Addition Of Gramicidin Research Articles

Nature of proton cycling during gramicidin uncoupling of oxidative phosphorylation

Addition of gramicidin D to liver mitochondria, incubated in low- or high-salt media, results in stimulation of respiration in the absence or presence of depression of delta muH, respectively. Gramicidin D concentrations 2 orders of magnitude higher are required in the low-salt media with full uncoupling at 1 nmol of gramicidin.mg-1. The stimulation of respiration is not accompanied by increased passive proton influx in low-salt media. In high-salt media, the extent of respiratory stimulation and the extent of delta muH depression differ according to the nature and concentration of cation. The flow-force relationship is very steep when gramicidin D induced uncoupling occurs in low-salt media and much less steep in high-salt media. A multiplicity of flow-force relationship, respiratory rate vs delta muH, is obtained, the slope of which depends on the nature and concentration of cation, and which can be reproduced by computer simulation by introducing a variable extent of proton cycling either in the membrane or in the pump. The apparent proton conductance, as analyzed in the relationship of Je/delta muH vs delta muH, increases in the so-called ohmic and nonohmic regions according to whether gramicidin D is added in high-salt or low-salt media, respectively. Titration with antimycin of the respiratory control ratio (RCR) in gramicidin D treated mitochondria leads to a depression of the RCR in high-salt but not in low-salt media. The view is discussed that in low-salt media the gramicidin D induced uncoupling is due to a cycling of protons within a proton domain operationally located at or near the proton pump.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous optical measurement of osmotic and diffusional water permeability in cells and liposomes

A quantitative description of transmembrane water transport requires specification of osmotic (Pf) and diffusional (Pd) water permeability coefficients. Methodology has been developed to measure Pf and Pd simultaneously on the basis of the sensitivity and rapid response of the fluorophore aminonaphthalenetrisulfonic acid (ANTS) to solution H2O/D2O content. Cells loaded with ANTS in an H2O buffer were subjected to an inward osmotic gradient with a D2O buffer in a stopped-flow apparatus. The time courses of cell volume (giving Pf) and H2O/D2O content (giving Pd) were recorded with dual photomultiplier detection of scattered light intensity and ANTS fluorescence, respectively. The method was validated by using sealed red cell ghosts and artificial liposomes reconstituted with the pore-forming agent gramicidin D. At 25 degrees C, red cell ghost Pf was 0.021 cm/s with Pd 0.005 cm/s (H2O/D2O exchange time 7.9 ms). Pf and Pd were inhibited by 90% and 45% upon addition of 0.5 mM HgCl2. The activation energy for Pd increased from 5.1 kcal/mol to 10 kcal/mol with addition of HgCl2 (18-35 degrees C). In 90% phosphatidylcholine (PC)/10% cholesterol liposomes prepared by bath sonication and exclusion chromatography, Pf and Pd were 5.1 X 10(-4) and 6.3 X 10(-4) cm/s, respectively (23 degrees C). Addition of gramicidin D (0.1 micrograms/mg of PC) resulted in a further increment in Pf and Pd of 7 X 10(-4) and 3 X 10(-4) cm/s, respectively. These results validate the new methodology and demonstrate its utility for rapid determination of Pf/Pd in biological membranes and in liposomes reconstituted with water channels.

Relationship between gramacidin conformation dependent induction of phospholipid transbilayer movement and hexagonal H II phase formation in erythrocyte membranes

Addition of gramicidin in sufficient concentration from dimethylsulfoxide or trifluoroethanol to isolated erythrocyte membranes induces hexagonal H II phase formation for the phospholipids. In contrast, addition from ethanol does not change the overall bilayer organization despite a similar extent of peptide incorporation. The same solvent dependence is observed for the enhancement of transbilayer reorientation of lysophospholipids and unspecific leak formation in intact erythrocytes at lower gramicidin concentrations. These results indicate that the (β 6.3) conformation of the peptide is essential for all three membrane perturbing effects.

A model for gramicidin A?-phospholipid interactions in bilayers

A model is proposed for the effect of gramicidin A' on the order and structure of phospholipid dispersions. According to this model, the addition of gramicidin A' influences the surrounding lipids via two independent mechanisms. The first arises from a drop in surface pressure for those lipids substantially bounded by gramicidin A'. The second mechanism arises from the increase in the phospholipid headgroup spacing due to the small polar region of the polypeptide. The model provides an explanation for the currently available NMR, X-ray diffraction and Langmuir monolayer results. The model also suggests mechanisms for the ability of gramicidin A' to trigger a transition of the lipid from the lamellar to hexagonal II phase, the dependence of this transition on the lipid chain length and the formation of a lamellar phase with lysophosphatidylcholine.

Pyridine nucleotide oxidation by a plasma membrane fraction from red beet ( Beta vulgaris L.) storage tissue

The potential role of pyridine nucleotide oxidation in the energization and/or regulation of membrane transport was examined using sealed plasma membrane vesicles isolated from red beet ( Beta vulgaris L.) storage tissue. In this system, pyridine nucleotide oxidation, which was enhanced in the presence of ferricyanide, occurred. In the presence or absence of ferricyanide, the oxidation of NADH was several-fold greater than the oxidation of NADPH, indicating that it was the preferred substrate for oxidation in this system. Ferricyanide reduction coupled to NADH oxidation did not require the transmembrane movement of reducing equivalents since ferricyanide incorporated inside the vesicles could not be reduced by NADH added externally to the vesicles, unless the vesicles were made leaky by the addition of 0.05% (v/v) Triton X-100. Using fluorescent probes for the measurement of transmembrane pH gradients and membrane potentials, it was determined that NADH oxidation did not result in the production of a proton electrochemical gradient or have any effect upon the proton electrochemical gradient produced by the plasma membrane H +-ATPase. The oxidation of NADH in the presence of ferricyanide did result in the acidification of the reaction medium. This acidification was unaffected by the addition of Gramicidin D and stimulated by the addition of 0.05% (v/v) Triton X-100, suggesting a scalar (nonvectorial) production of protons in the oxidation/reduction reaction. The results of this study suggest that the oxidation of pyridine nucleotides by plasma membrane vesicles is not related to energization of transport at the plasma membrane or modulation of the activity of the plasma membrane H +-ATPase.

A Comparison of the Effects of Chilling on Thylakoid Electron Transfer in Pea (Pisum sativum L.) and Cucumber (Cucumis sativus L.)

Experiments comparing the photosynthetic responses of a chilling-resistant species (Pisum sativum L. cv Alaska) and a chilling-sensitive species (Cucumis sativus L. cv Ashley) have shown that cucumber photosynthesis is adversely affected by chilling temperatures in the light, while pea photosynthesis is not inhibited by chilling in the light. To further investigate the site of the differential response of these two species to chilling stress, thylakoid membranes were isolated under various conditions and rates of photosynthetic electron transfer were determined. Preliminary experiments revealed that the integrity of cucumber thylakoids from 25 degrees C-grown plants was affected by the isolation temperature; cucumber thylakoids isolated at 5 degrees C in 400 millimolar NaCl were uncoupled, while thylakoids isolated at room temperature in 400 millimolar NaCl were coupled, as determined by addition of gramicidin. The concentration of NaCl in the homogenization buffer was found to be a critical factor in the uncoupling of cucumber thylakoids at 5 degrees C. In contrast, pea thylakoid membranes were not influenced by isolation temperatures or NaCl concentrations. In a second set of experiments, thylakoid membranes were isolated from pea and cucumber plants at successive intervals during a whole-plant light period chilling stress (5 degrees C). During wholeplant chilling, thylakoids isolated from cucumber plants chilled in the light were uncoupled even when the membranes were isolated at warm temperatures. Pea thylakoids were not uncoupled by the whole-plant chilling treatment. The difference in integrity of thylakoid membrane coupling following chilling in the light demonstrates a fundamental difference in photosynthetic function between these two species that may have some bearing on why pea is a chilling-resistant plant and cucumber is a chilling-sensitive plant.

Effect of arachidonic acid, fatty acids, prostaglandins, and leukotrienes on volume regulation in Ehrlich ascites tumor cells.

Arachidonic acid inhibits the cell shrinkage observed in Ehrlich ascites tumor cells during regulatory volume decrease (RVD) or after addition of the Ca ionophore A23187 plus Ca. In Na-containing media, arachidonic acid increases cellular Na uptake under isotonic as well as under hypotonic conditions. Arachidonic acid also inhibits KCl and water loss following swelling in Na-free, hypotonic media even when a high K conductance has been ensured by addition of gramicidin. In isotonic, Na-free medium arachidonic acid inhibits A23187 + Ca-induced cell shrinkage in the absence but not in the presence of gramicidin. It is proposed that inhibition of RVD in hypotonic media by arachidonic acid is caused by reduction in the volume-induced Cl and K permeabilities as well as by an increase in Na permeability and that reduction in A23187 + Ca-induced cell shrinkage is due to a reduction in K permeability and an increase in Na permeability. The A23187 + Ca-activated Cl permeability in unaffected by arachidonic acid. PGE2 inhibits RVD in Na-containing, hypotonic media but not in Na-free, hypotonic media, indicating a PGE2-induced Na uptake. PGE2 has no effect on the volume-activated K and Cl permeabilities. LTB4, LTC4 and LTE4 inhibit RVD insignificantly in hypotonically swollen cells. LTD4, moreover, induces cell shrinkage in steady-state cells and accelerates the RVD following hypotonic exposure. The effect of LTD4 even reflects a stimulating effect on K and Cl transport pathways. Thus none of the leukotrienes show the inhibitory effect found for arachidonic acid on the K and Cl permeabilities. The RVD response in hypotonic, Na-free media is, on the other hand, also inhibited by addition of the unsaturated oleic, linoleic, linolenic and palmitoleic acid, even in the presence of the cationophor gramicidin. The saturated arachidic and stearic acid had no effect on RVD. It is, therefore, suggested that a minor part of the inhibitory effect of arachidonic acid on RVD in Na-containing media is via an increased synthesis of prostaglandins and that the major part of the arachidonic acid effect on RVD in Na-free media, and most probably also in Na-containing media, is due to the inhibition of the volume-induced K and Cl transport pathways, caused by a nonspecific detergent effect of an unsaturated fatty acid.

Study of sidedness and tightness to H + of corn root plasmalemma vesicles: preparation of a fraction enriched in inside-out vesicles

The latency of the Mg 2+-ATPase of corn root plasmalemma was measured using Triton X-100, Zwittergent 3–14, sodium dodecyl sulfate and lysophosphatidylcholine. Lysophosphatidylcholine was the only permeabilizing agent which did not inhibit the enzyme. It gave a higher latency than the other surfactants. The sidedness of plasmalemma vesicles obtained after sucrose gradient purification was estimated from: (i) the Mg 2+-ATPase latency; (ii) the fraction of Mg 2+-ATPase resistant to trypsin attack; (iii) the effect of the specific binding of concanavalin A on the microelectrophoretic mobility of the vesicles; and (iv) the separation of the vesicles by chromatography on a Sepharose-Con A column. These four kinds of independent data were consistent. They led to the conclusion that this membrane preparation contained only sealed vesicles (impermeable to MgATP), the majority of which (approx. 65%) were oriented right-side out. Addition of 0.25 M sucrose in the same way inhibited the MgATP hydrolysis measured with or without lysophosphatidylcholine, so that the latency of the activity remained unchanged. The inhibition was higher in the presence of 0.25 M inositol and trehalose, again without modifying the latency. Finally, this preparation contained no significant proportion of tightly sealed active vesicles, as indicated by the absence of quinacrine quenching after addition of MgATP. Tightly sealed vesicles, displaying quinacrine quenching, were obtained according to De Michelis and Spanswick ((1986) Plant Physiol. 81, 542–547). The sidedness in this vesicle preparation was the same as the previous one (approx. 65% right-side out). Washing with Triton X-100 described by Clement et al. ((1986) Physiol. Vég. 24, 25–35) was applied. The simultaneous measurements of quinacrine quenching and Mg 2+-ATPase latency suggested a general inversion of the vesicle sidedness (approx. 65% of inside-out vesicles) after 0.1% treatment with (w / v) Triton X-100. Furthermore, the total Mg 2+-ATPase activity was recovered in spite of protein loss of approx. 60%, and the specific activity increased by approx. 150% in the presence of lysophosphatidylcholine. In the absence of lysophosphatidylcholine, coupling of Mg 2+-ATPase activity with H + transport was indicated by a 30% increase of MgATP hydrolysis upon addition of gramicidin or NH 4Cl.

Tetradecanoylphorbol acetate and terbutaline stimulate surfactant secretion in alveolar type II cells without changing the membrane potential

Alveolar type II cells were isolated from adult rat lungs after tissue dissociation with elastase. The effect of known secretagogues on transmembrane potential was examined in freshly isolated cells (day 0 cells) and in cells after one day of primary culture (day 1 cells). Freshly isolated type II cells were incubated with 3,3′-dipentyloxacarbocyanine (di-O-C 5(3)) or 3,3′-dipropylthiadicarbocyanine (di-S-C 3(5)), dyes whose intracellular fluorescence intensity is a direct function of the cellular transmembrane potential. Fluorescence was continuously recorded by fluorescence spectrophotometry. Type II cells rapidly incorporated the dyes, and the addition of gramicidin (1 μg/ml) depolarized the cells as indicated by a change in fluorescence. Neither 12- O-tetradecanoylphorbol 13-acetate (TPA) nor terbutaline plus 3-isobutyl-1-methylxanthine (IBMX), which stimulate surfactant secretion from isolated alveolar type II cells, changed the transmembrane potential. The lipophilic cation triphenylmethylphosphonium (TPMP +) was used to quantitate the transmembrane potential of type II cells cultured for one day. Addition of TPA or terbutaline plus IBMX induced surfactant secretion but did not alter the transmembrane potential. To study further the relationship of secretion to the transmembrane potential, secretion was also determined in the presence of high extracellular potassium which depolarizes the cells and in the presence of choline in place of sodium. High potassium enhanced the basal secretion of phosphatidylcholine from 1.8% to 3.4% ( P < 0.01, n = 7). Substitution of sodium chloride by choline chloride had no effect on basal secretion but enhanced TPA-induced secretion ( P < 0.01). We conclude that high extracellular potassium induces membrane depolarization and stimulates surfactant secretion, but TPA or terbutaline plus IBMX stimulates secretion without detectable membrane depolarization and stimulation of secretion by TPA does not require extracellular sodium.

Human platelet diffusional water permeability measured by nuclear magnetic resonance.

To examine whether water transport in human platelets is mediated by an aqueous pore or channel, the ratio of osmotic to diffusional water permeability coefficients (Pf/Pd) was measured. Pd was measured from protein spin-lattice relaxation times in a dense platelet suspension (approximately 20% intracellular exchangeable water) using 20 mM solution Mn as a paramagnetic quencher. The decay of magnetization was biexponential with time constants of 1.3 and 6.9 ms (10 MHz, 37 degrees C), corresponding to a Pd of (2.9 +/- 0.2) X 10(-3) cm/s (SE; n = 8). Pd did not depend on concentrations of Mn (6-20 mM) or of platelets (2-4 X 10(10) platelets/ml), but increased to 4.5 X 10(-3) cm/s with addition of gramicidin (6 micrograms/10(10) platelets). 54Mn uptake studies showed less than 1% of equilibrium uptake of Mn into platelets in 30 min at 37 degrees C. The activation energies (Ea) for Pd were 4.5 kcal/mol (less than 28 degrees C) and 16.3 kcal/mol (greater than 28 degrees C). Pf was measured by a stopped-flow light scattering technique as reported previously [M. M. Meyer and A. S. Verkman, Human platelet osmotic water and nonelectrolyte transport, Am. J. Physiol. 251 (Cell Physiol. 20): C549-C557, 1986], in which the time course of platelet volume was measured in response to a 100 mM inwardly directed sucrose gradient. At 37 degrees C, Pf was (2.7 +/- 0.2) X 10(-3) cm/s and independent of [Mn]. The measured platelet Pf/Pd of 0.93 +/- 0.1 suggests that unlike water transport in erythrocytes, platelet water transport is not associated with an aqueous channel.

Separate, Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K+ and Cl- transport pathways. RVD is accelerated when a parallel K+ transport pathway is provided by addition of gramicidin, indicating that the K+ conductance is rate limiting. Addition of ionophore A23187 plus Ca2+ also activates separate K+ and Cl- transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K+ and Cl- conductance is increased 14- and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl- conductance is rate limiting. An A23187-induced activation of 42K and 36Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: inhibited by quinine which blocks the Ca2+-activated K+ channel, unaffected by substitution of NO-3 or SCN- for Cl-, and inhibited by the anti-calmodulin drug pimozide. When the K+ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl- conductance. The Cl- conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl- transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca2+-free media (probably by release of Ca2+ from internal stores) is also transient, whereas the activation is persistent in Ca2+-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl- transport pathway. These findings suggest that a transient increase in free cytosolic Ca2+ may account for the transient activation of the Cl- transport pathway. The activated anion transport pathway is unselective, carrying both Cl-, Br-, NO-3, and SCN-. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl- transport pathway and also blocks the activation of the K+ transport pathway. This is demonstrated directly by 42K flux experiments and indirectly in media where the dominating anion (SCN-) has a high ground permeability. A comparison of the A23187-induced K+ conductance estimated from 42K flux measurements at high external K+, and from net K+ flux measurements suggests single-file behavior of the Ca2+-activated K+ channel. The number of Ca2+-activated K+ channels is estimated at about 100 per cell.

Effect of linear gramicidin on sporulation and intracellular ATP pools of Bacillus brevis

When Bacillus brevis ATCC 8185 was subjected to nutritional shiftdown from a rich medium to one completely devoid of a nitrogen source, sporulation could be stimulated by the addition of linear gramicidin. Gramicidin-induced sporulation occurred after a considerably longer lag period than the earlier described tyrocidine-induced process (Ristow and Paulus 1982) but involved similar associated biochemical changes, such as extracellular protease production, rapid incorporation of radioactive precursors into RNA, and dipicolinate synthesis. The increased incorporation of [3H]leucine into tyrocidine was a characteristic element in gramicidin-induced sporulation, not being observed when spore formation was accelerated by limited nitrogen supplementation. Nitrogen supplementation (0.02-0.01% nutrient broth) caused a slow and gradual increase in dipicolinate production, in contrast to the sudden, rapid rise of dipicolinate synthesis provoked by the addition of gramicidin or tyrocidine. The induction of sporulation by gramicidin occurred at very low peptide concentrations (0.03 microM), which also brought about an acute depletion of intracellular ATP. In sporulation accelerated by nutrient broth, no depression of ATP level was observed and nonionophoric analogues of gramicidin were unable to substitute for gramicidin in inducing sporulation.

Dication and trication which can increase the permeability of Escherichia coli outer membrane

Recent success in the preparation of the monomer, dimer and trimer in compound 48 80 prompted us to investigate the action of these compounds on Escherichia coli cells. It was found that compound 48 80 inhibited growth of E. coli cells, while the monomer, dimer and trimer in 48 80 did not. However, the following experiments showed that the dimer and trimer disrupted the permeability barrier of the outer membrane of E. coli. First, addition of the dimer or trimer in cell suspension stimulated the uptake of tetraphenylphosphonium cation. Second, the synergistic effect of the dimer on the action of gramicidin caused the efflux of K +. In experiments using isolated cytoplasmic membrane vesicles, addition of gramicidin alone caused the efflux of K +. Thus, it was speculated that, with whole cells, the dimer formed some defect structure in the outer membrane, through which gramicidin reached the cytoplasmic membrane and increased the K + permeability. The temperature dependence of efflux of K + showed that the dimer in 48 80 rendered the outer membrane permeable to gramicidin at temperatures above the phase transition of the outer membrane.

The influence of gramicidin S on hydrophobicity of germinating Bacillus brevis spores

Gramicidin S is known to prolong the outgrowth stage of spore germination in the producing culture. Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared with respect to cell-surface hydrophobicity and germination of their spores. Parental spores were hydrophobic as determined by adhesion to hexadecane, whereas mutant spores showed no affinity to hexadecane. Addition of gramicidin S to mutant spores resulted in a high cell surface hydrophobicity and a delay in germination outgrowth. The hydrophobicity of parental spores was retained throughout most of the germination period. Hydrophobicity was lost as outgrowing spores entered into the stage of vegetative growth. The data indicate that gramicidin S is responsible for the hydrophobicity of B. brevis spores. It is suggested that in making spores hydrophobic, the antibiotic plays a role in concentrating the spores at interfaces where there is a higher probability of finding nutrients for germination and growth.

Ethylene formation in plant mitochondria. Dependence on the transport of 1‐aminocyclopropane‐1‐carboxylic acid across the membrane

The mitochondrial fraction isolated from plumular hooks of etiolated pea seedlings (Pisum sativum L. cv. Kelvedon Wonder) displayed a ten‐fold higher rate of ethylene formation from 1‐aminocyclopropane‐1‐carboxylic acid [ACC; 3.2 nmol C2H4 (mg protein −1)h−1], than the tissue from which it was isolated. When the ionophores valinomycin or nigericin were added, a 60‐ to 70‐fold increase in activity in intact mitochondria over the activity in plumular hooks was obtained for ethylene formation under the same conditions, and a 20‐fold increase was obtained upon addition of gramicidin. The addition of ionophores did not affect the rate of ethylene formation in submitochondrial particles (55% inside‐out as determined by cytochrome oxidase latency) which already exhibited a 2–3‐fold higher specific activity than intact mitochondria. Low concentrations of the detergents cholate and deoxycholate increased mitochondrial ethylene formation activity and had no effect on the rate of the reaction in submitochondrial particles. These results support the suggestion that ACC conversion to ethylene is associated with the inner side of the inner mitochondrial membrane and that transport across the intact mitochondrial membrane is rate‐limiting in the reaction.

Germination-initiated spores of Bacillus brevis Nagano retain their resistance properties.

Initiated spores and vegetative cells of the gramicidin S-producing Bacillus brevis Nagano were compared with respect to their resistance to various forms of stress (osmotic shock-starvation, exposure to ethanol, sonic oscillation, and heat). The resistance of initiated spores to all of these stress situations was considerably greater than that of vegetative cells and approached that of dormant spores. The period during which the initiated spores remained resistant to heat was extended by addition of gramicidin S. The antibiotic may therefore be of survival value to the species in nature by slowing down the development of initiated spores in the outgrowth phase of germination, thereby extending the period during which the cells are resistant to environmental stress.

Slow reversible bleaching and fluorescence quenching caused by carotenoid absorption in chromatophores and cells of Chromatium vinosum

Light absorbed by carotenoids in Chromatium can result in photobleaching of bacteriocholorophyll and quenching of B 890 fluorescence, whereas light of the same intensity absorbed by bacteriochlorophyll has no such effect. Photobleching and fluorescence quenching are partly and slowly reversible in the dark. They are prevented by removal of oxygen or by addition of various reductants and decreased by addition of NaN3− histidine or tryptophane. This suggests the participants of singlet excited oxygen in the measured phenomena. As change in temperature between 30° and –30°C and addition of gramicidin S, which changes the permeability of the membranes, does not affect photobleaching or fluorescence quenching markedly, enzymatic or structural properties do not seem to be involved. The results suggest that light absorbed by carotenoids is partly transferred to bacteriochlorophyll and partly used to excite carotenoids to triplet states. The latter process will counteract the function that carotenoids have in protecting chromatophore components against photobleaching.

Na+ requirement for growth, photosynthesis, and pH regulation in the alkalotolerant cyanobacterium Synechococcus leopoliensis.

We have found that Na+ is required for the alkalotolerance of the cyanobacterium Synechococcus leopoliensis. Cell division did not occur at any pH in the absence of Na+, but cells inoculated into Na+-free growth medium at pH 6.8 did continue metabolic activity, and over a period of 48 h, the cells became twice their normal size. Many of these cells remained viable for at least 59 h and formed colonies on Na+ -containing medium. Cells grown in the presence of Na+ and inoculated into Na+ -free growth medium at pH 9.6 rapidly lost viability. An Na+ concentration of ca. 0.5 milliequivalents X liter-1 was required for sustained growth above pH 9.0. The Na+ requirement could be only partially met by Li+ and not at all by K+ or Rb+. Cells incubated in darkness in growth medium at pH 6.8 had an intracellular pH near neutrality in the presence or absence of Na+. When the external pH was shifted to 9.6, only cells in the presence of Na+ were able to maintain an intracellular pH near 7.0. The membrane potential, however, remained high (-120 mV) in the absence or presence of Na+ unless collapsed by the addition of gramicidin. Thus, the inability to maintain a neutral intracellular pH at pH 9.6 in the absence of Na+ was not due to a generalized disruption of membrane integrity. Even cells containing Na+ still required added Na+ to restore photosynthetic rates to normal after the cells had been washed in Na+ -free buffer at pH 9.6. This requirement was only partially met by Li+ and was not met at all by K+, Rb+, Cs+ Mg2+, or Ca2+. The restoration of photosynthesis by added Na+ occurred within 30 s and suggests a role for extracellular Na+. Part of our results can be explained in terms of the operation of an Na+/H+ antiporter activity in the plasma membrane, but some results would seem to require other mechanisms for Na+ action.

Mechanism of Ca 2+ resistance in adult heart cells isolated with trypsin plus Ca 2+

Ca 2+-resistant heart cells prepared with trypsin and Ca 2+ leak Na + and K + more slowly than Ca 2+-susceptible cells prepared without trypsin and Ca 2+. The two preparations show similar leak rates for amino acids and nucleotides. Cells prepared with Ca 2+ alone show low ion leak rates, but the yield of rod-shaped cells is less than half that when trypsin is present. Cells prepared with trypsin alone show high ion leak rates. The Na +-K + ATPase activity of Ca 2+-susceptible cells appears to be approximately three-fold greater than that of Ca 2+-resistant cells. Imposing a Na +-K + leak by the addition of gramicidin D causes no stimulation of Na +-K + ATPase in Ca 2+-susceptible cells, but stimulates the activity of Ca 2+-resistant cells up to that of the Ca 2+-susceptible cells. Ca 2+-resistant cells appear to contain more K + and less Na + than Ca 2+-susceptible cells. Treatment of Ca 2+-resistant cells with ouabain (1 m m) for 5 min changes the Na + K + balance to approximately that of the Ca 2+-susceptible cells, and induces a similar degree of Ca 2+ susceptibility. We therefore conclude that treatment with trypsin plus Ca 2+ confers Ca 2+ resistance by keeping the permeability of the sarcolemma to Na + and K + sufficiently low to allow the Na +-K + ATPase and Na + Ca + exchanger to maintain normal gradients of Na +, K + and Ca 2+. The agent responsible for maintaining low ion permeability appears to be Ca 2+ itself, while trypsin increases the yield and purity of the Ca 2+-resistant cells.

The appearance of single-ion channels in unmodified lipid bilayer membranes at the phase transition temperature.

Ion-specific channels in artificial membranes have been formed by the addition of gramicidin A, alamethicin, polyene antibiotics and some proteins to the solution surrounding the bilayer lipid membrane. Until now there have been no reports of single-ion channels in unmodified lipid membranes. We have now studied the electrical conductance of planar lipid bilayers membranes made of synthetic distearoylphosphorylcholine (DSPC). Current fluctuations of amplitude approximately 1pA and duration approximately 1 s have been discovered at phase transition temperature, which shows that the appearance of ionic channels may be the result of lipid domain interactions. This would explain the dramatic increase in ion permeability observed in liposomes during phase transition. We suggest that these channels could conduct the transmembrane ionic current in biological membranes without the involvement of peptides and proteins.