H+ translocation in lysozyme-treated cells of the blue-green alga Plectonema boryanum. Difference between isotonically and hypotonically treated preparations and effects of divalent cations.

Lysozyme-treated cells of a blue-green alga, Plectonema boryanum, had an internal pH of 7.3+/-0.2 under isotonic and hypotonic conditions. This value was similar to that of untreated cells. The CCCP-induced biphasic H+ change seen in the isotonic cells was not observed in the hypotonically treated cells. The biphasic time course remained in the hypotonic preparation if CaCl2 or MgCl2 was added prior to the osmotic shock. It is suggested that the cells have two compartments of H+ concentration. The outer region may be more acidic than the inner region. A light-induced H+ efflux was observed under isotonic conditions and an influx of H+ under hypotonic conditions. The H+ influx was not observed when lysozyme-treated cells were incubated with CaCl2 or MgCl2 prior to the hypotonic treatment. Two types of effects of divalent cations, one on the rigidity of the outer membrane and another on the permeability characteristics of the inner photosynthetic membrane, are indicated. Rearrangement of the photosynthetic membranes and an apparent inversion of the H+ pump by hypotonic shock are also suggested.     

Fluorimetric pH measurement in whole cells of dark aerobic and anaerobic cyanobacteria

9-Aminoacridine and atebrin fluorescence quenching by dark aerobic and anaerobic suspensions of Anacystis nidulans, Plectonema boryanum and Gloeobacter violaceus was determined at external pH 7–9. Individual pH values in cytosol and thylakoid compartments were calculated from the simultaneously different intracellular enrichment factors of the monovalent and the divalent base. Concomitantly, at external pH 4–7 the cytosolic pH was measured with fluorescein diacetate which is taken up and decomposed in the cell by cytosolic hydrolases. The pH-dependent fluorescence of the free fluorescein, which remains trapped in the cell, monitors the cytosolic pH. The fact that the latter was higher in aerobic than in anaerobic cells, and insensitive to saturating concentrations of dicyclohexylcarbodiimide aerobically, was taken to support the concept of a proton-translocating respiratory chain in the plasma membrane.     

Kinetic properties of electrogenic Na+/H+ antiport in membrane vesicles from an alkalophilic Bacillus sp.

The effects of imposed proton motive force on the kinetic properties of the alkalophilic Bacillus sp. strain N-6 Na+/H+ antiport system have been studied by looking at the effect of delta psi (membrane potential, interior negative) and/or delta pH (proton gradient, interior alkaline) on Na+ efflux or H+ influx in right-side-out membrane vesicles. Imposed delta psi increased the Na+ efflux rate (V) linearly, and the slope of V versus delta psi was higher at pH 9 than at pH 8. Kinetic experiments indicated that the delta psi caused a pronounced increase in the Vmax for Na+ efflux, whereas the Km values for Na+ were unaffected by the delta psi. As the internal H+ concentration increased, the Na+ efflux reaction was inhibited. This inhibition resulted in an increase in the apparent Km of the Na+ efflux reaction. These results have also been observed in delta pH-driven Na+ efflux experiments. When Na(+)-loaded membrane vesicles were energized by means of a valinomycin-induced inside-negative K+ diffusion potential, the generated acidic-interior pH gradients could be detected by changes in 9-aminoacridine fluorescence. The results of H+ influx experiments showed a good coincidence with those of Na+ efflux. H+ influx was enhanced by an increase of delta psi or internal Na+ concentration and inhibited by high internal H+ concentration. These results are consistent with our previous contentions that the Na+/H+ antiport system of this strain operates electrogenically and plays a central role in pH homeostasis at the alkaline pH range.     

Respiration and photosynthesis in energy-transducing membranes of cyanobacteria

Cyanobacteria are photolithotrophic organisms exhibiting oxygenic photosynthesis. In the dark they satisfy their need for energy with respiration. These reactions occur in the same compartment and probably on the same energy-transducing membranes. The characterization of the electron transport chain in the light and in the dark, photophosphorylation and oxidative phosphorylation, as well as possible common pathways in photosynthesis and respiration, are discussed.Abbreviations: DCUM, 3-(3,4-dicholrophenyl)-1,1-dimethylurea; LDAO, lauryldimethylamine oxide; SDS-PAGE, Na-dodecyl sulfate polyacrylamide gel electrophoresis; DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; TTFA, 5-thenoxyltrifluoroacetone;m-CLAM,m-chlorobenzhydroxamic acid; DCCD,N,N-dicyclohexylcarbodiimide. Systematical Notes:Plectonema boryanum = Phormidium luridum; Anacystis nidulans = Synechococcus sp.; Mastigocladus laminosus = Fischerella sp.     

Endogenous energy supply to the plasma membrane of dark aerobic cyanobacterium Anacystis nidulans: ATPase-independent efflux of H+ and Na+ from respiring cells

The ejection of protons from oxygen-pulsed cells and the gradients of Na+ concentration (Na+o/Na+i at 150 mM external NaCl) and proton electrochemical potential (delta mu H+) across the plasma membrane of Anacystis nidulans were studied in response to dark endogenous energy supply. Saturating concentrations of the F0F1-ATPase inhibitors dicyclohexylcarbodiimide (F0) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (F1) eliminated oxidative phosphorylation and lowered the ATP level from 2.6 +/- 0.15 to 0.7 +/- 0.1 nmol/mg dry wt while overall O2 uptake and delta mu H+ were much less affected. H+ efflux was inhibited only 60 to 75%. Aerobic Na+o/Na+i ratios (5.9 +/- 0.6) under these conditions remained 50% above the anaerobic level (2.1 +/- 0.2). Increasing concentrations of the electron transport inhibitors CO and KCN depressed H+ efflux and O2 uptake in parallel, with a pronounced discontinuity of the former at inhibitor concentrations, which reduced ATP levels from 2.6 to 0.8 nmol/mg dry wt, resulting in an abrupt shift of the apparent H+/O ratios from 4.0 +/- 0.3 to 1.9 +/- 0.2. Similarly, with KCN and CO the Na+o/Na+i ratios paralleled decreasing respiration rates more closely than decreasing ATP pool sizes. Ejection of protons also was observed when intact spheroplasts were pulsed with horse heart ferrocytochrome c or ferricyanide; the former reaction was inhibited, the latter was increased, by 1 mM KCN. Measurements of the proton motive force (delta mu H+) across the plasma membrane showed a strong correlation with respiration rates rather than ATP levels. It is concluded that the plasma membrane of intact A. nidulans can be directly energized by proton-translocating respiratory electron transport in the membrane and that part of this energy may be used by a Na+/H+ antiporter for the active exclusion of Na+ from the cell interior.     

Altered stoichiometry of the human leucocyte Na+/H+ antiport with decreasing pH.

The Na+/H+ antiport is an important regulator of cellular volume, pH and Na+ concentration in mammalian cells. The stoichiometry of this antiporter has previously been shown to be a 1:1 exchange of internal H+ for external Na+. We have investigated this stoichiometry in human leucocytes by using a novel intracellular pH-clamping technique and measuring 22Na+ influx and H+ efflux in the same cells. As internal pH was lowered, the stoichiometry of H+/Na+ exchange rose to a mean +/- S.D. of 2.23 +/- 0.69. This mechanism allows a higher H+ efflux in the face of intracellular acid stress without causing excessive intracellular Na+ overload.     

INFLUENCE OF IRON LIMITATION AND NITROGEN SOURCE ON GROWTH AND SIDEROPHORE PRODUCTION BY CYANOBACTERIA1

To characterize the mobilization and uptake of iron by cyanobacteria, 14 species were screened for ability to scavenge iron in a competitive system. The cyanobacteria exhibited a range of growth responses to iron limitation which could be separated into three groups, and a representative species from each group was chosen for further study. Effects of iron-limitation on growth and siderophore production of Anacystis nidulans R2, Anabaena variabilis ATCC 29413, and Plectonema boryanum UTEX 581 were determined. Both A. nidulans R2 and A. variabilis showed a reduced rate of growth with decreased available iron concentration (PFe 17–19). Growth rates increased with further reduction in the level of available iron (pFe 20 to pFe 21). The increase in growth rate occurred at the same available iron concentration as the initiation of extracellular siderophore production. In contrast, the growth of P. boryanum decreased with decreasing available iron levels. No siderophore production was detected from P. boryanum cultures. The growth kinetics of siderophore-producing species differ from traditional nutrient-limited growth kinetics and clearly reflect the presence of a high affinity, siderophore-mediated iron transport system in A. nidulans R2 and A. variabilis. Iron-limited growth kinetics more similar to traditional nutrient-limited growth kinetics were found in P. boryanum. The available nitrogen source influenced amount of siderophore produced and concentration of available iron which induced siderophore production. Siderophores were produced at high iron concentrations (pFe 18) when A. variablilis cultures were grown in the absence of combined nitrogen source. When nitrate was supplied to the culture, iron concentrations had to be reduced to pFe 20 before siderophores were produced. Cells grown on nitrogen also produced greater than two times the amount of siderophore compared with nitrate grown cells. This may be indicative of an increased demand for iron by nitrogen fixing A. variabilis Cultures.     

Active extrusion of potassium in the yeast Saccharomyces cerevisiae induced by low concentrations of trifluoperazine

Trifluoperazine (TFP), the antipsychotic drug, induces substantial K+ efflux, membrane hyperpolarization and inhibition of H+-ATPase in the yeast Saccharomyces cerevisiae. Investigations on the mechanism of these effects revealed two different processes observed at different incubation conditions. At an acidic pH of 4.5 and an alkaline pH of 7.5, K+ efflux was accompanied by substantial proton influx which led to intracellular acidification and dissipation of delta psi formed by cation efflux. The results indicated nonspecific changes in membrane permeability. Similar results were also observed when cells were incubated at pH 5.5-6.0 with higher concentrations of TFP (above 75 microM). On the other hand, low concentrations of TFP (30-50 microM) at pH 5.5-6.0 caused marked membrane hyperpolarization and K+ efflux unaccompanied by the efflux of other cations and by H+ influx. Our experiments indicate that under these conditions K+ efflux was an active process. (1) K+ efflux proceeded only in the presence of a metabolic substrate and was inhibited by metabolic inhibitors. (2) When 0.3-0.9 mM-KCl was present in the medium at pH 6.0, the concentration of K+ within the cells (measured at the end of the incubation with TFP) was much lower than the theoretical concentration of Kin+ if the distribution of K+ between medium and cell water was at equilibrium (at zero electrochemical gradient). (3) Valinomycin decreased the net K+ efflux and decreased the membrane hyperpolarization induced by TFP, probably by increasing the flux of K+ into the cells along its electrochemical gradient. (4) Conditions which led to active K+ efflux also led to a marked decrease in cellular ATP level. The results indicate that under a specific set of conditions TFP induces translocation of K+ against its electrochemical gradient.     

Effects of pH on beta-hydroxybutyrate transport in rat erythrocytes and thymocytes.

Entry of beta-hydroxybutyrate into erythrocytes and thymocytes is facilitated by a carrier (C), as judged from temperature dependence, saturation kinetics, stereospecificity, competition with lactate and pyruvate, and inhibition by moderate concentrations of methylisobutylxanthine, phloretin, or alpha-cyanocinnamate. We studied the dependence of influx and efflux on internal and external pH and [beta-hydroxybutyrate]. Lowering external pH from 8.0 to 7.3 to 6.6 enhanced influx into erythrocytes by lowering entry Km from 29 to 16 to 10 mM, entry V being independent of external pH. Lowering external pH inhibited efflux. At low external pH, external beta-hydroxybutyrate enhanced efflux slightly. At high external pH, external beta-hydroxybutyrate inhibited efflux. Internal acidification inhibited influx and internal alkalization enhanced influx. Internal beta-hydroxybutyrate (betaHB) enhanced influx more in acidified than alkalized cells. These data are compatible with coupled betaHB-/OH- exchange, betaHB- and OH- competing for influx, C:OH- moving faster than C: betaHB-, empty C being immobile. They are also compatible with coupled betaHB-/H+ copermeation, empty C moving inward faster than H+:C:betaHB-, H+:C being immobile, and C:betaHB- (without H+) being so unstable as not to be formed in significant amounts (relative to C, H+:C, and H+:C:betaHB-).     

Temperature dependence on the delayed fluorescence of chlorophyll a in blue-green algae.

1. The delayed fluorescence of chlorophyll a was measured with a phosphoroscope by changing the temperature in a range of room temperatures in intact cells of blue-green algae, Anacystis nidulans, two strains of Anabaena variabilis and Plectonema boryanum, and other kinds of algae, Cyanidium caldarium and Chlorella pyrenoidosa. The induction of delayed fluorescence remarkably depended on the temperature of measurment. Nevertheless, the induction pattern was characterized by three levels of intensity; the initial rise level at the onset of excitation light, the maximum level after a period of excitation and the steady-state level after 10 min of excitation. 2. In A. nidulans and a strain of A. variabilis grown at various temperatures, close relationship was found between the phase transition of membrane lipids and the initial rise and the steady-state levels of delayed fluorescence. The initial rise level showed the maximum at the temperature of phase transition between the liquid crystalline and the mixed solid-liquid crystalline states, The steady-state levels showed a remarkable change from a high in the liquid crystalline state to a low level in the mixed solid-liquid crystalline state. 3. The millisecond decay kinetics of the delayed fluorescence measured at the steady-state level in A. nidulans grown at 38 degrees C consisted of two components with different decay rates. The half-decay time of the fast component was about 0.17 ms and was constant throughout the temperature range of measurement. The half decay time of slow component ranged from 0.6 to 1.5 ms, depending on the temperature of measurment.     

Amiloride-sensitive sodium transport in lamprey red blood cells: evidence for two distinct transport pathways

To determine Na+/H+ exchange in lamprey erythrocyte membranes, the cells were acidified to pH(i) 6.0 using the K+/H+ ionophore nigericin. Incubation of acidified erythrocytes in a NaCl medium at pH 8.0 caused a considerable rise in 22Na+ influx and H+ efflux during the first 1 min of exposure. In addition, exposure of acidified red cells to NaCl medium was associated with rapid elevation of intracellular Na+ content. The acid-induced changes in Na+ influx and H+ efflux were almost completely inhibited by amiloride and dimethylamiloride. In native lamprey erythrocytes, amiloride-sensitive Na+ influx progressively increased as the osmolality of incubation medium was increased by addition of 100, 200, or 300 mmol/l sucrose. Unexpectedly, the hypertonic stress induced a small, yet statistically significant decrease in intracellular Na+ content in these cells. The reduction in the cellular Na+ content increased with hypertonicity of the medium. The acid- and shrinkage-induced Na+ influxes were inhibited by both amiloride and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) in a dose-dependent manner. For both blockers, the half-maximal inhibitory values (IC50) were much greater for the shrinkage-induced (44 and 15 micromol/l for amiloride and EIPA, respectively) than for the acid-induced Na+ influx (5.1 and 3.3 micromol/l, respectively). The data obtained are the first demonstration of the presence of a Na+/H+ exchanger with high activity in acidified (pH(i) 6.0) lamprey red blood cells (on average, 512 +/- 56 mmol/l cells/h, n = 13). The amiloride-sensitive Na+ influxes produced by hypertonic cell shrinkage and acid load are likely to be mediated by distinct ion transporters in these cells.     

Toxicity of ammonia to algae in sewage oxidation ponds.

Ammonia, at concentrations over 2.0 mM and at pH values over 8.0, inhibits photosynthesis and growth of Scenedesmus obliquus, a dominant species in high-rate sewage oxidation ponds. Photosynthesis of Chlorella pyrenoidosa, Anacystis nidulans, and Plectonema boryanum is also susceptible to ammonia inhibition. Dark respiration and cell morphology were unaffected by any combination of pH and ammonia concentrations tested, thus limiting the apparent effect to inhibition of the normal function of the chloroplasts. Methylamine had the same effect as ammonia, and its penetration into the cells was found to be pH dependent. Therefore, the dependence of toxicity of amines to algae on pH apparently results from the inability to penetrate the cell membrane in the ionized form. When operated at 120-h detention time of raw wastewater, the high-rate oxidation pond maintained a steady state with respect to algal growth and oxygen concentration, and the concentration of ammonia did not exceed 1.0 mM. Shifting the pond to 48-h detention time caused an increase in ammonia concentration in the pond water to 2.5 mM, and the pond gradually turned anaerobic. Photosynthesis, which usually elevates the pH of the pond water to 9.0 to 10.0, could not proceed beyond pH 7.9 because of the high concentration of ammonia, and the algal population was washed out and reduced to a concentration that could maintain a doubling time of 48 h without photosynthesis bringing the pH to inhibitory levels. Under these conditions, the pH of the bond becomes a factor that limits the operational efficiency of the oxidation pond.     

Solubilization and Reconstitution of the Mg2+/2H+ Antiporter of the Lutoid Tonoplast from Hevea brasiliensis Latex

The Mg2+/2H+ antiporter recently described on lutoid membrane (Z. Amalou, R. Gibrat, C. Brugidou, P. Trouslot, J.d'Auzac [1992] Plant Physiol 100: 255-260) was solubilized by octylglucoside and reconstituted into soybean liposomes using the detergent dilution method. Magnesium efflux or influx experiments were used to generate a H+ influx or efflux, respectively, monitored with the fluorescent probe 9-amino-6-chloro-2-methoxyacridine. Both experiments gave saturable H+ fluxes as a function of internal or external Mg2+ concentrations with similar kinetic parameters Km and Vmax. The Km value for Mg2+ (about 2 mM) was identical to that previously found in lyophilized-resuspended lutoid (reference therein), whereas the Vmax value was 14-fold higher. Since only 10% of the initial proteins were recovered in proteoliposomes, and electrophoretic patterns of the two kinds of vesicles differed significantly, it was inferred that the increase in Vmax was due essentially to an enrichment of the protein antiporter in the reconstituted fraction, owing to a selective effect of octylglucoside at both solubilization and reconstitution steps. None of the various divalent cations used could dissipate the pH gradient of control liposomes of soybean lipids, unless the divalent/H+ exchanger A23187 was added, whereas a rapid dissipation of the pH gradient was observed with reconstituted proteoliposomes from lutoid proteins, with the cation selectivity sequence Zn2+ > Cd2+ > Mg2+ in the millimolar concentration range. The divalent ions Ca2+, Ba2+, and Mn2+ were incapable of generating a H+ efflux in reconstituted proteoliposomes, whereas both Mg2+/H+ and Ca2+/H+ exchanges were observed in lyophilized-resuspended lutoids. Therefore, the lutoid membrane seems to contain separate Mg2+/H+ and Ca2+/H transport systems, the latter being eliminated during the solubilization/reconstitution of lutoid membrane proteins.     

Cation coupling to melibiose transport in Salmonella typhimurium.

Melibiose transport in Salmonella typhimurium was investigated. Radioactive melibiose was prepared and the melibiose transport system was characterized. Na+ and Li+ stimulated transport of melibiose by lowering the Km value without affecting the Vmax value; Km values were 0.50 mM in the absence of Na+ or Li+ and 0.12 mM in the presence of 10 mM NaCl or 10 mM LiCl. The Vmax value was 140 nmol/min per mg of protein. Melibiose was a much more effective substrate than methyl-beta-thiogalactoside. An Na+-melibiose cotransport mechanism was suggested by three types of experiments. First, the influx of Na+ induced by melibiose influx was observed with melibiose-induced cells. Second, the efflux of H+ induced by melibiose influx was observed only in the presence of Na+ or Li+, demonstrating the absence of H+-melibiose cotransport. Third, either an artificially imposed Na+ gradient or membrane potential could drive melibiose uptake in cells. Formation of an Na+ gradient in S. typhimurium was shown to be coupled to H+ by three methods. First, uncoupler-sensitive extrusion of Na+ was energized by respiration or glycolysis. Second, efflux of H+ induced by Na+ influx was detected. Third, a change in the pH gradient was elicited by imposing an Na+ gradient in energized membrane vesicles. Thus, it is concluded that the mechanism for Na+ extrusion is an Na+/H+ antiport. The Na+/H+ antiporter is a transformer which converts an electrochemical H+ gradient to an Na+ gradient, which then drives melibiose transport. Li+ was inhibitory for the growth of cells when melibiose was the sole carbon source, even though Li+ stimulated melibiose transport. This suggests that high intracellular Li+ may be harmful.     

Toxicity of ammonia to algae in sewage oxidation ponds.

Ammonia, at concentrations over 2.0 mM and at pH values over 8.0, inhibits photosynthesis and growth of Scenedesmus obliquus, a dominant species in high-rate sewage oxidation ponds. Photosynthesis of Chlorella pyrenoidosa, Anacystis nidulans, and Plectonema boryanum is also susceptible to ammonia inhibition. Dark respiration and cell morphology were unaffected by any combination of pH and ammonia concentrations tested, thus limiting the apparent effect to inhibition of the normal function of the chloroplasts. Methylamine had the same effect as ammonia, and its penetration into the cells was found to be pH dependent. Therefore, the dependence of toxicity of amines to algae on pH apparently results from the inability to penetrate the cell membrane in the ionized form. When operated at 120-h detention time of raw wastewater, the high-rate oxidation pond maintained a steady state with respect to algal growth and oxygen concentration, and the concentration of ammonia did not exceed 1.0 mM. Shifting the pond to 48-h detention time caused an increase in ammonia concentration in the pond water to 2.5 mM, and the pond gradually turned anaerobic. Photosynthesis, which usually elevates the pH of the pond water to 9.0 to 10.0, could not proceed beyond pH 7.9 because of the high concentration of ammonia, and the algal population was washed out and reduced to a concentration that could maintain a doubling time of 48 h without photosynthesis bringing the pH to inhibitory levels. Under these conditions, the pH of the bond becomes a factor that limits the operational efficiency of the oxidation pond.     

Modulation of the voltage-sensitive Na+/H+ exchange in sea urchin spermatozoa through membrane potential changes induced by the egg peptide speract

Sea urchin sperm motility can be activated by alkalinization of the internal pH, and previous studies have shown that the internal pH can be regulated by a voltage-sensitive Na+/H+ exchanger present in the flagellar plasma membrane. In this study, the effects of speract, a peptide purified from egg conditioned media, on the Na+/H+ exchange were investigated. Evidence presented indicates that speract activates K+ channels in the flagellar membrane and modulates the Na+/H+ exchange activity through resultant changes in membrane potential. In the presence of tetraphenylphosphonium, a lipophilic ion, or high external Na+, the isolated flagella were depolarized, and Na+/H+ exchanger was inhibited. Speract and valinomycin, a K+ ionophore, were able to reactivate 22Na+ uptake, H+ efflux, and alkalinization of intraflagellar pH under either of the depolarizing conditions. Membrane potential measurements using 3,3'-dipropylthiodicarbocyanide iodide indicated repolarization by either speract or valinomycin. The speract-induced voltage changes did not require Na+ but were sensitive to [K+]. Thus, speract induced a slight depolarization in Na+-free seawater with 10 mM K+ but a hyperpolarization with 2 mM K+. Further support for the activation of K+ channels in the flagella was the 2-5-fold stimulation of K+ efflux induced by speract as measured with a K+ electrode. The ionic selectivity of the speract-activated channel assessed by voltage measurements was K+ greater than Rb+ greater than Cs+. The half-maximally effective concentration of speract was about 0.2 nM. That the H+ and K+ efflux in response to peptide was receptor-mediated was confirmed by the use of speract or resact on intact sea urchin spermatozoa, where the peptides were found to stimulate K+ efflux and to reverse the tetraphenylphosphonium inhibition on H+ efflux only in the homologous spermatozoa. Modulation of the voltage-sensitive Na+/H+ exchange by egg peptides, therefore, appears to be indirect and is coupled through its action on membrane potential.     

Characterization of the Bacillus subtilis motile system driven by an artificially created proton motive force.

Transient swimming was induced in energy-depleted cells of Bacillus subtilis by an artificial proton motive force, which was created by valinomycin addition and a pH reduction. This system did not require any ions except protons in the medium. The size of the induced motility was strongly influenced by changes in the size of either the K+ diffusion potential or the pH gradient. A rough estimation indicated that a proton motive force higher than -100 mV was required for induction of translational swimming of the cell. Corresponding with the transient appearance of swimming, a rapid but transient efflux of K+ and influx of H+ were observed. With decreases in the rate of H+ influx, the amount of motility decreased. A rate of H+ influx higher than 0.2 mumol/s per ml of cell water gave translational swimming. These results suggest direct coupling of H+ influx to rotation of bacterial flagella.     

Phosphate metabolism in blue-green bacteria. V. Factors affecting phosphate uptake in Plectonema boryanum.

Plectonema boryanum requires approximately 5 days of exposure to a culture medium lacking phosphorus to induce the "polyphosphate overplus" phenomenon. At pH9, phosphate uptake is greatest both from normal culture medium and from dilute salt solutions. Phosphate uptake from dilute salt solutions was greatest when Na+ or K+ are combined with Ca2+ or Ca2+ and Mg2+. Cells starved of phosphorus in the presence of a high concentration of K+ or Ca2+ in the medium, and then allowed to take up phosphorus under the same conditions, assimilate more phosphorus than with other major ions.     

Glycolate metabolism in cyanobacteria

A comparative analysis of glycolate excretion in 11 cyanobacteria showed that 8 strains, although grown and assayed in air, excreted glycolate. The largest quantities were excreted by the filamentous strains Plectonema boryanum 73110 and Anabaena cylindrica (Lemm). The carbon lost by excretion was at most 9% of the net fixed carbon in air for heterocystous cyanobacteria but increased (up to 60%) in some strains under a high pO₂ (0.03 kPa CO₂ in pure O₂). A. cylindrica excreted glycolate at a maximum level of 2 and 10 μmol (mg chl a )⁻¹ h⁻¹ in air and at high pO₂, respectively. The excretion continued for several hours. Increases in light intensity and pO₂ and a shift in pH from 7 to 9 increased the amount of glycolate excreted. A. cylindrica also showed the most O₂-sensitive fixation of CO₂. In vitro activity of phosphoglycolate phosphatase (EC 3.1.3.18) was found in all strains tested, with the highest activities noted for Gloeobacter violaceus 7.82 and Gloeothece 6909 and for young cultures of A. cylindrica . The lowest activities were found in Anabaena 7120 and Anacystis nidulans 625, strains excreting no or only minor quantities of glycolate.     

Polarized distribution of the Na+/H+ exchange system in a renal cell line (LLC-PK1) with characteristics of proximal tubular cells

Studies of Na+ and H+ transport by confluent monolayers of the epithelial cell line LLC-PK1 were performed to verify the presence of a Na+/H+ exchange system. The presence of an outwardly directed H+ gradient produced a large stimulation of Na+ influx measured under net flux conditions. Amiloride (10(-3) M) completely inhibited Na+ influx stimulated by the H+ gradient and part of the Na+ influx measured in the absence of a pH gradient. Half-maximal inhibition of the Na+ influx stimulated by a pH gradient at 143 mM Na was observed at 5 microM amiloride. The presence of an inwardly oriented proton gradient also stimulated Na+ efflux from Na+-loaded cells. The stimulation was completely inhibited by the presence of 10(-3) M amiloride in the washout medium. These results indicate that this system could operate in the opposite direction depending on the orientation of the Na+ and H+ gradient. Incubation in Na+-free medium or in the presence of 10(-3) M ouabain resulted in a dramatic decrease of H+ release from LLC-PK1 cells. This H+ release was largely, although not completely, inhibited by 10(-4) M amiloride. Neither chloride substitution by the impermeable anion isethionate nor incubation in the presence of the ionophore valinomycin in high K+ medium affected Na+ influx by stimulated by a pH gradient. Inhibition of the Na+ influx by amiloride occurred only from the apical side of the monolayer. These results indicate that the Na+/H+ exchange system in LLC-PK1 monolayers is specifically localized in the apical membrane of the epithelial cells.