Membrane permeabilisation and antimycoplasmic activity of the 18-residue peptaibols, trichorzins PA.

The membrane permeabilisation properties of six linear natural 18-residue peptaibols, termed trichorzins PA, have been assessed on liposomes and on mollicutes (trivial name, mycoplasmas), a class of parasitic bacteria characterized by a small genome, the lack of a cell wall, a minute cell size, and the incorporation in their plasma membrane of exogenously supplied cholesterol. The trichorzins PA used in this study (PA II, PA IV-VI, PA VIII, and PA IX) differ between them by amino acid or amino alcohol substitutions at positions 4, 7, and 18, and form slightly amphipathic alpha-helices. They proved bactericidal for mollicutes belonging to the genera Acholeplasma, Mycoplasma, and Spiroplasma, with minimal inhibitory concentrations (3.12

Ionophoric activity of the antibiotic peptaibol trichorzin PA VI: a 23Na- and 35Cl-NMR study.

Trichorzin PA VI (Ac Aib1 Ser Ala Aib Iva Gln Aib Val Aib Gly10 Leu Aib Pro Leu Aib Aib Gln Pheol18) is one of the seven main peptaibols forming the natural antibiotic 18-residue peptide mixture biosynthesised by a Trichoderma harzianum strain. Trichorzins exhibit antimycoplasmic activity resulting from membrane permeability perturbations. The membrane permeabilisation process by trichorzin PA VI has been examined in egg yolk phosphatidylcholine large unilamellar vesicles (LUV) and under conditions of ionic equilibrium by 23Na- and 35Cl-NMR experiments conducted in the presence of a chemical shift reagent and a relaxation agent, respectively. In such conditions, trichorzin PA VI exchanges both cations and anions across the vesicle bilayers, indicating the absence of ion- and charge-selectivity, in contrast to antibiotic ionophores, such as monensin or nigericin; the Na+ exchange is not influenced by the ionic strength. The kinetics of the Na+ exchange have been found to be third to fourth order with respect to the peptide concentration. The permeabilisation process of liposomes has been shown to be due to the formation of aggregates of three to four helical peptide monomers arranged into a supramolecular complex including presumably lipid molecules and forming a badly-defined pore in the bilayer. The major mechanism by which ions may exchange through the bilayer involves a long-lasting opening of the pores allowing complete exchange of the internal and external media in an 'all or nothing mode'.

The uptake of Li+ into human 1321 N1 astrocytomas using 7Li NMR spectroscopy.

The uptake of Li+ ions into human 1321 N1 astrocytomas cultured on the surface of microcarrier beads was followed by 7Li NMR spectroscopy. The intracellular and extracellular 7Li resonances were separated by the use of dysprosium tripolyphosphate as a shift reagent. Excellent spectra were obtained from which the uptake of Li+ was found to be approximately ten times faster than that into human erythrocytes using the same technique and a steady-state intracellular Li+ concentration was observed within 60 min. The low intracellular Li+ concentration attained, relative to the extracellular concentration, indicates the presence of an efflux mechanism in astrocytomas that actively transports Li+ against its concentration gradient. The intracellular volume was estimated by quantitative 23Na NMR spectroscopy and the viability of the cells was confirmed by 31P NMR spectroscopy.

The effect of lithium therapy upon the composition of the human erythrocyte membrane.

The membrane phospholipids and membrane proteins from the erythrocytes of manic depressive patients undergoing lithium therapy and of normal controls were examined by high performance TLC and gel electrophoresis, respectively. The phospholipid composition of the erythrocyte membranes of the normal controls was close to that reported previously. The lipid composition of the erythrocyte membranes of the patients was very similar but differed from the normal controls in that the proportion of sphingomyelin and cholesterol appeared to be lower, whereas phosphatidylserine appeared to be higher in the patients by amounts comparable with the sums of the 95% confidence limits on the measurements. No major differences in the protein composition of the erythrocyte membranes were detected.

Mn2+ as a contrast reagent for NMR studies of 35Cl- and 81Br- transport through model biological membranes.

One major problem in using NMR to study halide ions in biological and model biological systems has been to find a contrast reagent to differentiate between halide ions in different compartments. Mn2+ is shown to be a very efficient NMR relaxation agent for the halide ions chloride and bromide and preferable to Co2+ at high magnetic fields. Its use is demonstrated in experiments in which halide ions are exchanged across the membranes of egg yolk phosphatidylcholine vesicles by the phase transfer catalysts tetrabutylammonium ion and benzyltributylammonium ion. Benzyltributylammonium ion is shown to be the more rapid anion transporter through the membrane. Valinomycin is found to cotransport ammonium ions with chloride as an ion pair at a faster rate than the phase transfer catalysts.

Cesium uptake studies on human erythrocytes.

The uptake of Cs+ ions into the erythrocytes of abstemious volunteers and of alcoholic patients was followed using 133Cs NMR. The uptake rates are approximately linear with a rate of 0.33 mM.h-1 at an extracellular Cs+ concentration of 10 mM. There is no discernible difference in the uptake rate between the two classes of subject despite earlier reports that Cs+ distribution is different and consequently that Cs+ transport might be anomalous in alcoholics. There is no evidence of saturation of the input rate and Cs(+)-loaded cells retain their Cs+ when incubated in a Cs(+)-free buffer, strongly suggesting that there is no transport mechanism for the removal of Cs+ from the erythrocyte. Experiments designed to ascertain which intracellular ion is being replaced by Cs+ indicate that it predominantly displaces K+.

Lithium uptake rate and lithium: lithium exchange rate in human erythrocytes at a nearly pharmacologically normal level monitored by 7Li NMR.

7Li NMR was used to follow the rate of uptake of Li+ and Li+:Li+ exchange rates in human erythrocytes at an external lithium concentration of 2 mM, marginally higher than used in therapeutic applications of lithium. The rate of Li+:Li+ exchange is approximately 16 times faster than the rate of Li uptake from the medium. The results are in agreement with lithium-sodium countertransport being the dominant mode for lithium uptake into erythrocytes and for the countertransport system having a greater affinity for Li+ than for Na+.

The transport of Na+ and K+ ions through phospholipid bilayers mediated by the antibiotics salinomycin and narasin studied by 23Na- and 39K-NMR spectroscopy.

Addition of the ionophoric antibiotics salinomycin or narasin to preparations of large unilamellar vesicles made from egg yolk phosphatidylcholine in sodium or potassium chloride solutions gives rise to dynamic effects in the 23Na- and 39K-NMR spectra. The dynamic spectra arise from the ionophore-mediated transport of the metal ions through the membrane. The kinetics of the transport are followed as a function of the concentrations of ionophore and the metal ion and are compatible in all cases with a model in which one ionophore molecule transports one metal ion. For both ionophores the transport of potassium ions is appreciably faster than that of sodium and in both cases the rate-limiting step for sodium transport is dissociation of the ionophore-metal complex. Assuming dissociation to be rate limiting in all four cases it is shown that the transport rate differences between the pairs of complexes of each metal arise solely from differences in the rates of formation. The stability constants for ionophore-metal complex formation in the membrane/water interface are evaluated.

Surface charge effects upon membrane transport processes: the effects of surface charge on the monensin-mediated transport of lithium ions through phospholipid bilayers studied by 7Li-NMR spectroscopy.

Addition of monensin to preparations of large unilamellar vesicles prepared from egg-yolk phosphatidylcholine (egg PC) or egg PC containing 5% phosphatidylserine (PS-) or cetylpyridinium (CP+) ions in lithium chloride solution allows the transport of Li+ ions to be monitored by an NMR magnetisation transfer technique. The kinetics of the transport are followed as a function of the metal ion and monensin concentrations and are compatible with a model in which one monensin molecule transports one Li+ ion. The data allow the extraction of the rate constants for the association and dissociation of the monensin-Li+ complex in the water/membrane interfaces and the evaluation of the stability constants for complex formation in the interfaces. Placing positive charge (CP+) on the membrane surface reduces the formation rate by a factor of about three but hardly alters the dissociation rate. Placing negative charge (PS-) on the membrane surface hardly alters the formation rate but speeds the dissociation rate by about a factor of three. Data from relaxation times of 7Li+ inside the vesicles and from the total enclosed volumes as the vesicles are formed, point to appreciable Li+ surface interactions that increase as the charge on the surface is made more negative. The size of the vesicles formed by the dialytic detergent removal technique increases with the surface charge. The results support a view that enzyme-phospholipid or substrate-phospholipid interactions could play an important role in determining the efficacity of action of membrane bound enzymes. The relevance of the results in the role of Li+ in the control of manic depression is also discussed.

The monensin-mediated transport of Na+ and K+ through phospholipid bilayers studied by 23Na- and 39K-NMR.

Addition of monesin to preparations of large unilamellar vesicles made from egg yolk phosphatidylcholine (EPC) in sodium or potassium chloride solution and from dioleoylphosphatidylcholine (DOPC) in sodium chloride solutions gives rise to dynamic 23Na- and 39K-NMR spectra. The dynamic spectra arise from the monensin-mediated transport of the metal ions through the membrane. The kinetics of the transport are followed as a function of monensin and metal ion concentrations and are compatible with a model in which one monensin molecule transports one metal ion. Rate constants for the association and dissociation of the monensin-metal complex in the membrane/water interface are extracted and the stability constants for complex formation are evaluated. The rate constants in DOPC are similar to those in EPC, confirming that diffusion is not rate-limiting in the transport process and that dissociation of the complex is the rate-limiting step. Although potassium on its own is transported more rapidly, sodium forms the more stable complex and is therefore transported preferentially in competition with potassium.

The monensin-mediated transport of sodium ions through phospholipid bilayers studied by 23Na-NMR spectroscopy.

The monensin-mediated transport of sodium ions through the walls of large unilamellar vesicles of egg phosphatidylcholine was studied using 23Na-NMR and aqueous shift reagents. The transport is dynamic on the NMR time-scale and is strictly first order in monensin over the concentration ranges studied indicating that transport occurs by a 1:1 Na+-ionophore complex. Transport appears to be inhibited by increasing concentrations of Na+.

Measurement of intracellular potassium ion concentrations by n.m.r.

39K n.m.r. was used to detect and quantify K+ within human erythrocytes. A shift reagent consisting of an anionic complex of dysprosium(III) with sodium tripolyphosphate permitted a distinction to be made between K+ inside and outside erythrocytes. Intracellular K+ concentrations determined by this method were similar to values obtained by flame photometry.