under ABA treatment the expression of theRic 1 mRNA had already reached its maximum after 1 h of incubation. Transferring submerged thalli onto an agar surface resulted in a slower induction. TheRic 1 gene product shows homology to an embryo-specific polypeptide of carrot seeds and to the group 3 of late-embryogenesis-abundant (LEA) proteins. Interestingly, ABA treatment improved the desiccation tolerance of the submerged thalli. Additionally, ABA stimulated the synthesis of a protein which is immunologically related to a tonoplast protein. This finding, together with the fact that the ABA-induced landform exhibits an increased activity of several vacuolar enzymes, may indicate a special role of the tonoplast and the vacuole during ABA-induced conversion of the thallus from the submerged to the terrestrial form.Abbreviations ABA abscisic acid - 2-D PAGE two-dimensional polyacrylamide gel electrophoresis - F_o, F_m initial and maximal chlorophylla fluorescence - LEA late embryogenesis abundant - Q _a primary quinone acceptor of PS II We are grateful to the Deutsche Forschungsgemeinschaft for financial support SFB 251, SFB 176 to Prof. O.H. Volk (Institut für Biowissenschaften, Universität Würzburg, Germany) for generous supply ofRiccia fluitans thalli and many useful discussions and to Mrs B. Arbinger and Mrs B. Dierich for skilful technical assistance.     

Iron(III) hydroxamate transport across the cytoplasmic membrane ofEscherichia coli

Summary Transport of iron(III) hydroxamates across the inner membrane into the cytoplasm ofEscherichia coli is mediated by the FhuC, FhuD and FhuB proteins and displays characteristics typical of a periplasmic-binding-protein-dependent transport mechanism. In contrast to the highly specific receptor proteins in the outer membrane, at least six different siderophores of the hydroxamate type and the antibiotic albomycin are accepted as substrates. AfhuB mutant (deficient in transport of substrates across the inner membrane) which overproduced the periplasmic FhuD 30-kDa protein, bound [⁵⁵Fe] iron(III) ferrichrome. Resistance of FhuD to proteinase K in the presence of ferrichrome, aerobactin, and coprogen indicated binding of these substrates to FhuD. FhuD displays significant similarity to the periplasmic FecB, FepB, and BtuE proteins. The extremely hydrophobic FhuB 70-kDa protein is located in the cytoplasmic membrane and consists of two apparently duplicated halves. The N-and C-terminal halves [FhuB(N) and FhuB(C)] were expressed separately infhuB mutants. Only combinations of FhuB(N) and FhuB(C) polypeptides restored sensitivity to albomycin and growth on iron hydroxamate as a sole iron source, indicating that both halves of FhuB were essential for substrate translocation and that they combined to form an active permease. In addition, a FhuB derivative with a large internal duplication of 271 amino acids was found to be transport-active, indicating that the extra portion did not disturb proper insertion of the active FhuB segments into the cytoplasmic membrane. A region of considerable similarity, present twice in FhuB, was identified near the C-terminus of 20 analyzed hydrophobic proteins of periplasmic-binding-protein-dependent systems. The FhuC 30 kDa protein, most likely involved in ATP binding, contains two domains representing consensus sequences among all peripheral cytoplasmic membrane proteins of these systems. Amino acid replacements in domain I (LysGlu and Gln) and domain II (AspAsn and Glu) resulted in a transport-deficient phenotype.     

Day-to-night variations of cytoplasmic pH in a crassulacean acid metabolism plant

Summary In crassulacean acid metabolism (CAM) large amounts of malic acid are redistributed between vacuole and cytoplasm in the course of night-to-day transitions. The corresponding changes of the cytoplasmic pH (pH_cyt) were monitored in mesophyll protoplasts from the CAM plantKalanchoe daigremontiana Hamet et Perrier by ratiometric fluorimetry with the fluorescent dye 2′,7′-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein as a pH_cyt indicator. At the beginning of the light phase, pH_cyt was slightly alkaline (about 7.5). It dropped during midday by about 0.3 pH units before recovering again in the late-day-to-early-dark phase. In the physiological context the variation in pH_cyt may be a component of CAM regulation. Due to its pH sensitivity, phosphoenolpyruvate carboxylase appears as a likely target enzyme. From monitoring ΔpH_cyt in response to loading the cytoplasm with the weak acid salt K-acetate a cytoplasmic H⁺-buffer capacity in the order of 65 mM H+ per pH unit was estimated at a pH_cyt of about 7.5. With this value, an acid load of the cytoplasm by about 10 mM malic acid can be estimated as the cause of the observed drop in pH_cyt. A diurnal oscillation in pH_cyt and a quantitatively similar cytoplasmic malic acid is predicted from an established mathematical model which allows simulation of the CAM dynamics. The similarity of model predictions and experimental data supports the view put forward in this model that a phase transition of the tonoplast is an essential functional element in CAM dynamics.     

Malate and malate-channel antibodies inhibit electrogenic and ATP-dependent citrate transport across the tonoplast of citrus juice cells

Citrus juice cells accumulate high levels of citric acid in their vacuoles when compared to other organic ions including malate. Uptake of citrate into tonoplast vesicles from Citrus juice cells was investigated in the presence of malate, and after incubation with antibodies raised against the vacuolar malate-specific channel of Kalancho? diagremontiana leaves. Antibodies against the vacuolar malate channel immunoreacted with a protein of similar size in tonoplast extracts from three Citrus varieties differing in citric acid content. Malate channel antibodies inhibited both delta MicroH(+)-dependent and delta MicroH(+)-independent ATP-dependent citrate transport, indicating common domains in both transport systems and to the malate-specific channel of Kalancho? diagremontiana leaves. Malate strongly inhibited electrogenic citrate transport, whereas ATP-dependent citrate uptake was less affected. Kinetic analysis of citrate transport in the presence of malate confirmed the existence of two citrate transport mechanisms and indicated that both citrate and malate share a common transport channel across the tonoplast of Citrus juice cells.     

A general amino-acid permease is inducible in Chlorella vulgaris

Glucose or non-metabolizable glucose analogues induce two systems of amino-acid transport in Chlorella vulgaris: an arginine-lysine system and a proline system. An additional third system of amino-acid transport is induced when glucose and an inorganic nitrogen source are present during glucose induction. The transport rates in glucose-NH ₄ ⁺ -treated cells are 10 to 80 times higher than in untreated cells. The transport system shows a rather broad specificity and catalyses the transport of at least ten neutral and acidic amino acids. Three of these amino acids (l-alanine, l-serine and glycine) are transported by the proline system as well. The system is specific for l-amino acids and has a pH optimum between 5 and 6. Transport by this system seems to be active, since amino acids are accumulated inside the cells.     

Protonmotive force in freshwater sulfate-reducing bacteria,and its role in sulfate accumulation in Desulfobulbus propionicus

The protonmotive force in several sulfate-reducing bacteria has been determined by means of radiolabelled membrane-permeant probes (tetraphenyl-phosphonium cation, TPP⁺, for , and benzoate for pH). In six of ten freshwater strains tested only the pH gradient could be determine, while the membrane potential was not accessible due to nonspecific binding of TPP⁺. The protonmotive force of the other four strains was between –110 and –155 mV, composed of a membrane potential of –80 to –140 mV and a pH gradient between 0.25 and 0.8 (inside alkaline) at pH_out=7. In Desulfobulbus propionicus the pH gradient decreased with rising external pH values. This decrease, however, was compensated by an increasing membrane potential. Sulfate, which can be highly accumulated by the cells, did not affect the protonmotive force, if added in concentrations of up to 4 mM. The highest sulfate accumulation observed (2500-fold), which occurred at external sulfate concentrations below 5 M, could be explained by a symport of three protons per sulfate, if equilibrium with the protonmotive force was assumed. At higher sulfate concentrations the accumulation decreased and suggested an electroneutral symport of two protons per sulfate. At sulfate concentrations above 500 M, the cells stopped sulfate uptake before reaching an equilibrium with the protonmotive force.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - MOPS morpholinopropanesulfonic acid - TPP⁺ tetraphenylphosphonium cation - EDTA ethylenediaminetetraacetic acid - pH transmembrane pH gradient (pH_in-pH_out) - transmembrane electrical potential difference     

Effects of starvation on valine and alanine transport across the intestinal mucosal border in sea bass,Dicentrarchus labrax

Summary kinetics of intestinal transport of l-alanine and l-valine (substrates of the A-system and the L-system, respectively, in mammals) across the brush-border membrane in sea bass, Dicentrarchus labrax, were studied on intact mucosa using a short-term uptake technique. When fish were starved for 4–8 weeks, total influx (mucosa-to-cell) of valine fell owing to disappearance or modification of the diffusion component. The maximum influx rate of saturable component increased but its affinity (reflected by the Michaelis constant) decreased. Alanine transport by Na⁺-dependent and diffusion pathways was unchanged after starvation Fasting also induced an almost 20% decrease in the length of intestinal microvilli.Abbreviations K _d diffusional constant - K _m Michaelis constant - V _max maximum influx rate     

Bioenergetics of lactic acid bacteria: cytoplasmic pH and osmotolerance

Abstract Lactic acid bacteria maintain a cytoplasm that is more alkaline than the medium, but whose pH decreases as the medium is acidified during growth and fermentation. Streptococci generally acidify the cytoplasm from approximately pH 7.6 to 5.7 (external pH 4.5) before growth and then fermentation cease. The internal enzyme machinery of these anaerobic fermenters thus tolerates a fairly wide range in internal proton concentration. Lactobacilli tolerate a significantly more acidic cytoplasmic pH of 4.4 (external pH 3.5). However, when the cytoplasmic pH decreases below a threshold pH, which depends on the organism cellular functions are inhibited. Fermentation end-products, such as organic acids or alcohols, exert their deleterious effects by bringing about acidification of the cytoplasm below the permissible pH. Organic acids, which act as protonophores, or solvents, which perturb membrane phospholipids, at high concentrations increase the inward leak of H⁺ so that H⁺ efflux is not rapid enough to alkalinize the cytoplasm. The membrane pH gradient is thus dissipated.
A specific strain of Lactobacillus acidophilus has been found to be unusually osmotolerant. The osmoresistance is due to the cells' capacity to accumulate glycine betaine by a transport carrier that is activated, but not induced, by high medium osmotic pressure.     

Manipulating cytoplasmic pH under anoxia: A critical test of the role of pH in the switch from aerobic to anaerobic metabolism

Ethanol production by maize (Zea mays L.) root tips, measured by an enzymic assay of the suspending medium, was correlated with changes in the cytoplasmic pH, determined by in-vivo ³¹P nuclear magnetic resonance (NMR) spectroscopy, following the onset of anoxia. Strong evidence for the role of the cytoplasmic pH in triggering the switch to ethanol production under anoxia was obtained by: (i) varying the pH of the suspending medium between pH 4 and pH 10; and (ii) using the permeant weak base methylamine to combat the acidification of the cytoplasm induced by the anoxic conditions. Experimentally, it proved to be much easier to manipulate the cytoplasmic pH under anoxia after the pH had stabilised, rather than during the initial rapid acidification that occurred following the onset of anoxia, and in the presence of methylamine, it was possible to impose a normal aerobic cytoplasmic pH value on tissue that was metabolising anaerobically. By this means it was possible to demonstrate the reversibility of the pH effect on ethanol production under anoxia and thus to provide good evidence in support of the biochemical pH-stat model of the anoxic response. The NMR measurement of the cytoplasmic pH in the presence of methylamine was achieved by using a manganese pretreatment technique to eliminate interference between the cytoplasmic and vacuolar P_i signals, and it seems likely that the experimental approach used here will have further applications in studies of the metabolic response to anoxia.Abbreviations Caps 3-(cyclohexylamino)-1-propane sulphonic acid - Mes 2-(N-morpholino)-ethane sulphonic acid - NMR nuclear magnetic resonance - Pi inorganic phosphate We acknowledge the financial support of the Agricultural and Food Research Council and G.G.F. acknowledges the receipt of a Research Fellowship from the Royal Commission for the Exhibition of 1851.     

Active transport of oxalate by Pseudomonas oxalaticus OX1

Membrane vesicles isolated from oxalategrown cells of Pseudomonas oxalaticus accumulated oxalate by an inducible transport system in unmodified form against a concentration gradient. This accumulation was dependent on the presence of a suitable electron donor system such as ascorbate-phenazinemethosulphate. In the presence of this energy source, steady state levels of accumulation of oxalate were 10–20-fold higher than in its absence. The oxalate transport system involved showed a high affinity for oxalate (K _m =11 M) and was highly specific. Oxalate transport was not affected by the presence of other dicarboxylic acids, such as malate, succinate and fumarate and only partly inhibited by acetate. The energy requirement for oxalate transport is discussed and it is concluded that this requirement is most likely equivalent to 1 mole of ATP per mole of oxalate.Abbreviation PMS phenazinemethosulphate