Mechanisms of phosphate uptake into brush-border membrane vesicles from goat jejunum

This study concerns the uptake of inorganic phosphate into brush-border membrane vesicles prepared from jejunal tissues of either control or Ca-and/or P-depleted goats. The brush-border membrane vesicles showed a time-dependent accumulation of inorganic phosphate with a typical overshoot phenomenon in the presence of an inwardly directed Na⁺ gradient. The Na⁺-dependent inorganic phosphate uptake was completely inhibited by application of 5 mmol·l^-1 sodium arsenate. Half-maximal stimulation of inorganic phosphate uptake into brush-border membrane vesicles was found with Na⁺ concentrations in the order of 5 mmol·l^-1. Inorganic phosphate accumulation was not affected by a K⁺ diffusion potential (inside negative), suggesting an electroneutral transport process. Stoichiometry suggested an interaction of two or more Na ions with one inorganic phosphate ion at pH 7.4. Na⁺-dependent inorganic phosphate uptake into jejunal brush-border membrane vesicles from normal goats as a function of inorganic phosphate concentration showed typical Michaelis-Menten kinetic with V _max=0.42±0.08 nmol·mg^-1 protein per 15 s^-1 and K _m=0.03±0.01 mmol·l^-1 (n=4, x ±SEM). Long-term P depletion had no effect on these kinetic parameters. Increased plasma calcitriol concentrations in Ca-depleted goats, however, were associated with significant increases of V _max by 35–80%, irrespective of the level of P intake. In the presence of an inwardly directed Na⁺ gradient inorganic phosphate uptake was significantly stimulated by almost 60% when the external pH was decreased to 5.4 (pH_out/pH_in=5.4/7.4). The proton gradient had no effect on inorganic phosphate uptake in absence of Na⁺. In summary, in goats Na⁺ and calcitriol-dependent mechanisms are involved in inorganic phosphate transport into jejunal brush-border membrane vesicles which can be stimulated by protons.Abbreviations AP activity of alkaline phosphatase - BBMV brush-border membrane vesicles - EGTA ethyleneglycol-triacetic acid - n _app apparent Hill coefficient - P _i inorganic phosphate - PTH parathyroid hormone     

Ca binding to the human red cell membrane: Characterization of membrane preparations and binding sites

Summary Inside out and right side out vesicles were used to study the sidedness of Ca binding to the human red cell membrane. It was shown that these vesicles exhibited only a limited permeability to Ca, enabling the independent characterization of Ca binding to the extracellular and cytoplasmic membrane surfaces. Ca binding was studied in 10 mM Tris HCl at pH 7.4, 22±2°C and was shown to be complete in under 5 min. Scatchard plots were made from Ca binding data obtained at free Ca concentrations in the range of 10^–6 to 10^–3M. Under these conditions inside out vesicles exhibit two independent binding sites for Ca with association constants of 1×10⁵ and 6×10³ M^–1, and right side out vesicles exhibit three independent binding sites with association constants of 2×10⁵, 1.4×10⁴ and 3×10²M^–1. Upon the addition of 0.1M KCl a third, high affinity site was found on inside out vesicles with an association constant of 3×10⁵, (in 0.1 M KCl). Ca binding to inside out vesicles increased nearly linearly with pH in the, range of pH 4 to pH 11, while binding to right side out vesicles remained practically unchanged in the range of pH 7 to pH 9. Progressive increase of the ionic strength of the medium by the addition of K, Mg or Tris decreased Ca binding to inside out vesicles as did the addition of ATP. Comparison of a series of cation competitors for Ca binding sites on inside out vesicles at 0.003 mM Ca showed that La was the most effective competitor of all while Cd was the most effective divalent cation competitor of those tested. Our findings suggest that the effects of low concentrations of Ca at the inner surface of the red cell membrane are mediated primarily through Ca binding to site 1 (and, possibly site 2) of inside out vesicles of which there are approximately 1.6×10⁵ per equivalent cell.     

NMR study of rapid water diffusion across lipid bilayers in dipalmitoyl lecithin vesicles

A method for measurement of rapid diffusional exchange between external and internal water in lecithin vesicles is described. Paramagnetic ions were inserted inside DPL vesicles and the NMR relaxation times for water protons were measured as a function of temperature. It was found that water diffusion rate is described by a single activation energy of 15±1 kcal/mole in the temperature range 16 – 35°C and exhibits a maximum at 44°C. The permeability of DPL vesicles to water was calculated to 16–18 × 10^?4 cm/s at 44°C and 1.7 × 10^?4 cm/s at 20°C.     

Measurement of proton-linked transport activities in pyranine loaded chloroplast inner envelope vesicles

A method has been devised for loading chloroplast inner envelope vesicles prepared from pea (Pisum sativum L. var Progress No. 9) or spinach (Spinacia oleracea L.) with 8-hydroxypyrene-1,3,6-trisulfonate (pyranine), a membrane impermeant, fluorescent pH indicator. Two known proton-linked transport activities of the inner envelope, glycolate/H⁺ co-transport and phosphate/phosphoglycerate exchange have been shown to cause quenching of the internal pyranine fluorescence. This represents the first demonstration that these vesicles are sealed and competent for transport measurements. The technique, as it now stands, is essentially qualitative. It does, however, offer advantages over transport measurements with intact chloroplasts, for example compatibility with rapid mixing techniques and accessibility of the transport proteins to antibodies.     

Observation of the phosphatidyl ethanolamine amino proton magnetic resonance in phospholipid vesicles: inside/outside ratios and proton transport.

The amino proton resonance of phosphatidyl ethanolamine in sonicated mixed phospholipid vesicles is observed 3.3 ppm downfield from H₂O. Above pH ～ 5 it is broadened beyond detectability as a result of exchange with H₂O protons. In low salt, resonances of amino protons inside the vesicles appear to persist as the pH is raised, while those on the outside disappear. Solvent catalized proton conduction along the surface is proposed, with an effective -NH₂ to -NH₃ transfer rate of about 8 × 10⁵ sec^?1 at 25°C.     

Combined strategies for liposome characterization during in vitro digestion

Three types of pyranine (HPTS)-containing liposomes were prepared by high-pressure homogenization under optimized conditions. At 37°C, they were 1) fluid-state vesicles made from soybean phosphatidylcholine (SPC), 2) gel-state liposomes made from hydrogenated SPC (HSPC), and 3) solid-disordered membranes obtained from HSPC and cholesterol (HSPC-Chol). These liposome formulations were characterized before, during, and after in vitro digestion, which involved the presence of pH gradients, enzymes, and bile salts. Mean sizes and size distributions of the vesicles were determined by DLS; ³¹P-NMR (nuclear magnetic resonance) was used to quantify lyso-PC forms; internal pH was monitored throughout digestion with two different fluorescent pH probes; and changes in bilayer permeability and HPTS encapsulation were determined by size-exclusion chromatography and fluorimetry. Differential scanning calorimetry analysis was also performed in order to study the effect of digestion on HSPC vesicles. SPC liposomes were physically stable during digestion; they presented 8% lyso-forms and an HPTS encapsulation around 85% after in vitro digestion. However, they were extremely permeable to ions, so that the internal pH immediately equilibrated with the bulk pH. HSPC liposomes were the most affected by the digestive process. Even though they were chemically stable, as inferred from the low lyso-PC content, very important changes in their size distribution were observed. A final 50% HPTS leakage was quantified after in vitro digestion. Nevertheless, they were the least permeable to protons under pH gradients. HSPC-Chol vesicles presented intermediate permeability to protons, having their internal pH decreased from approximately 6.8 to 4.6 after 1 hour of incubation at pH 2. This was the most chemically stable formulation and showed the highest encapsulation, even after in vitro digestion. Therefore, HSPC-Chol liposomes would be the most adequate choice for the design of lipid products for oral administration.     

Permeability of large unilamellar digalactosyldiacylglycerol vesicles for protons and glucose – influence of α-tocopherol, β-carotene,zeaxanthin and cholesterol

The permeability of large unilamellar vesicles formed from digalactosyldiacylglycerol for glucose and protons was measured. The vesicle composition was modified by addition of different terpenoids: α-tocopherol, cholesterol, zeaxanthin and β-carotene. The digalactosyldiacylglycerol species composition was dominated by the species 18:2/18:2 and 18:1/18:2. Using the self-quenching properties of the fluorescent probe 6-carboxyfluorescein trapped in the aqueous space of the vesicles, the permeability for glucose was determined with a glucose gradient of 800 mM. The calculated permeability coefficient for glucose was in the range of 4.85.10^–10 to 1.12.10^–9 cm.s^–1. For proton permeability measurements, the pH-sensitive fluorescent dye pyranine was used. The proton permeability was measured with a pH gradient of 0.6 pH units from 7.0 to 7.6 with valinomycin present to dissipate any diffusion potential across the membrane. The permeability coefficients for protons were in the range of 3.5.10^–8 to 1.0.10^–7 cm.s^–1. Digalactosyldiacylglycerol vesicles with 5 % α-tocopherol or 10 % cholesterol or 2 % zeaxanthin reduced the permeability for protons, the two latter significantly as compared to digalactosyldiacylglycerol vesicles. α-Tocopherol (5 %) decreased the permeability for glucose remarkably and so did cholesterol (10 %). β-Carotene (< than 1 %) and zeaxanthin (2 %) in galactolipid vesicles, however, increased the permeability. The significance of these results is discussed in relation to the physiological functions of galactolipids and terpenoids in chloroplast membranes.     

Energy transformation coupled to formate oxidation during anaerobic fermentation

A correlation between the rate of ATP synthesis by F₀F₁ ATP synthase and formate oxidation by formate hydrogen lyase (FHL) has been found in inside-out membrane vesicles of the Escherichia coli mutant JW 136 (Δhya/Δhyb) with double deletions of hydrogenases 1 and 2, grown anaerobically on glucose in the absence of external electron acceptors at pH 6.5. ATP synthesis was suppressed by the H⁺-ATPase inhibitors N,N′-dicyclohexylcarbodiimide, sodium azide, and the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Copper ions inhibited formate-dependent hydrogenase and ATP-synthase activities but did not affect the ATPase activity of the vesicles. The maximal rate of ATP synthesis (0.83 μmol/min per mg protein) was determined at simultaneous application of sodium formate, ADP, and inorganic phosphate, and was stimulated by K⁺ ions. The results confirm the assumption of a dual role of hydrogenase 3, the formate hydrogen lyase subunit that can couple the reduction of protons to H₂ and their translocation through membrane with chemiosmotic synthesis of ATP.     

Localized or delocalized protons in photophosphorylation? On the accessibility of the thylakoid lumen for ions and buffers

The deposition of protons inside thylakoids after flash excitation was measured photometrically with neutral red as pH indicator. In continuation of previous work (Junge, W., Ausländer, W., McGeer, A. and Runge, T. (1979) Biochim. Biophys. Acta 546, 121–141), we studied the influence of salts on neutral red binding and on the pK of the heterogeneous protonation-deprotonation of inside-bound neutral red as a function of salts. With freeze-thawed (cryoprotective dimethyl sulphoxide) or aged chloroplasts, we observed that the heterogeneous pK of inside-bound neutral red was salt dependent in a way which suggested that neutral red was bound close to the plane of negative fixed charges and that the adjacent inner aqueous phase could accommodate an extended ionic double layer. This, together with the known extremely rapid proton exchange between surface layer and adjacent bulk phase, led us to conclude that inside-deposited protons rapidly reached an aqueous inner bulk phase. This conclusion was corroborated by the observation that extremely hydrophilic buffers like phosphate quenched the transient internal acidification independent of whether proton deposition was due to water oxidation or to plastohydroquinone oxidation. Very different behaviour was observed for freshly prepared chloroplasts with broken outer envelope. Here, inside-bound neutral red was seemingly unaffected by salts and hydrophilic buffers failed to quench the internal acidification. The electrical conductivity and proton permeability of the thylakoid membrane, on the other hand, were as usual. We attributed the seeming inaccessibility of the internal phase to the failure to accommodate a sufficiently extended ionic cloud between the tightly appressed membranes. In such material we observed hindered lateral mobility of protons at the outer side of the thylakoid membrane. This was tentatively attributed to multiple binding-debinding at buffering groups. The consequences for the chemiosmotic theory are: There is one type of damaged chloroplast material, which is competent in photophosphorylation and where protons are deposited into an internal aqueous bulk phase in the orthodox sense. In more intact material, however, the internal space lacks the characteristic properties of an aqueous bulk phase and there is evidence for lateral diffusion limitation for protons. Here, the thermodynamics of photophosphorylation may be inadequately described by the proton-motive force between two aqueous phases which are each isopotential.     

Control of the Ca2+-triggered bioluminescence of Veretillum cynomorium lumisomes

Calcium ions can trigger an emission of light from Veretillum cynomorium lumisomes (bioluminescent vesicles) under conditions where they are not lysed. This process does not require a metabolically-linked source of energy, but is dependent upon the nature of the ions present inside and outside the vesicles. The Ca²⁺-triggered bioluminescence is stimulated by an asymmetrical distribution of cations or anions. Either high internal sodium or high external chloride is required for the maximal effect. When sodium is present outside the structure and potassium inside, the slow inward diffusion of calcium is decreased. Unbalanced diffusion of internal cations also stimulates the bioluminescence, suggesting control of the calcium influx by an electrochemical gradient. It is assumed that rapid outward diffusion of sodium or inward diffusion of chloride generates an electrical potential difference (inside negative) which drives the Ca²⁺-influx. With purified lumisomes it has been shown that Ca²⁺-triggered bioluminescence and calcium uptake (presumably net uptake) were correlated. In two instances uptake of the lipophilic cation dibenzyldimethylammonium has given direct evidence for the existence of a potential difference. With NaCl-loaded vesicles, it has not been possible to demonstrate an uptake of lipophilic cations but experiments with ²²Na and ⁴²K indicated a higher rate of sodium efflux, in accord with the proposed hypothesis.     

Solubilization and reconstitution of the oat root vacuolar h/ca exchanger

Calcium is sequestered into vacuoles of oat (Avena sativa L.) root cells via a H⁺/Ca²⁺ antiporter, and vesicles derived from the vacuolar membrane (tonoplast) catalyze an uptake of calcium which is dependent on protons (pH gradient [ΔpH] dependent). The first step toward purification and identification of the H⁺/Ca²⁺ antiporter is to solubilize and reconstitute the transport activity in liposomes. The vacuolar H⁺/Ca²⁺ antiporter was solubilized with octylglucoside in the presence of soybean phospholipids and glycerol. After centrifugation, the soluble proteins were reconstituted into liposomes by detergent dilution. A ΔpH (acid inside) was generated in the proteoliposomes with an NH₄Cl gradient (NH₄⁺_in » NH₄⁺_out) as determined by methylamine uptake. Fundamental properties of ΔpH dependent calcium uptake such as the K_m for calcium (~15 micromolar) and the sensitivity to inhibitors such as N,N′-dicyclohexylcarbodiimide, ruthenium red, and lanthanum, were similar to those found in membrane vesicles, indicating that the H⁺/Ca²⁺ antiporter has been reconstituted in active form.     

Proton/Phosphate Stoichiometry in Uptake of Inorganic Phosphate by Cultured Cells of Catharanthus roseus (L.) G. Don

Upon absorption of phosphate, cultured cells of Catharanthus roseus (L.) G. Don caused a rapid alkalinization of the medium in which they were suspended. The alkalinization continued until the added phosphate was completely exhausted from the medium, at which time the pH of the medium started to drop sharply toward the original pH value. Phosphate exposure caused the pH of the medium to increase from pH 3.5 to values as high as 5.8, while the rate of phosphate uptake was constant throughout (10-17 micromoles per hour per gram fresh weight). This indicates that no apparent pH optimum exists for the phosphate uptake by the cultured cells. The amount of protons cotransported with phosphate was calculated from the observed pH change up to the maximum alkalinization and the titration curve of the cell suspension. Proton/phosphate transport stoichiometry ranged from less than unity to 4 according to the amount of phosphate applied. At low phosphate doses, the stoichiometries were close to 4, while at high phosphate doses, smaller stoichiometries were observed. This suggests that, at high phosphate doses, activation of the proton pump is induced by the longer lasting proton influx acidifying the cytoplasm. The increased H⁺ efflux due to the proton pump could partially compensate protons taken up via the proton-phosphate cotransport system. Thus, the H⁺/H₂PO₄⁻ stoichiometry of the cotransport is most likely to be 4.     

Computational modeling predicts ephemeral acidic microdomains in the glutamatergic synaptic cleft

At chemical synapses, synaptic vesicles release their acidic contents into the cleft, leading to the expectation that the cleft should acidify. However, fluorescent pH probes targeted to the cleft of conventional glutamatergic synapses in both fruit flies and mice reveal cleft alkalinization rather than acidification. Here, using a reaction-diffusion scheme, we modeled pH dynamics at the Drosophila neuromuscular junction as glutamate, ATP, and protons (H⁺) were released into the cleft. The model incorporates bicarbonate and phosphate buffering systems as well as plasma membrane calcium-ATPase activity and predicts substantial cleft acidification but only for fractions of a millisecond after neurotransmitter release. Thereafter, the cleft rapidly alkalinizes and remains alkaline for over 100 ms because the plasma membrane calcium-ATPase removes H⁺ from the cleft in exchange for calcium ions from adjacent pre- and postsynaptic compartments, thus recapitulating the empirical data. The extent of synaptic vesicle loading and time course of exocytosis have little influence on the magnitude of acidification. Phosphate but not bicarbonate buffering is effective at suppressing the magnitude and time course of the acid spike, whereas both buffering systems are effective at suppressing cleft alkalinization. The small volume of the cleft levies a powerful influence on the magnitude of alkalinization and its time course. Structural features that open the cleft to adjacent spaces appear to be essential for alleviating the extent of pH transients accompanying neurotransmission.     

Anion- and proton-dependent gating of ClC-4 anion/proton transporter under uncoupling conditions

ClC-4 is a secondary active transporter that exchanges Cl⁻ ions and H⁺ with a 2:1 stoichiometry. In external SCN⁻, ClC-4 becomes uncoupled and transports anions with high unitary transport rate. Upon voltage steps, the number of active transporters varies in a time-dependent manner, resembling voltage-dependent gating of ion channels. We here investigated modification of the voltage dependence of uncoupled ClC-4 by protons and anions to quantify association of substrates with the transporter. External acidification shifts voltage dependence of ClC-4 transport to more positive potentials and leads to reduced transport currents. Internal pH changes had less pronounced effects. Uncoupled ClC-4 transport is facilitated by elevated external [SCN⁻] but impaired by internal Cl⁻ and I⁻. Block by internal anions indicates the existence of an internal anion-binding site with high affinity that is not present in ClC channels. The voltage dependence of ClC-4 coupled transport is modulated by external protons and internal Cl⁻ in a manner similar to what is observed under uncoupling conditions. Our data illustrate functional differences but also similarities between ClC channels and transporters.     

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot : II. Evidence for a Sucrose/H-Antiport

The process of sucrose transport was investigated in sealed putative tonoplast vesicles isolated from sugarbeet (Beta vulgaris L.) taproot. If the vesicles were allowed to develop a steady state pH gradient by the associated transport ATPase and 10 millimolar sucrose was added, a transient flux of protons out of the vesicles was observed. The presence of an ATPase produced pH gradient allowed [¹⁴C]sucrose transport into the vesicles to occur at a rate 10-fold higher than the rate observed in the absence of an imposed pH gradient. Labeled sucrose accumulated into the sealed vesicles could be released back to the external medium if the pH gradient was dissipated with carbonylcyanide-m-chlorophenyl hydrazone (CCCP). When the kinetics of ATP dependent [¹⁴C]sucrose uptake were examined, the kinetic profile followed the simple Michaelis-Menten relationship and a Michaelis constant of 12.1 millimolar was found. When a transient, inwardly directed sucrose gradient was imposed on the vesicles in the absence of charge compensating ions, a transient interior negative membrane potential was observed. This membrane potential could be prevented by the addition of CCCP prior to sucrose or dissipated by the addition of CCCP after sucrose was added. These results suggest that an electrogenic H⁺/sucrose antiport may be operating on the vesicle membrane.     

Characterization of H+/OH− currents in phospholipid vesicles

In pure phospholipid vesicles, the conductivity of H⁺/OH^– ions exceeds that for other simple inorganic ions. Protons achieve electrochemical equilibrium across egg phosphatidylcholine vesicles within tens of minutes. When pH gradients are established across vesicles, transmembrane potentials develop. Conversely, the establishment of transmembrane potentials leads to the formation of pH gradients. When the phenomenological permeability of H⁺/OH^– ions in vesicles is estimated, values are obtained that are much greater (six orders of magnitude larger) than those for Na⁺ or K⁺. A wide range in the values for this permeability has been reported; however, much of the discrepancy can be attributed to differences in the vesicle systems and experimental conditions. The H⁺/OH^– current appears to be modulated by changes in membrane dielectric constant. However, the dependence of this current on the pH gradient and on the membrane voltage argues against simple diffusion mechanisms as the source of the H⁺/OH^– current. In addition, in vesicle systems the H⁺/OH^– current shows a surprising invariance to changes in the membrane dipole potential, an observation that argues against the role of simple carriers for H⁺ and OH^– ions.     

Root exudates as mediators of mineral acquisition in low-nutrient environments

Plant developmental processes are controlled by internal signals that depend on the adequate supply of mineral nutrients by soil to roots. Thus, the availability of nutrient elements can be a major constraint to plant growth in many environments of the world, especially the tropics where soils are extremely low in nutrients. Plants take up most mineral nutrients through the rhizosphere where micro-organisms interact with plant products in root exudates. Plant root exudates consist of a complex mixture of organic acid anions, phytosiderophores, sugars, vitamins, amino acids, purines, nucleosides, inorganic ions (e.g. HCO₃ ^–, OH^–, H⁺), gaseous molecules (CO₂, H₂), enzymes and root border cells which have major direct or indirect effects on the acquisition of mineral nutrients required for plant growth. Phenolics and aldonic acids exuded directly by roots of N₂-fixing legumes serve as major signals to Rhizobiaceae bacteria which form root nodules where N₂ is reduced to ammonia. Some of the same compounds affect development of mycorrhizal fungi that are crucial for phosphate uptake. Plants growing in low-nutrient environments also employ root exudates in ways other than as symbiotic signals to soil microbes involved in nutrient procurement. Extracellular enzymes release P from organic compounds, and several types of molecules increase iron availability through chelation. Organic acids from root exudates can solubilize unavailable soil Ca, Fe and Al phosphates. Plants growing on nitrate generally maintain electronic neutrality by releasing an excess of anions, including hydroxyl ions. Legumes, which can grow well without nitrate through the benefits of N₂ reduction in the root nodules, must release a net excess of protons. These protons can markedly lower rhizosphere pH and decrease the availability of some mineral nutrients as well as the effective functioning of some soil bacteria, such as the rhizobial bacteria themselves. Thus, environments which are naturally very acidic can pose a challenge to nutrient acquisition by plant roots, and threaten the survival of many beneficial microbes including the roots themselves. A few plants such as Rooibos tea (Aspalathus linearis L.) actively modify their rhizosphere pH by extruding OH^– and HCO₃ ^– to facilitate growth in low pH soils (pH 3 – 5). Our current understanding of how plants use root exudates to modify rhizosphere pH and the potential benefits associated with such processes are assessed in this review.     

Pore water dynamics in the sediment of a shallow and hypertrophic lake

Seasonal variations in pore water with main stress on pH and phosphate were investigated in the sediment of the shallow and hypertrophic Lake Søbygaard, Denmark. The purpose was to evaluate factors affecting the internal phosphorus loading. Pore water was obtained by in situ incubation of ceramic cups, sampled anaerobicaly from a fixed position in the sediment. The method is evaluated. During summer, pH and phosphate concentrations increased in the upper 8–10 cm of the sediment. Increased pH was most pronounced in the upper 5 cm, where pH increased to between 9 and 10. This is believed to be caused by the photosynthetically elevated pH in the above lake water. Phosphate concentrations increased with depth, from 0–2 mg P 1^–1 in the upper 5 cm to 3–6 mg P 1^–1 in 6–10 cm depth. Average phosphate gradient in the upper 6–8 cm was 1.0 mg P 1^–1 cm ^–1 in the summer decreasing to 0.2 mg P 1^–1 cm ^t1 in the autumn/winter. In spite of low redox potential, Fe(II) was not present in the upper 20 cm. The seasonal variation in pore water phosphate is believed mainly to be due to the variations in pore water pH inducing a substitution of phosphate ions with hydroxyl ions on ironhydroxides during summer. A considerable sedimentation of organic bound phosphorus and decomposition in the sediment is also considered important. Phosphorus release from the sediment is facilitated by bio- and gas turbation and by the frequent occurrence of resuspension caused by windaction. Net release rate is highly variable during the season. The summer average is 40 mg P m^–2 d^–1.     

Nonequilibrium Statistical Model of Active Transport of Ions and ATP Production in Mitochondria

A model of the active transport of ions through internal membranes of mitochondria is proposed. If concentrations of ions in a cell are known, this model allows calculating concentrations of all main ions (H⁺, Ca⁺², K⁺, Mg²⁺, Na⁺, Cl⁻) in the mitochondrion matrix and the resting potential across the membrane. The theoretical values satisfactorily agree with available experimental data on the concentrations and the potentials, including different operating regimes of the adenosine triphosphate (ATP) synthetase (the main regime, short circuiting or ATP synthetase blocking). The active transport of Mg²⁺ ions in exchange for protons was assumed. In accordance with the model, the ATP synthetase operation is possible only if the stoichiometric coefficient of protons is 3.     

Direct measurement of k channels in thylakoid membranes by incorporation of vesicles into planar lipid bilayers

Light-driven electron transfer reactions cause the active accumulation of protons inside thylakoids, yet at steady state the electrical potential difference across the thylakoid membrane is very small; therefore, there must be a flux of other ions to balance the charge that would otherwise be built up by the net movement of H⁺. This paper presents direct measurements of ion movements through channels in the thylakoid membrane. These were made possible by fusing thylakoid vesicles from spinach (Spinacia oleracea L.) into planar lipid bilayers, using techniques developed originally to study sarcoplasmic reticulum. No Mg²⁺ current was found, but voltage-dependent channels have been characterized, these being somewhat selective for K⁺ over Cl⁻. The data are consistent with a role for these channels in charge balance during light-driven H⁺ movements.