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R Benz

Publications and source records attributed to R Benz.

At least 217 records · Page 12Linked to original sources
PubMed1986

Effects of hydrostatic pressure on lipid bilayer membranes. II. Activation and reaction volumes of carrier mediated ion transport.

Measurements of voltage relaxations following brief charge-pulses applied to lipid bilayers have been performed at different hydrostatic pressures in the presence of the neutral carriers cyclo (D-Val-L-Pro-L-Val-D-Pro)3(PV) and valinomycin. From double-exponential relaxations observed in membranes containing PV-K+ complexes estimates were obtained of the amount of membrane absorbed complexes, NMS, and of the rate of complex translocation, kMS. The pressure dependence of kMS corresponded to an activation volume for translocation of approximately 12 cm3/mol independent of ionic strength and K+ concentration. The pressure dependence of NMS strongly varied with K+-concentration suggesting a major role of ion-complexation in solution which is estimated to involve a reaction volume of 25.5 cm3/mol, while the volume of absorption of a PV-K+ complex by the membrane was estimated -7.5 cm3/mol. The relaxations observed in the presence of valinomycin contained three exponentials and could be used to estimate four rate constants and one absorption parameter which characterize the valinomycin-mediated transport. When the transport of Rb+ was tested, the rate constant for the complex dissociation, kD, and the total concentration of free and complexed carriers in the membrane, No, were found to be pressure insensitive. The translocation rates for the complex, kMS and for the free carrier, kS, were instead markedly pressure dependent according to estimated activation volumes in the range of 11 to 18 cm3/mol. The recombination rate constant kR was also pressure dependent according to an activation volume of 12-14 cm3/mol. The study of the valinomycin-K+ transport yielded similar results as far as N.,ks, and kms are concerned, but in this case kR was pressure independent, while kD was increased by pressure. The net volume change associated with the transfer of a free ion to the membrane in the form of a valinomycin-ion complex was nevertheless very similar for K+ and Rb+. It is concluded that pressure affects the transmembrane mobility of liposoluble molecules, whether charged or not, mostly by increasing the effective viscosity of the hydrocarbon core of the bilayer. The pressure dependence of the membrane uptake of amphipathic compounds seems also to obey the general rule: that of involving a negative volume change. However, when the compounds arise from a complexation reaction in solution or at the membrane solution interface possible positive volumes of complexation may make effective uptake to be reversed rather than increased by pressure.

Biological Transport, Active↗
PubMed1986

Pore formation by LamB of Escherichia coli in lipid bilayer membranes.

Lipid bilayer experiments were performed in the presence of different Escherichia coli LamB preparations. These LamB preparations formed two types of pores in the membranes. Large pores, which had a single-channel conductance of 2.7 nS and comprised about 1 to 6% of the total pores, were presumably contaminants which might have been induced together with LamB. LamB itself formed small pores with a single-channel conductance of 160 pS in 1 M KCl. These pores could be completely blocked by the addition of maltose and maltodextrins. Titration of the pore conductance with maltotriose suggested that there was a binding site inside the pores with a Ks of 2.5 X 10(-4) M for maltotriose. On the basis of our data we concluded that the structure of the LamB channels is quite different from the structures of the channels of general diffusion porins, such as OmpF and OmpC.

Bacterial Outer Membrane Proteins↗
PubMed1985

Properties of the leak permeability induced by a cytotoxic protein from Pseudomonas aeruginosa (PACT) in rat erythrocytes and black lipid membranes.

A cytotoxic protein, isolated from Pseudomonas aeruginosa (PACT), was tested on red blood cells of rats and on black lipid membranes for changes of membrane permeability. In rat erythrocytes PACT induces lysis indicative of the formation of a leak permeable to monovalent ions. The dose response curve for the PACT-induced hemolysis demonstrates that the rate of lysis as well as the fraction of lytic cells increases with increasing toxin concentration. Furthermore, the leak pathway discriminates hydrophilic non-electrolytes according to their molecular weight. The findings indicate formation by PACT of a pore with an apparent radius of about 1.2 nm. In pure lipid membranes PACT forms hydrophilic pathways with moderate selectivity for small cations over small anions. The presence of cholesterol is a prerequisite for the occurrence of these PACT-induced permeability changes.

Animals↗
PubMed1985

A water-soluble form of porin from the mitochondrial outer membrane of Neurospora crassa. Properties and relationship to the biosynthetic precursor form.

Mitochondrial porin, the outer membrane pore-forming protein, was isolated in the presence of detergents and converted into a water-soluble form. This water-soluble porin existed under nondenaturing conditions as a mixture of dimers and oligomers. The proportion of dimers increased with decreasing porin concentration during conversion. Water-soluble porin inserted spontaneously into artificial bilayers as did detergent-solubilized porin. Whereas the latter form had no specific requirements for the lipid composition of the bilayer, water-soluble porin inserted only into membranes containing a sterol, and only in the presence of very low concentrations of Triton X-100 (0.001% w/v) in the solution bathing the bilayer. The channels formed by water-soluble porin were indistinguishable from those formed by detergent-purified porin with respect to specific conductance and voltage dependence of conductance. Water-soluble porin bound tightly in a saturable fashion to isolated mitochondria. The bound form was readily accessible to added protease, indicating its presence on the mitochondrial surface. The number of binding sites was in the range of 5-10 pmol/mg of mitochondrial protein. Water-soluble porin apparently binds to a site on the assembly pathway of the porin precursor, since mitochondria whose binding sites were saturated with the water-soluble form did not import porin precursor synthesized in a cell-free system.

Bacterial Outer Membrane Proteins↗
PubMed1985

The 35 kDa DCCD-binding protein from pig heart mitochondria is the mitochondrial porin.

The protein which can be labelled by low concentrations of dicyclohexylcarbodiimide in the Mr region of 30 000-35 000 has been purified from pig heart mitochondria with a high yield and as a single band of apparent Mr 35 000 in dodecyl sulphate-containing gels. The protein is not identical with the phosphate carrier as suggested before, since the two proteins behave differently during isolation. Incorporation of the isolated 35 kDa dicyclohexylcarbodiimide-binding protein into lipid bilayer membranes causes an increase of the membrane conductance in definite steps, due to the formation of pores. The specific pore-forming activity increases during the purification procedure. The single pore conductance is about 4.0 nS, suggesting a diameter of 1.7 nm of the open pore. The pore conductance is dependent on the voltage across the membrane. Anion permeability of the pore is higher than cation permeability. These properties are similar to those described for isolated mitochondrial and bacterial porins. It is concluded that the 35 kDa dicyclohexylcarbodiimide-binding protein from pig heart mitochondria is identical with porin from outer mitochondrial membrane.

Amino Acids↗
PubMed1985

Optical and electrical properties of thin monoolein lipid bilayers.

Monoolein lipid bilayers were formed using a monolayer transfer technique and from dispersions of monoolein in squalene, triolein, 1-chlorodecane and 1-bromodecane. Measurements of optical reflectance and electrical capacitance were used to determine the thickness and dielectric constant of the bilayers. The thickness of the hydrocarbon region of the five bilayer systems ranged from 2.5 to 3.0 nm. Two of the bilayer systems (made from 1-chlorodecane and 1-bromodecane solvents) had a high dielectric constant (2.8 to 2.9) whereas the other bilayer systems had dielectric constants close to that of pure hydrocarbons (2.2). The charge-pulse technique was used to study the transport kinetics of three lipophilic ions and two ion carrier complexes in the bilayers. For the low dielectric constant bilayers, the transport of the lipophilic ions tetraphenylborate, tetraphenylarsonium and dipicrylamine was governed mainly by the thickness of the hydrocarbon region of the bilayer whereas the transport of the ion-carrier complexes proline valinomycin-K+ and valinomycin-Rb+ was nearly independent of thickness. This is consistent with previous studies on thicker monoolein bilayers. The transport of lipophilic anions across bilayers with a high dielectric constant was 20 to 50 times greater than expected on the basis of thickness alone. This agrees qualitatively with predictions based on Born charging energy calculations. High dielectric constant bilayers were three times more permeable to the proline valinomycin-K+ complex than were low dielectric constant bilayers but were just as permeable as low dielectric constant bilayers to the valinomycin-Rb+ complex.

Glycerides↗
PubMed1985

Ion selectivity of gram-negative bacterial porins.

Twelve different porins from the gram-negative bacteria Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Yersinia pestis were reconstituted into lipid bilayer membranes. Most of the porins, except outer membrane protein P, formed large, water-filled, ion-permeable channels with a single-channel conductance between 1.5 and 6 nS in 1 M KCl. The ions used for probing the pore structure had the same relative mobilities while moving through the porin pore as they did while moving in free solution. Thus the single-channel conductances of the individual porins could be used to estimate the effective channel diameters of these porins, yielding values ranging from 1.0 to 2.0 nm. Zero-current potential measurements in the presence of salt gradients across lipid bilayer membranes containing individual porins gave results that were consistent with the conclusions drawn from the single-channel experiments. For all porins except protein P, the channels exhibited a greater cation selectivity for less mobile anions and a greater anion selectivity for less mobile cations, which again indicated that the ions were moving inside the pores in a fashion similar to their movement in the aqueous phase. Three porins, PhoE and NmpC of E. coli and protein P of P. aeruginosa, formed anion-selective pores. PhoE and NmpC were only weakly anion selective, and their selectivity was dependent on the mobility of the ions. In contrast, cations were unable to enter the selectivity filter of the protein P channel. This resulted in a high anion selectivity for all salts tested in this study. The other porins examined, including all of the known constitutive porins of the four gram-negative bacteria studied, were cation selective with a 3- to 40-fold preference for K+ ions over Cl- ions.

Anions↗
PubMed1985

Porin from bacterial and mitochondrial outer membranes.

The outer membrane of gram-negative bacteria acts as a molecular filter with defined exclusion limit for hydrophilic substances. The exclusion limit is dependent on the type of bacteria and has for enteric bacteria like Escherichia coli and Salmonella typhimurium a value between 600 and 800 Daltons, whereas molecules with molecular weights up to 6000 can penetrate the outer membrane of Pseudomonas aeruginosa. The molecular sieving properties result from the presence of a class of major proteins called porins which form trimers of identical subunits in the outer membrane. The porin trimers most likely contain only one large but well-defined pore with a diameter between 1.2 and 2 nm. Mitochondria are presumably descendents of gram-negative bacteria. The outer membrane of mitochondria contains in agreement with this hypothesis large pores which are permeable for hydrophilic substances with molecular weights up to 6000. The mitochondrial porins are processed by the cell and have molecular weights around 30,000 Daltons. There exists some evidence that the pore is controlled by electric fields and metabolic processes.

Amino Acid Sequence↗
PubMed1984

Formation and properties of tetramers of band 3 protein from human erythrocyte membranes in planar lipid bilayers.

Lipid bilayer experiments were performed in the presence of solubilized band 3 protein from human red cell membranes. Band 3 protein increased the conductance of the lipid membranes by several orders of magnitude. Membrane conductance was found to be dependent on the fourth power of protein concentration. This shows that four band 3 subunits form an ion permeable pathway in the lipid bilayer membranes. It also shows that, in the membranes, the protein molecules undergo an association equilibrium which involves at least the monomer and the tetramer of the protein, relaxation towards equilibrium being rapid on the time scale of the experiment. The increase in bilayer conductance induced by the band 3 tetramer could be inhibited by pretreatment of the protein with several SH-reagents (pCMB, pCMBS, DTNB) which also inhibit water transport across the human red cell membrane. Other SH-reagents which do not influence water transport (iodoacetamide, N-ethylmaleimide) did not show any influence on the band 3 induced conductance increase. A band 3-mediated exchange of anions comparable to that in the erythrocyte membrane did not occur in the system studied by us. Our results suggest that, in the human erythrocyte membrane, a pore formed by the band 3 tetramer could be the pathway responsible for the protein-mediated part of water transport.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1984

Chemical modification of the anion selectivity of the PhoE porin from the Escherichia coli outer membrane.

The PhoE porin of Escherichia coli is induced by phosphate deprivation and when purified, forms moderately anion-selective channels in lipid bilayer membranes. To further investigate the basis of anion selectivity, PhoE was chemically acetylated with acetic anhydride. Acetylation modified the mobility and staining characteristics of the PhoE porin on SDS-polyacrylamide gel electrophoresis but the acetylated protein was still found in its normal trimeric state after solubilization in SDS at low temperatures. Furthermore, the acetylated PhoE porin retained its ability to reconstitute into lipid bilayer membranes and the single channel conductance in 1 M KCl was unaltered. Zero-current potential measurements demonstrated that whereas the native PhoE porin was anion-selective, a 30-40-fold increase in preference for cations upon acetylation resulted in the acetylated PhoE porin being cation-selective. Increasing the pH of KCl solutions bathing lipid bilayer membranes from pH 3 to pH 6 caused symmetrical 4-fold increases in the selectivity of both the native and acetylated PhoE proteins for cations. In contrast, increasing the pH from 7 to 9 caused a 2.5-fold increase in selectivity only for the native PhoE porin. These results suggest that the basis of anion selectivity in the native PhoE porin is fixed protonated amino groups (possibly on lysines) in or near the channel, and furthermore indicate that deprotonated carboxyl groups have a strong influence on ion selectivity.

Acetylation↗
PubMed1984

Outer-membrane protein PhoE from Escherichia coli forms anion-selective pores in lipid-bilayer membranes.

Porin PhoE of the outer membrane of Escherichia coli was isolated and purified. Reconstitution experiments with lipid bilayer membranes showed that this protein formed pores which had a single channel conductance of 210 pS at 0.1 M KCl. The PhoE pores were obviously not voltage-controlled or regulated. In contrast to pores formed by the OmpF porin from E. coli the PhoE channel was found to be anion-selective at neutral pH. Chloride is about three to ten times more permeable through the pore than alkali ions. On the basis of the observed pH dependence of the permeability ratio of anions and cations, this anionic selectivity is explained by the assumption that the PhoE pore contains an excess of fixed positive charges.

Anions↗
PubMed1984

Properties of chemically modified porin from Escherichia coli in lipid bilayer membranes.

Purified porin OmpF from Escherichia coli outer membrane was chemically modified by acetylation and succinylation of amino groups and by amidation of the carboxyl groups. Native and chemically modified porins were incorporated into lipid bilayer membranes and the permeability properties of the pores were studied. Acetylation and succinylation of the porin trimers had almost no influence on the single channel conductance in the presence of small cations and anions and the cation selectivity remained essentially unchanged as compared with the native porin. Amidation had also only little influence on the single channel conductance and changed the pore conductance at maximum by less than 50%, whereas the cation selectivity of the porin is completely lost after amidation. The results suggest that the structure of the porin pore remains essentially unchanged after chemical modification of the pores and that their cation selectivity is caused by an excess of negatively charged groups inside the pore and/or on the surface of the protein. Furthermore, it seems very unlikely that the pore contains any positively charged group at neutral pH.

Acetylation↗
PubMed1984

Different effects of mutagens on sister chromatid exchange induction in three Chinese hamster cell lines.

The sister chromatid exchange (SCE) induction of mutagens with different mechanisms of action was comparatively investigated on permanent cell lines of the Chinese hamster (CHO, V-79, and DON) with and without exogenous metabolic activation and with the use of various experimental protocols. CHO and V-79 cells were treated with ethylmethanesulfonate (EMS), a direct mutagen; with the two indirect mutagens cyclophosphamide (CP) and benzo[a]pyrene (BP); as well as with the radical former hydrogen peroxide (H2O2) and hydroxyurea (HU), an inhibitor of DNA synthesis. Aside from an increased basal SCE level and a higher bromodeoxyuridine (BrdUrd) sensitivity, there was no decisive difference between CHO and V-79 cells. However, there was a distinct relationship between SCE induction and the experimental protocol used, which was most pronounced after HU treatment. Neither cell line was able to metabolize the indirect mutagen BP. Only in CHO cells did CP lead to increased SCE frequencies. However, in all cases, the simultaneous application of S9 mix produced a distinct SCE induction. In contrast, BP caused SCE induction in DON cells, whereas CP was not metabolized. The reason for these findings must obviously be sought in the metabolization of CP and BP via different monooxygenase systems, whose activity can differ in these permanent cell lines. One notable finding was that the number of SCE induced by H2O2 could be distinctly reduced by the simultaneous application of S9 mix. This effect can be explained by the fact that S9 mix contains H2O2-degrading enzymes. The results indicate that closely related cell lines differ in their capability for inducing SCE and that investigations of SCE inductions performed on only one cell line do not necessarily produce a representative response.

Animals↗
PubMed1984

Extrinsic charge movement in the squid axon membrane. Effect of pressure and temperature.

The absorption of the lipophilic anions dipicrylamine (DPA-) and tetraphenylborate (TPhB-) by the lipid matrix of the squid axon membrane, and the kinetics of their translocation, were studied by the charge pulse relaxation technique. The axons were treated with tetrodotoxin (TTX) and 4-aminopyridine to block the ionic currents responsible for nerve excitation. At high enough concentrations of absorbed ions (approximately 10(-12) mol/cm2) the membrane voltage relaxation following a brief current pulse consisted mainly of two exponential components, whose time constants and relative amplitudes were used for estimating the translocation rate constant, K, and the density of absorbed ions, N. These measurements were performed at different hydrostatic pressures in the range 1-100 MPa (approximately 1,000 atm), and at different temperatures in the range 5 degrees C-20 degrees C. Both K and N were found to be little affected by pressure. The pressure dependence of K indicated that the translocation of lipophilic ions across the nerve membrane involves activation volumes of the order of 5 cm3/mol. In all experiments the passive membrane resistance was little affected by pressures up to 80 MPa. However, above 100 MPa it fell dramatically to low values, presumably because of phase separation phenomena between the membrane components. The temperature dependence of K, both for DPa- and TPhB-, implied an activation energy for ion translocation of the order of 60 kJ/mol, close to that measured in artificial lipid bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

The kinetic mechanism by which CCCP (carbonyl cyanide m-chlorophenylhydrazone) transports protons across membranes.

We demonstrate that a simple kinetic model describes the transport of protons across lipid bilayer membranes by the weak acid CCCP (carbonyl cyanide m-chlorophenylhydrazone). Four parameters characterize this model: the adsorption coefficients of the anionic and neutral forms of the weak acid onto the interface (beta A and beta HA) and the rate constants for the movement of A- and HA across the membrane (kappa A and kappa HA). These parameters were determined by equilibrium dialysis, electrophoretic mobility, membrane potential, membrane conductance, and spectrophotometric measurements. From these equilibrium and steady state measurements on diphytanoyl phosphatidylcholine/chlorodecane membranes we found that beta A = beta HA = 1.4 10(-3) cm, kappa A = 175 s-1 and kappa HA = 12,000 sec-1. These parameters and our model describe our kinetic experiments if we assume that the protonation reactions, which occur at the interfaces, remain at equilibrium. The model predicts a single exponential decay of the current in a voltage-clamp experiment. The model also predicts that the decay in the voltage across the membrane following an intense current pulse of short duration (approximately 50 nsec) can be described by the sum of two exponentials. The magnitudes and time constants of the relaxations that we observed in both voltage-clamp and charge-pulse experiments agree well with the predictions of the model for all values of pH, voltage and [CCCP].

Biological Transport↗