Amino Acid transport into membrane vesicles isolated from zucchini : evidence of a proton-amino Acid symport in the plasmalemma

Several lines of evidence with intact tissues suggest amino acid transport is mediated by a proton-amino acid symport (L Rheinhold, A Kaplan 1984 Annu Rev Plant Physiol 35: 45-83). However, biochemical studies of proton-coupled amino acid transport in isolated membrane vesicles have not been reported. In the experiments presented here, amino acid transport was studied in membrane vesicles isolated from zucchini (Cucurbita pepo L. cv Black Beauty) hypocotyls. An imposed pH gradient (basic interior) was used to energize isolated membrane vesicles and drive amino acid transport. Proton-coupled amino acid accumulation was demonstrated for alanine, glutamate, glutamine, leucine, and tabtoxinine-β-lactam. Alanine transport into the isolated membrane vesicles was studied in detail. Alanine transport was protonophore sensitive and accumulation ratios exceeding 10 times that predicted by diffusion alone were observed. ΔpH-Dependent alanine transport exhibited saturation kinetics, suggesting translocation was mediated via a carrier transport system. In support of that conclusion, 50 micromolar N,N′-dicyclohexylcarbodiimide, a hydrophobic modifier of protein carboxyls, completely inhibited proton-coupled alanine accumulation. Transport activity, equilibrated on a linear sucrose gradient, peaked at 1.16 grams per cubic centimeter and co-migrated with a plasmalemma marker (vanadate-sensitive K⁺-Mg²⁺-ATPase). These results provide direct evidence in support of a proton-amino acid symport in the plasmalemma of higher plants.     

DeltapH-Dependent Amino Acid Transport into Plasma Membrane Vesicles Isolated from Sugar Beet (Beta vulgaris L.) Leaves: II. Evidence for Multiple Aliphatic, Neutral Amino Acid Symports

Proton-coupled aliphatic, neutral amino acid transport was investigated in plasma membrane vesicles isolated from sugar beet (Beta vulgaris L., cv Great Western) leaves. Two neutral amino acid symport systems were resolved based on inter-amino acid transport competition and on large variations in the specific activity of each porter in different species. Competitive inhibition was observed for transport competition between alanine, methionine, glutamine, and leucine (the alanine group) and between isoleucine, valine, and threonine (the isoleucine group). The apparent K_m and K_i values were similar for transport competition among amino acids within the alanine group. In contrast, the kinetics of transport competition between these two groups of amino acids did not fit a simple competitive model. Furthermore, members of the isoleucine group were weak transport antagonists of the alanine group. These results are consistent with two independent neutral amino acid porters. In support of that conclusion, the ratio of the specific activity of alanine transport versus isoleucine transport varied from two- to 13-fold in plasma membrane vesicles isolated from different plant species. This ratio would be expected to remain relatively stable if these amino acids were moving through a single transport system and, indeed, the ratio of alanine to glutamine transport varied less than twofold. Analysis of the predicted structure of the aliphatic, neutral amino acids in solution shows that isoleucine, valine, and threonine contain a branched methyl or hydroxyl group at the β-carbon position that places a dense electron cloud close to the α-amino group. This does not occur for the unbranched amino acids or those that branch further away, e.g. leucine. We hypothesize that this structural feature of isoleucine, valine, and threonine results in unfavorable steric interactions with the alanine transport system that limits their flux through this porter. Hydrophobicity and hydrated volumes did not account for the observed differences in transport specificity.     

Proton-Coupled Sucrose Transport in Plasmalemma Vesicles Isolated from Sugar Beet (Beta vulgaris L. cv Great Western) Leaves

Sucrose is the predominant form of photosynthetically reduced carbon transported in most plant species. In the experiments reported here, an active, proton-coupled sucrose transport system has been identified and partially characterized in plasmalemma vesicles isolated from mature sugar beet (Beta vulgaris L. cv Great Western) leaves. The isolated vesicles concentrated sucrose fivefold in the presence of an imposed pH gradient (basic interior). The presence of carbonyl cyanide m-chlorophenylhydrazone, a protonophore, prevented sucrose accumulation within the vesicles. ΔpH-dependent sucrose transport exhibited saturation kinetics with an apparent K_m of 1.20 ± 0.40 millimolar, suggesting translocation was carrier-mediated. In support of that conclusion, two protein modifiers, diethyl pyrocarbonate and p-chloromercuribenzenesulfonic acid, were found to be potent inhibitors with 50% inactivation achieved at 750 and 30 micromolar, respectively. ΔpH-Dependent sucrose transport was not inhibited by glucose, fructose, raffinose, or maltose suggesting the transport system was specific for sucrose. Transport activity was associated with the plasmalemma because ΔpH-dependent sucrose transport equilibrated on a linear sucrose gradient at 1.17 grams per cubic centimeter and comigrated with a plasmalemma enzyme marker, vanadate-sensitive K⁺, Mg²⁺-ATPase. Taken together, these results provide the first In vitro evidence in support of a sucrose-proton symport in the plasmalemma of mature leaf tissue.     

Electrogenicity, pH-Dependence, and Stoichiometry of the Proton-Sucrose Symport

The electrogenicity, pH-dependence, and stoichiometry of the proton-sucrose symport were examined in plasma membrane vesicles isolated from sugar beet (Beta vulgaris L. cv Great Western) leaves. Symport mediated sucrose transport was electrogenic as demonstrated by the effect of membrane potential on ΔpH-dependent flux. In the absence of significant charge compensation, a low rate of sucrose transport was observed. When membrane potential was clamped at zero with symmetric potassium concentrations and valinomycin, the rate of sucrose flux was stimulated fourfold. In the presence of a negative membrane potential, transport increased six-fold. These results are consistent with electrogenic sucrose transport which results in a net flux of positive charge into the vesicles. The effect of membrane potential on the kinetics of sucrose transport was on V_max only with no apparent change in K_m. Sucrose transport rates driven by membrane potential only, i.e. in the absence of ΔpH, were comparable to ΔpH-driven flux. Both membrane potential and ΔpH-driven sucrose transport were used to examine proton binding to the symport and the apparent K_m for H⁺ was 0.7 micromolar. The kinetics of sucrose transport as a function of proton concentration exhibited a simple hyperbolic relationship. This observation is consistent with kinetic models of ion-cotransport systems when the stoichiometry of the system, ion:substrate, is 1:1. Quantitative measurements of proton and sucrose fluxes through the symport support a 1:1 stoichiometry. The biochemical details of protoncoupled sucrose transport reported here provide further evidence in support of the chemiosmotic hypothesis of nutrient transport across the plant cell plasma membrane.     

Interamino Acid Inhibition of Transport in Higher Plants : EVIDENCE FOR TWO TRANSPORT CHANNELS WITH ASCERTAINABLE AFFINITIES FOR AMINO ACIDS

Data from published experiments were analyzed to determine the number and specificities of amino acid transport channels in cells of higher plants. Each experiment measured the uptake of a labeled amino acid in the presence of unlabeled amino acids, used one at a time, in the incubating medium. The observed interamino acid inhibitions can be accounted for by two transport channels, each with characteristic affinities that were computed from the observed interamino acid inhibitions. The first channel is a general transport system with the following relative affinities for the amino acids: methionine 75, alanine 75, phenylalanine 64, tyrosine 64, leucine 63, cysteine 58, serine 57, glycine 56, tryptophan 54, glutamine 51, threonine 49, valine 44, isoleucine 44, glutamic acid 44, proline 43, histidine 33, lysine 32, asparagine 22, arginine 22, aspartic acid 18. The second channel is a basic amino acid tranport system with relative affinities for arginine, lysine, and histidine of 66, 39, and 21, respectively. The affinities for the other acids in the second channel are lower. Despite considerable diversity in the species, tissues, and solute concentrations employed in the experiments, multiple regression equations (Y = α + β₁X₁ + βX₂, in which Y is the observed transport inhibition and X₁ and X₂ are the relative transport affinities of the two channels) account for 50 to 99% of the variance in all but six experiments, five of which employed unusually high solute concentrations.     

Tabtoxinine-beta-Lactam Transport into Cultured Corn Cells : Uptake via an Amino Acid Transport System

Tabtoxinine-β-lactam (T-β-L), a unique amino acid, is a toxin produced by several closely related pathovars of Pseudomonas syringae. These chlorosis-inducing pathogens establish themselves in the apoplastic space of their hosts where they release the toxin. We have examined the transport of T-β-L into cultured corn (Zea mays cv Black Mexican) cells using [¹⁴C]T-β-L. The pH optimum of the uptake of the toxin was between 4.0 and 5.5 pH units. Toxin uptake was inhibited by the protonophore, carbonyl cyanide m-chlorophenyl hydrazone, and by the sulfhydryl re-agent, N-ethylmaleimide. Tabtoxinine-β-lactam transport exhibited saturation kinetics that were described by the Michaelis-Menton equation for toxin concentrations of 1 millimolar and less. However, the transport of toxin in concentrations greater than 1 millimolar was not described by Michaelis-Menten kinetics. Glutamate and alanine exhibited similar transport kinetics with a transition to non-Michaelis-Menten kinetics when the amino acid concentration exceeded 1 millimolar. Hill numbers for glutamate, alanine, and T-β-L ranged from 0.6 to 0.8. Methionine, alanine, tyrosine, glutamine, glutamate, and arginine were inhibitors of toxin transport. Alanine was a competitive inhibitor of the transport of T-β-L and of glutamate. The data are consistent with T-β-L being transported into the plant cell through an amino acid transport system.     

Amino Acid Composition of the Host-specific Toxin of Helminthosporium carbonum

The host-specific toxin of Helminthosporium carbonum (C₃₂H₅₀N₆O₁₀) was hydrolyzed by 6 n HCl to yield a number of α-amino acids. The common amino acids, proline and alanine, occurred in a ratio of 1:2. Two other unstable α-amino acids that produced lower color values with ninhydrin were also produced. One of these was tentatively identified as 2-amino-2,3-dehydro-3-methylpentanoic acid by electrolytic reduction to isoleucine. Additional ninhydrin-reacting substances were produced in low yield and probably represented secondary hydrolysis products of the unstable amino acids. The finding of an α,β-unsaturated linkage in H. carbonum toxin explains the instability of the compound and may also account for its specific toxicity.     

Effect of pH and Monensin on Glucose Transport by Fibrobacter succinogenes, a Cellulolytic Ruminal Bacterium

Fibrobacter succinogenes S85, a cellulolytic ruminal bacterium, required sodium for growth and glucose uptake. Cells which were deenergized with iodoacetate (500 μM) could not take up [¹⁴C]glucose. However, deenergized cells which were treated with valinomycin, loaded with potassium, and diluted into sodium or sodium plus potassium to create an artificial electrical gradient (ΔΨ) plus a chemical gradient of sodium (ΔpNa) or ΔpNa alone transported glucose at a rapid rate. Cells which were loaded with potassium plus sodium and diluted into sodium (ΔΨ with sodium, but no ΔpNa) also took up glucose at a rapid rate. Potassium-loaded cells that were diluted into buffers which did not contain sodium (ΔΨ without sodium) could not take up glucose. An artificial ZΔpH which was created by acetate diffusion could not drive glucose transport even if sodium was present. The maximum rate and affinity of glucose transport (pH 6.7) were 62.5 nmol/mg of protein per min and 0.51 mM, respectively. S85 was unable to grow at a pH of less than 5.5, and there was little glucose transport at this pH. When the extracellular pH was decreased, the glucose carrier was inhibited, intracellular pH declined, the cells were no longer able to metabolize glucose, and ΔΨ declined. Monensin (1 μM) or lasalocid (5 μM) decreased intracellular ATP and dissipated both the ΔΨ and ΔpNa. Since there was no driving force for transport, glucose transport was inhibited. These results indicated that F. succinogenes used a pH-sensitive sodium symport mechanism to take up glucose and that either a ΔΨ or a ΔpNa was required for glucose transport.     

Amino Acid Metabolism of Lemna minor L. : II. Responses to Chlorsulfuron

Chlorsulfuron, an inhibitor of acetolactate synthase (EC 4.1.3.18) (TB Ray 1984 Plant Physiol 75: 827-831), markedly inhibited the growth of Lemna minor at concentrations of 10⁻⁸ molar and above, but had no inhibitory effects on growth at 10⁻⁹ molar. At growth inhibitory concentrations, chlorsulfuron caused a pronounced increase in total free amino acid levels within 24 hours. Valine, leucine, and isoleucine, however, became smaller percentages of the total free amino acid pool as the concentration of chlorsulfuron was increased. At concentrations of chlorsulfuron of 10⁻⁸ molar and above, a new amino acid was accumulated in the free pool. This amino acid was identified as α-amino-n-butyrate by chemical ionization and electron impact gas chromatography-mass spectrometry. The amount of α-amino-n-butyrate increased from undetectable levels in untreated plants, to as high as 840 nanomoles per gram fresh weight (2.44% of the total free pool) in plants treated with 10⁻⁴ molar chlorsulfuron for 24 hours. The accumulation of this amino acid was completely inhibited by methionine sulfoximine. Chlorsulfuron did not inhibit the methionine sulfoximine induced accumulations of valine, leucine, and isoleucine, supporting the idea that the accumulation of the branched-chain amino acids in methionine sulfoximine treated plants is the result of protein turnover rather than enhanced synthesis. Protein turnover may be primarily responsible for the failure to achieve complete depletion of valine, leucine, and isoleucine even at concentrations of chlorsulfuron some 10⁴ times greater than that required to inhibit growth. Tracer studies with ¹⁵N demonstrate that chlorsulfuron inhibits the incorporation of ¹⁵N into valine, leucine, and isoleucine. The α-amino-n-butyrate accumulated in the presence of chlorsulfuron and [¹⁵N]H₄⁺ was heavily labeled with ¹⁵N at early time points and appeared to be derived by transamination from a rapidly labeled amino acid such as glutamate or alanine. We propose that chlorsulfuron inhibition of acetolactate synthase may lead to accumulation of 2-oxobutyrate in the isoleucine branch of the pathway, and transamination of 2-oxobutyrate to α-amino-n-butyrate by a constitutive transaminase utilizing either glutamate or alanine as α-amino-N donors.     

Type 1 Sodium-dependent Phosphate Transporter (SLC17A1 Protein) Is a Cl?-dependent Urate Exporter

SLC17A1 protein (NPT1) is the first identified member of the SLC17 phosphate transporter family and mediates the transmembrane cotransport of Na⁺/P_i in oocytes. Although this protein is believed to be a renal polyspecific anion exporter, its transport properties are not well characterized. Here, we show that proteoliposomes containing purified SLC17A1 transport various organic anions such as p-aminohippuric acid and acetylsalicylic acid (aspirin) in an inside positive membrane potential (Δψ)-dependent manner. We found that NPT1 also transported urate. The uptake characteristics were similar to that of SLC17 members in its Cl⁻ dependence and inhibitor sensitivity. When arginine 138, an essential amino acid residue for members of the SLC17 family such as the vesicular glutamate transporter, was specifically mutated to alanine, the resulting mutant protein was inactive in Δψ-dependent anion transport. Heterologously expressed and purified human NPT1 carrying the single nucleotide polymorphism mutation that is associated with increased risk of gout in humans exhibited 32% lower urate transport activity compared with the wild type protein. These results strongly suggested that NPT1 is a Cl⁻-dependent polyspecific anion exporter involved in urate excretion under physiological conditions.     

The Proton Electrochemical Transmembrane Gradients Generated by the Transfer Cells of the Haustorium of Polytrichum formosum and Their Use in the Uptake of Amino Acids

The epidermal cells of the sporophyte haustorium of Polytrichum formosum are modified into transfer cells. These cells are located in a strategic place allowing them to control the exchanges between the two generations. Their plasmalemma creates proton gradients (Δψ and ΔpH) which increase during the development of the sporophyte. As the sporophyte grows from 2 to 4 cm long, the pH of the incubation medium of the haustoria decreases from 5.2 to 4.3, and the transmembrane potential difference (PD) hyperpolarizes form −140 to −210 millivolts. These gradients become rapidly larger than that generated by the plasmalemma of the basal cells of the sporophyte. They are used to energize the uptake of the solutes present in the apoplast of the gametophyte, particularly the amino acids. Below 20 micromolar α-aminoisobutyric acid uptake in the transfer cells is mediated by a saturable system and is optimal at acidic pH (4.0 and 4.5). It is strongly inhibited by compounds dissipating both Δψ and ΔpH (10 micromolar carbonylcyanide-m-chlorophenyl hydrazone) or only Δψ (0.1 molar KCl). The absorption of α-aminoisobutyric acid and of the other neutral amino acids tested induces an alkalinization of the medium and a depolarization of membrane potential difference which is concentration dependent. These data show that the uptake of amino acids by the transfer cells of the haustorium is a secondary translocation (proton-amino acid symport) energized by a primary translocation (proton efflux). More particularly, they show that transfer cells possess a membrane enzymic equipment particularly efficient to achieve the uptake of the solutes leaked in the apoplast from other cell types.     

Active transport of nonpolar amino acids in Chromatium vinosum

The photosynthetic purple sulfur bacterium, Chromatium vinosum, takes up the amino acids, L-phenylalanine and L-leucine, via two apparently different electrogenic, H+/amino acid symports. Na+ serves as an allosteric modulator for leucine transport, lowering the Km for leucine from 66 to 15 microM. C. vinosum cells also contain a system that transports both isoleucine and valine. The isoleucine/valine system has the attributes of a H+/amino acid symport at pH less than 7.5 but appears to function as a H+/Na+ (Li+)/amino acid symport at pH greater than or equal to 7.5. Na+ gradients produce an allosteric lowering of the Km values for both isoleucine and valine, from 14 to 7 microM and from 34 to 17 microM, respectively. C. vinosum also accumulates D-alanine in an energy-dependent reaction. The transport process appears to involve the electrogenic cotransport of D-alanine and Na+. The Km value for D-alanine was determined to be 9 microM. Unlike the previously characterized C. vinosum L-alanine/Na+ symport, Na+ gradients did not affect the Km for D-alanine transport. L-Alanine and glycine, but not alpha-aminoisobutyric acid, act as competitive inhibitors for D-alanine transport.     

A Phosphate-Bond-Driven Dipeptide Transport System in Streptococcus cremoris Is Regulated by the Internal pH

The uptake of amino acids and peptides by Streptococcus cremoris is mediated by different highly specific transport systems. The leucine transport system has a high affinity only for leucine, isoleucine, and valine and no affinity for leucyl-peptides. The transport system for leucyl-leucine is strongly inhibited by several dipeptides with hydrophobic, neutral, N-terminal amino acids but not by leucine. The leucyl-leucine transport system has a high affinity for dipeptides containing β-methyl groups in the side chain; the C terminus of the dipeptide affects the affinity to a much lower extent. Leucyl-leucine transport in whole cells was studied as a function of the internal pH at different external pH values in the presence and absence of nigericin. The internal pH was shown to be an important controlling factor in leucyl-leucine uptake, but the ΔpH was not involved as a driving force. At increasing external pH values, the affinity of the transport system for leucyl-leucine decreased. Uptake of leucyl-leucine was also studied in the presence of arsenate, which inhibited ATP synthesis by substrate-level phosphorylation. The rate of leucyl-leucine transport appeared to be dependent on the intracellular ATP concentrations. These results indicate that the energy for the leucyl-leucine transport is directly supplied by ATP.     

Energetics of Sodium Transport in Frog Skin : II. The effects of electrical potential on oxygen consumption

Studies were made of the dependence of the rate of oxygen consumption, J_r, on the electrical potential difference, Δψ, across the frog skin. After the abolition of sodium transport by ouabain the basal oxygen consumption was independent of Δψ. In fresh skins J_r was a linear function of Δψ over a range of at least ±70 mv. Treatment with aldosterone stimulated the short-circuit current, I_o, and the associated rate of oxygen consumption, J_ro, and increased their stability; linearity was then demonstrable over a range of ±160 mv. Brief perturbations of Δψ (±30–200 mv) did not alter subsequent values of I_o. Perturbations for 10 min or more produced a "memory" effect both with and without aldosterone: accelerating sodium transport by negative clamping lowered the subsequent value of I_o; positive clamping induced the opposite effect. Changes in J_ro were more readily detectable in the presence of aldosterone; these were in the same direction as the changes in I_o. The linearity of J_r in Δψ indicates the validity of analysis in terms of linear nonequilibrium thermodynamics—brief perturbations of Δψ appear to produce no significant effect on either the phenomenological coefficients or the free energy of the metabolic driving reaction. Hence it is possible to evaluate this free energy.     

Identification of Genes Encoding Conjugated Bile Salt Hydrolase and Transport in Lactobacillus johnsonii 100-100

Cytosolic extracts of Lactobacillus johnsonii 100-100 (previously reported as Lactobacillus sp. strain 100-100) contain four heterotrimeric isozymes composed of two peptides, α and β, with conjugated bile salt hydrolase (BSH) activity. We now report cloning, from the genome of strain 100-100, a 2,977-bp DNA segment that expresses BSH activity in Escherichia coli. The sequencing of this segment showed that it contained one complete and two partial open reading frames (ORFs). The 3′ partial ORF (927 nucleotides) was predicted by BLAST and confirmed with 5′ and 3′ deletions to be a BSH gene. Thermal asymmetric interlaced PCR was used to extend and complete the 948-nucleotide sequence of the BSH gene 3′ of the cloned segment. The predicted amino acid sequence of the 5′ partial ORF (651 nucleotides) was about 80% similar to the C-terminal half of the largest, complete ORF (1,353 nucleotides), and these two putative proteins were similar to several amine, multidrug resistance, and sugar transport proteins of the major facilitator superfamily. E. coli DH5α cells transformed with a construct containing these ORFs, in concert with an extracellular factor produced by strain 100-100, demonstrated levels of uptake of [¹⁴C]taurocholic acid that were increased as much as threefold over control levels. [¹⁴C]Cholic acid was taken up in similar amounts by strain DH5α pSportI (control) and DH5α p2000 (transport clones). These findings support a hypothesis that the ORFs are conjugated bile salt transport genes which may be arranged in an operon with BSH genes.     

Decrease of pH Gradients in Tonoplast Vesicles by NO(3) and Cl: Evidence for H-Coupled Anion Transport

Chloride or nitrate decreased a pH gradient (measured as [¹⁴C]methylamine accumulation) in tonoplast-enriched vesicles. The ΔpH decrease was dependent on the anion concentration. These effects are independent of the anion-sensitive H⁺-ATPase of the tonoplast, since the pH gradient (acid inside) was imposed artificially using a pH jump or a K⁺ gradient and nigericin. 4,4′-Diisothiocyano-2,2′-stilbene disulfonic acid partially prevented the decrease in pH gradient induced by Cl⁻. Two possible models to account for this anion-dependent decrease of ΔpH are: (a) H⁺ loss is accompanied by Cl⁻ or NO₃⁻ efflux from the vesicles via H⁺/anion symport systems on the tonoplast and (b) H⁺ loss is accompanied by Cl⁻ or NO₃⁻ uptake into the vesicles via H⁺/anion antiport systems. Depending on the requirements and conditions of the cell, these two systems would serve to either mobilize Cl⁻ and NO₃⁻ stored in the vacuole for use in the cytoplasm or to drive anions into the vacuole. Chloride or nitrate also decreased a pH gradient in fractions containing plasma membrane and Golgi, implying that these membranes may have similar H⁺-coupled anion transport systems.     

Stimulation of Lysine Decarboxylase Production in Escherichia coli by Amino Acids and Peptides

A commercial hydrolysate of casein stimulated production of lysine decarboxylase (EC 4.1.1.18) by Escherichia coli B. Cellulose and gel chromatography of this hydrolysate yielded peptides which were variably effective in this stimulation. Replacement of individual, stimulatory peptides by equivalent amino acids duplicated the enzyme levels attained with those peptides. There was no indication of specific stimulation by any peptide. The peptides were probably taken up by the oligopeptide transport system of E. coli and hydrolyzed intracellularly by peptidases to their constituent amino acids for use in enzyme synthesis. Single omission of amino acids from mixtures was used to screen them for their relative lysine decarboxylase stimulating abilities. Over 100 different mixtures were evaluated in establishing the total amino acid requirements for maximal synthesis of lysine decarboxylase by E. coli B. A mixture containing all of the common amino acids except glutamic acid, aspartic acid, and alanine increased lysine decarboxylase threefold over an equivalent weight of casein hydrolysate. The nine most stimulatory amino acids were methionine, arginine, cystine, leucine, isoleucine, glutamine, threonine, tyrosine, and asparagine. Methionine and arginine quantitatively were the most important. A mixture of these nine was 87% as effective as the complete mixture. Several amino acids were inhibitory at moderate concentrations, and alanine (2.53 mM) was the most effective. Added pyridoxine increased lysine decarboxylase activity 30%, whereas other B vitamins and cyclic adenosine 5′-monophosphate had no effect.     

Components of Sodium and Chloride Flux Across Toad Bladder

The effect of transepithelial potential difference (ψ) on Na and Cl flux across toad bladder was assessed by measuring isotopic flux between identical media at various values of ψ. The contribution of edge damage to ionic permeability was eliminated, resulting in relatively high spontaneous ψ (-97 ±4 mv) and low electrical conductance g. Bidirectional Na fluxes were measured simultaneously. Unidirectional Cl fluxes were measured in paired hemibladders at ψ = 0 mv or -97 mv. Net Na flux J_Na, at ψ = 0 mv, was slightly less than short-circuit current (SCC). At ψ = -97 mv, J_Na averaged 17% of SCC, and was sometimes zero. ΔJ_Na/Δψ (= g₊) averaged 60% of g between -97 mv and +75 mv; at -150 mv, g₊ fell, indicating rectification. Analysis of unidirectional Na fluxes indicates low passive conductance (1.5 μmho/mg wet weight), a bidirectional, electrically neutral flux of approximately 0.13 μa/mg, and relatively large conductance of the active transport path at ψ ≥ -97 mv. The absence of appreciable transstimulation of serosal (S)-to-mucosal (M) Na flux (in response to increasing mucosal Na concentration) indicates that the electrically neutral flux is not exchange diffusion in the usual sense. Analysis of Cl fluxes indicates similar values for passive conductance and neutral flux, suggesting linked neutral flux of Na and Cl. Either the electromotive force of the Na pump E, its conductance g_a, or both are strong functions of ψ. The product of these two quantities, Eg_a, is a measure of the “transport capacity” at any given value of ψ, independent of the direct effect of ψ on J_Na through the pump path. Eg_a varies with ψ. Hence estimation of the net Na flux or current at any one value of ψ, including ψ = 0, fails to reveal the maximal transport capacity of the pump, its resting electromotive force (when J_Na = 0 through the pump), or the dependence of transport capacity on potential.     

Relationship between Intracellular Phosphate, Proton Motive Force, and Rate of Nongrowth Energy Dissipation (Energy Spilling) in Streptococcus bovis JB1

When the rate of glucose addition to nongrowing Streptococcus bovis cell suspensions was increased, the fermentation was homolactic, fructose-1,6-diphosphate (FDP) increased, intracellular inorganic phosphate (P_i) declined, and the energy-spilling rate increased. ATP and ADP were not significantly affected by glucose consumption rate, but the decrease in P_i was sufficient to cause an increase in the free energy of ATP hydrolysis (ΔG′p). The increase in ΔG′p was correlated with an increase in proton motive force (Δp). S. bovis continuous cultures (dilution rate of 0.65 h⁻¹) that were provided with ammonia as the sole nitrogen source also had high rates of lactate production and energy spilling. When Trypticase was added as a source of amino acids, lactate production decreased; a greater fraction of the glucose was converted to acetate, formate, and ethanol; and the energy-spilling rate decreased. Trypticase also caused a decrease in FDP, an increase in P_i, and a decrease in Δp. The change in Δp could be explained by P_i-dependent changes in the ΔG′p. When P_i declined, ΔG′p and Δp increased. The ratio of ΔG′p to Δp (millivolt per millivolt) was always high (>4) at low rates of energy spilling but declined when the energy-spilling rate increased. Based on these results, it appears that Δp and the energy-spilling rate are responsive to fluctuations in the intracellular P_i concentration.     

Transport of methionine and proline by rat liver slices and the effect of certain hormones

1. Transport characteristics of l-methionine and l-proline in rat liver slices in vitro were studied. 2. Intracellular concentration gradients for methionine were obtained. 3. Methionine uptake was inhibited by iodoacetate, dinitrophenol, Na⁺-free media and also by glycine, lysine, cysteine and dithiothreitol but not by α-aminoisobutyrate. 4. The rate of methionine metabolism in the slice was slow. 5. Puromycin inhibited methionine incorporation into protein, but not methionine uptake. 6. Methionine inhibited the transport of α-aminoisobutyrate but not of cystine. 7. Efflux and exchange diffusion of methionine was studied. 8. Amino acid transport in rat liver slices was not affected by thyroidectomy. 9. Addition of insulin, glucagon, adrenaline or cortisol did not affect the transport of methionine. 10. Addition of 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate increased methionine transport after a 120min incubation period in some experiments. 11. Studies of l-proline transport were invalidated because of the rapid evolution of CO₂ from the substrate.