Effect of ionophores on intralysosomal pH.

1. The effect of ionophores on the intralysosomal pH (as estimated from the distribution of a weak acid or base), on the distribution of 42K+ across the lysosomal membrane, and on the intralysosomal degradation of 125I-labelled bovine serum albumin has been studied. 2. Nigericin and X537A equilibrate both 42K+ and H+ across the lysosomal membrane. Gramicidin equilibrates H+ across the lysosomal membrane, this equilibration being more effective in a NaCl than in a KCl medium. Thus all three ionophores exhibit the same ion specificity as in other membranes. 3. The effect of the exchange-diffusion ionophores cannot be imitated by the combination of valinomycin with an uncoupler. Valinomycin by itself also has no effect. 4. X537A and nigericin inhibit the intralysosomal degradation of 125I-labelled albumin only when potassium is present. In a sucrose-containing medium no effect is found. Similar results were obtained with gramicidin. 5. These data suggest that the lysosomal membrane is impermeable to monovalent cations at 25 or 37 degrees C, and that the transport of protons is organised in such a way that electroneutrality is maintained.     

Effect of calcium ionophores on early development in fucoid algae

Although much studied, the role of Ca²⁺ in early fucoid development remains unclear. One technique to investigate Ca²⁺ function that has not been fully exploited is the use of ionophores to drastically increase cytosolic Ca²⁺ activity. We have therefore conducted an analysis of the effects of uniform application of the Ca²⁺ ionophores, A23187 and ionomycin, on early development of fucoid algae. Both ionophores had substantially the same effects. Cell adhesive secretion, rhizoid growth and negative phototropism were reduced but not abolished by ionophore treatment, and germination was delayed. One plausible interpretation of these data is that secretion is partially compromised in the treated zygotes. Surprisingly, photopolarization and cytokinesis were unaffected, indicating that Ca²⁺ homeostasis may not be required for these processes.     

Effect of monovalent cation ionophores on lymphocyte cellular metabolism

The effect of valinomycin, nigericin and gramicidin on the cellular O2 consumption and on ATP content has been investigation. It has been found that while valinomycin and nigericin interfere with mitochondrial functions, gramicidin D does not show any appreciable effect. These results are explained in terms of the differing abilities of ionophores to redistribute among intracellular membranes.     

Phosphatidate and oxidized fatty acids are calcium ionophores. Studies employing arsenazo III in liposomes

Liposomes which have entrapped the metallochromic dye, arsenazo III, constitute a sensitive assay system for ionophoresis of divalent cations. By this means we have compared known calcium ionophores (A23187, ionomycin) with membrane phospholipids, fatty acids, prostanoids, and retinoids. Added at micromolar concentrations to preformed multilamellar liposomes (phosphatidylcholine 7:dicetyl phosphate 2: cholesterol 1) both A23187 and ionomycin, as well as phosphatidic acid and products derived from linoleic acid, linolenic acid, and two eicosatrienoic acids provoked Ca influx (e.g. phosphatidic acid: 0.13 mol of Ca2+/mol of membrane lipid/5 min). A variety of other phospholipids (e.g. phosphatidylinositol), fatty acids (e.g. arachidonic acid), prostanoids (e.g. PGE1) retinoids (e.g. retinoic acid), and glyceryl ether phosphorylcholines ("platelet-activating factors") were without effect. Phosphatidic acid and oxidized fatty acids translocated divalent cations selectively, demonstrating the same rank order as A23187 or ionomycin: Mn greater than Ca greater than Sr much greater than Mg. Membrane lysis did not contribute to the perceived translocation; the liposomes remained impermeable to EDTA, EGTA, arsenazo III, or Mg. Liposomes with phosphatidic acid or oxidized trienoic acids preincorporated at 1-5 mole % of total lipids also permitted translocation of Ca but not Mg. Reduction of ionophoretic fatty acids or ionomycin with stannous chloride abolished their ionophoretic activity. Release of Ca from liposomes which had entrapped arsenazo III-Ca complexes into a medium rich in EGTA permitted calculation of efflux induced by ionophores, whether these were added to the outside of liposomes or preincorporated. Data suggest that phosphatidic acid and oxidized di- and trienoic fatty acids, which act as calcium ionophores in model bilayers, could serve as "endogenous ionophores" in cells.     

Effect of carboxylic ionophores on lysosomal protein degradation in rat hepatocytes

Three different carboxylic ionophores (monensin, nigericin and lasalocid) were each found capable of causing a relatively complete block of the lysosomal (i.e., methylamine-sensitive) protein degradation in isolated rat hepatocytes. Monensin was found to be the most specific in action, as it had no effect on non-lysosomal degradation and did not bring about any substantial inhibition of protein synthesis. Morphometric examination of electron micrographs revealed that monensin causes an accumulation of early forms of autophagic vacuoles and blocks the swelling of lysosomes seen in the presence of methylamine. The results indicate that monensin inhibits lysosomal protein degradation by affecting lysosomal pH.     

Effect of 2,4-dichlorophenol on DPPC/water liposomes studied by X-ray and freeze-fracture electron microscopy

The effect of 2,4-dichlorophenol (DCP) was studied on the fully hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)--water liposomes. The structure and the thermotropic phase behaviour of the liposomes was examined in the presence of DCP (DCP/DPPC molar ratio, varied from 2x10(-2) up to 1) using small- and wide-angle X-ray scattering (SAXS, WAXS) and freeze-fracture electron microscopy. The structural behaviour of the DPPC/DCP/water system was strongly dependent on the concentration of the DCP. In the pretransition range the DCP molecules (at 2x10(-2) DCP/DPPC molar ratio) induced the interdigitated phase beside the parent (gel and rippled gel) phases, locally which can be form at higher DCP concentration. When the DCP/DPPC molar ratio was increased the pretransition disappeared and the main transition was shifted to lower temperatures. In the molar ratio range from 2x10(-1) up to 5x10(-1), a coexistence of different phases was observed in the wide temperature range from 20 up to 40 degrees C. With a further increase of the DCP/DPPC molar ratio (6x10(-1) to 1) only the interdigitated gel phase occurred below 25 degrees C. A schematic phase diagram of DPPC/DCP/water system was constructed to summarise the results.     

A study on electron transport-driven proton translocation in Desulfovibrio desulfuricans

Proton translocation by washed cells of the sulfate-reducing bacterium Desulfovibrio desulfuricans strain Essex 6 was studied by means of pH and sulfide electrodes. Reversible extrusion of protons could be induced either by addition of electron acceptors to cells incubated under hydrogen, or by addition of hydrogen to cells incubated in the presence of an appropriate electron acceptor. Proton translocation was increased in the presence of ionophores that dissipate the membrane potential (thiocyanate, methyl triphenylphosphonium cation, but not valinomycin) and was sensitive to the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). Upon micromolar additions of H₂, usually sulfide was formed in stoichiometric amounts, and extrapolated H⁺/H₂ ratios were 1.8±0.5 with sulfate, 2.3±0.3 with sulfite and 0.5±0.1 with thiosulfate. In several experiments hydrogen pulses caused increased proton extrusion not associated with sulfide production. This was a hint that sulfite might be reduced via intermediates. In the absence of H₂S formation, extrapolated H⁺/H₂ ratios were 3.1±0.8 with sulfate, 3.4±1.1 with sulfite, 4.4±0.8 with thiosulfate and 6.3±1.2 with oxygen. Micromolar pulses of electron acceptors to cells incubated under H₂ caused less proton translocation than H₂ pulses in presence of excess of electron acceptor; extrapolated H⁺/H₂ ratios were 1.3±0.4 with sulfite, 3.3±0.9 with nitrite and 4.2±0.5 with oxygen. No proton translocation was observed after micromolar pulses of sulfate, thiosulfate or nitrate to cells incubated under hydrogen in the presence of thiocyanate. Inhibition experiments with CO and CuCl₂ revealed that the hydrogenase activity was localized in the intracellular space, and that no periplasmic hydrogenase was present. The results indicate that D. desulfuricans can generate a proton gradient by pumping protons across the cytoplasmic membrane.Abbreviations APS adenosine 5-phosphosulfate - CCCP carbonyl cyanide m-chlorophenylhydrazone - MTTP⁺ methyl triphenylphosphonium cation     

Cation ionophores A23187 and valinomycin enhance protein-mediated transfer of rat liver microsomal phosphatidylinositol to liposomes

Summary A standard reaction mixture has been established in which partially purified rat liver phosphatidylinositol exchange proteins sustain a maximal rate of phosphatidylinositol transfer from rat liver microsomes to liposomes. Determination of the transfer kinetics confirms the findings of Brophy et al. (Biochem J. 174:413–420, 1918) that under such conditions a maximum 70–80% of the homogenously radiolabeled, microsomal phosphatidylinositol is exchanged with biphasic kinetics. The phosphatidylinositol exchange proteins thus indicate the presence of three microsomal phosphatidylinositol pools: One pool is not subject to protein-mediated exchange; the other two pools are both exchangeable but are exchanged with significantly different half-lives. Both the divalent cation ionophore, A23187, and the monovalent cation ionophore, valinomycin, significantly enhance phosphatidylinositol transfer in the standard reaction mixture at concentrations 1 to 2 orders of magnitude greater than those sufficient for the ionophores to facilitate cation transport across membranes. The stimulatory effect of each ionophore, however, is not a function of the ionophore/microsome mass ratio in the reaction miture. Although both ionophores increase the relative amount of exchangeable phosphatidylinositol, neither ionophore results in all of the exchangeable phosphatidylinositol being transferred with singlestate kinetics. The evidence demonstrates that A23187 and valinomycin are the first substances found to markedly enhance the reactivity of a microsomal phospholipid class with phospholipid exchange proteins.     

Modification of phospholipid liposomes by electron dense labels

Phosphatidilethanolamine (PE) of liposome was modified by electron-density label, trichlortriazin being used as an intermediate reagent. The electron microscopic study of pure PE liposomes has revealed the presence of typical lamellar structure which also has been visualized on PE liposomes modified by electron-density label. In case of liposomes which consist of PE and phosphatidilcholine cluster distribution of modified PE molecules was observed.     

Alterations of the carrier-mediated transport of an anionic solute,methotrexate, by charged liposomes in Ehrlich ascites tumor cells

Summary Interaction of positively charged liposomes with Ehrlich ascites tumor cells increases the bidirectional transmembrane fluxes of the anionic folic acid analog, methotrexate. Negative liposomes reduce methotrexate influx. Stimulation of methotrexate influx by positively charged liposomes is time and concentration dependent, requiring at least a 5-min incubation with 2.5mm phosphatidylcholine containing 20% stearylamine for maximum effect. Stimulation is not appreciably reversed by washing the cells. Similar increases are observed for influx and efflux so that there is no change in the steady-state methotrexate electrochemical-potential difference across the cell membrane. The increase in influx appears to be a stimulation of the carrier-mediated transport process for methotrexate since both control and stimulated influx are abolished by the competitive inhibitor, 5-formyltetrahydrofolate or the sulfhydryl group inhibitor,p-chloromercuriphenylsulfonic acid and the Q₁₀ of the system remains unchanged. Influx of 5-methyltetrahydrofolate, which shares the same transport carrier as methotrexate, is also stimulated. However, the transport of folic acid, which is structurally similar to methotrexate but does not utilize the carrier, is unaffected. The kinetic change induced by positively charged liposomes is an increase in theV _ma ⁱⁿ , while theK _t ⁱⁿ remains unchanged. Trans-stimulation of methotrexate influx by 5-formyltetrahydrofolate occurs to the same extent in the presence or absence of positively charged liposomes. The liposomes have no apparent effect on the intracellular water, the extracellular space, or the chloride distribution ratio. The data suggest that interaction of positively charged liposomes with Ehrlich ascites tumor cells accelerates the rate of transposition of the membrane carrier system for methotrexate, altering the kinetics of transport without a change in transport thermodynamics.     

Effect of 2,4-dinitrophenol and ionophores on growth and methanogenesis inMethanobacterium thermoautotrophicum

2,4-Dinitrophenol and gramicidin D completely inhibited growth and methanogenesis inMethanobacterium thermoautotrophicum. At low K⁺ concentrations valinomycin inhibited growth and methanogenesis relatively slightly, at high K⁺ concentrations (0.1m KCl) growth was inhibited completely and methanogenesis by about 50%. Monensin and nigericin inhibited growth completely; methanogenesis was inhibited like with valinomycin at high K⁺ concentrations. The results can be interpreted in terms of Mitchell’s chemiosmotic theory as follows. The protonmotive force inM. thermoautotrophicum is the basic source of energy for endergonic processes. Dissipation of the electrical component of protonmotive force may probably be compensated by an increased generation of the proton gradient. However, the osmotic component is essential for growth ofM. thermoautotrophicum.     

Effect of membrane composition on lipid oxidation in liposomes

To study the effect of membrane composition on the oxidation of liposomes, different systems were prepared by adding one component at time to phosphatidylcholine (Epikuron 200). In particular, the effect of cholesterol and its ester, cholesterol stearate, on membrane structure and oxidation was studied. A first screening of the structure and net charge of the different preparation was made by means of z-potential and size measurements. Then the liposomes were oxidized by using a hydrophilic radical initiator, the (2,2-azobis(2-amidinopropane) hydrochloride, AAPH, which thermally decomposes to give a constant radical flux in water. The oxidation of liposomes, monitored by following the absorbance of the primary products of oxidation at 234 nm, was shown to be dependent on the composition of the liposomal bilayer and so on its biophysical properties. In addition, size and z-potential measurements gathered in the time course of the peroxidation reaction, revealed that the oxidation induced a modification of the superficial characteristics of the membrane bilayer so as to change its charge at the shear plane (z-potential). This behaviour was shared by all liposomal preparations independent of the composition. The change in sizes of the different liposomal preparation, instead, followed different trends, being more stable both in control samples and in oxidized ones when cholesterol was present. From the analysis of the results, it can be concluded that cholesterol affects the oxidation induced by hydrophilic radical initiator of model membranes by changing the biophysical properties of the phospholipid bilayer. The rigidity induced by cholesterol at temperatures above the T_m makes the membrane more resistant to radical attack from an external aqueous phase and this in turn delays the start of the reaction. The decrease of z-potential of the liposomal particles induced by the oxidation process can be an important clue to understand the mechanisms involved in the etiology of important diseases.