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Biomedical subjects

R Benz

Publications and source records attributed to R Benz.

At least 253 records · Page 14Linked to original sources
PubMed1980

Determination of ion permeability through the channels made of porins from the outer membrane of Salmonella typhimurium in lipid bilayer membranes.

The three types of porin (matrix-proteins) from Salmonella typhimurium with molecular weights of 38,000, 39,000 and 40,000 were reconstituted with lipid bilayer membranes either as a trimer or as an oligomer (complex I). The specific conductance of the membranes increased several orders of magnitude after the addition of the porins into the aqueous phase bathing the membranes. A linear relationship between protein concentration in the aqueous phase and membrane conductance was found. In the case of lower protein concentrations (10)(-12)M), the conductance increased in a stepwise fashion with a single conductance increment of 2.3 nS in 1 M KC1. For a given salt the conductance increment was found to be largely independent of the particular porin (38 K, 39 K or 40 K) and on the state of aggregation, although porin oligomers showed an up to 10 times smaller conductance increase in macroscopic conductance measurements. The conductance pathway has an ohmic current voltage characteristic and a poor selectivity for different alkali ions. Further information on the structure of the pores formed by the different porins from Salmonella was obtained from the selectivity for various ions. From the permeability of the pore for large ions (Tris+, glucosamine+, Hepes-) a minimum pore diameter of 0.8 nm is estimated. This value is in agreement with the size of the pore as calculated from the conductance data for 1 M KC1 (1.4 nm for a pore length of 7.5 nm). The pore diameter may well account for the sugar permeability which has been found in reconstituted vesicles. The findings reported here are consistent with the assumption that the different porins form large aqueous channels in the lipid bilayer membranes and that the single conductance unit is a trimer. In addition, it is suggested that one trimer contains only one pore rather than a bundle of pores.

Bacterial Proteins↗
PubMed1980

Pulse-length dependence of the electrical breakdown in lipid bilayer membranes.

Charge-pulse experiments were performed on artificial lipid bilayer membranes with charging times in the range between 10 ns and 10 mus. If the membranes are charged to voltages in the order of 100 mV, the membrane voltage at the end of the charge pulse is a linear function of the injected charge. However, if the membranes are charged to voltages in the range of 1V, this relationship no longer holds and a reversible high conductance state occurs. This state is defined as an electrical breakdown and it does not allow the membranes to charge to higher voltages than the breakdown voltage, Vc. Between charging times of 300 ns and 5 mus at 25 degrees C and between 100 ns and 2 mus at 40 degrees C, Vc showed a strong dependence on the charging time of the membrane and decreased from 1.2 to 0.5 V (25 degrees C) and from 1 to 0.4 V (40 degrees C). For other charging times below and above these ranges, the breakdown voltage seemed to be constant. The results indicate that the breakdown phenomenon occurs in less than 10 ns. The pulse-length dependence of the breakdown voltage is consistent with the interpretation of the electrical breakdown mechanism in terms of the electromechanical model. However, it seems possible that below a charging time of the membrane of 300 ns (25 degrees C) and 100 ns (40 degrees C) other processes (such as the Born energy) become possible.

Cell Membrane↗
PubMed1980

Effects of lipid structure on the kinetics of carrier-mediated ion transport.

The mechanism of alkali-ion transport mediated by valinomycin (or similar macrocyclic carriers) may be studied using artificial planar bilayer membranes. The rate constants of the single transport steps (association and dissociation of the ion-carrier complex, translocation of free and complexed carrier) can be determined from electrical relaxation experiments. The turnover number of valinomycin which may be calculated from the rate constants is found to be 10(4)-10(5) s-1. Carriers of the valnomycin-type offer the possibility of studying the relationship between membrane structure and transport kinetics. Increasing the chain-length of the lipid strongly reduces the translocation rate constants of the free and the loaded carrier, and also (in the case of lecithin membranes) the association rate constant. Increasing the number of double bonds in the fatty-acid residue of the lipid leads to an inrease of the translocation rate constants. These effects are discussed in terms of microviscosity of the membrane. Addition of cholesterol to monoglyceride membranes seems to affect both the microviscosity and the dipolar potential at the membrane-solution interface.

Carrier Proteins↗
PubMed1979

Reversible electrical breakdown of lipid bilayer membranes: a charge-pulse relaxation study.

Charge-pulse experiments were performed with lipid bilayer membranes from oxidized cholesterol/n-decane at relatively high voltages (several hundred mV). The membranes show an irreversible mechanical rupture if the membrane is charged to voltages on the order of 300 mV. In the case of the mechanical rupture, the voltage across the membrane needs about 50-200 musec to decay completely to zero. At much higher voltages, applied to the membrane by charge pulses of about 500 nsec duration, a decrease of the specific resistance of the membranes by nine orders of magnitude is observed (from 10(8) to 0.1 omega cm2), which is correlated with the reversible electrical breakdown of the lipid bilayer membrane. Due to the high conductance increase (breakdown) of the bilayer it is not possible to charge the membrane to a larger value than the critical potential difference Vc. For 1 M alkali ion chlorides Vc was about 1 V. The temperature dependence of the electrical breakdown voltage Vc is comparable to that being observed with cell membranes. Vc decreases between 2 and 48 degrees C from 1.5 to 0.6 V in the presence of 1 M KCl. Breakdown experiments were also performed with lipid bilayer membrane composed of other lipids. The fast decay of the voltage (current) in the 100-nsec range after application of a charge pulse was very similar in these experiments compared with experiments with membranes made from oxidized cholesterol. However, the membranes made from other lipids show a mechanical breakdown after the electrical breakdown, whereas with one single membrane from oxidized cholesterol more than twenty reproducible breakdown experiments could be repeated without a visible disturbance of the membrane stability. The reversible electrical breakdown of the membrane is discussed in terms of both compression of the membrane (electromechanical model) and ion movement through the membrane induced by high electric field strength (Born energy).

Cell Membrane↗
PubMed1979

Ionic selectivity of pores formed by the matrix protein (porin) of Escherichia coli.

Incorporation of the matrix protein (porin) from the outer membrane of Escherichia coli into black lipid films results in the formation of ion-permeable pores with a single-pore conductance of the order of 2 nS (in 1 M KCl). Information on the structure of this pore has been obtained by determining the selectivity for various species differing in charge and size. From the permeability of the pore for large organic ions (Tris+, glucosamine+, Hepes-) a minimum pore diameter of 0.8 nm is estimated. At neutral pH the pore is two to four times more permeable for alkali ions than for chloride. On the basis of the observed pH dependence of permeability, this cationic selectivity is explained by the assumption that the pore contains fixed negative charges.

Bacterial Proteins↗
PubMed1979

Receptor for bacteriophage lambda of Escherichia coli forms larger pores in black lipid membranes than the matrix protein (porin).

The receptor for phage lambda in Escherichia coli was isolated by cholate extraction and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands corresponding to the monomer and the dimer were eluted from the gel and tested for their activity to inactivate phage lambda and to form pores in black lipid membranes. It was found that only the dimer inactivated phage lambda, whereas both the monomer and the dimer were active in forming pores. The pore characteristics were similar to those exhibited by the matrix protein (porin) (R. Benz, K. Janko, W. Boos, and P. Läuger, Biochim. Biophys. Acta 511:305--319, 1978). In comparison, the lambda receptor showed a somewhat higher degree of cation specificity, and its pore size was larger. Assuming that the thickness of the outer membrane is 7.5 nm and that the pore is an ideal hydrophilic channel, the pore diameter in vivo was estimated to be 1.6 nm for the lambda receptor and 1.2 nm for the matrix protein.

Bacterial Proteins↗
PubMed1978

Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli.

One of the major proteins of the outer membrane of Escherichia coli, the matrix protein (porin), has been isolated by detergent solubilisation. When the protein is added in concentrations of the order 10 ng/cm3 to the outer phases of a planar lipid bilayer membrane, the membrane conductance increases by many orders of magnitude. At lower protein concentrations the conductance increases in a stepwise fashion, the single conductance increment being about 2 nS (1 nS = 10(-9) siemens = 10(-9) omega -1) in 1 MKCl. The conductance pathway has an ohmic current vs. voltage character and a poor selectivity for chloride and the alkali ions. These findings are consistent with the assumption that the protein forms large aqueous channels in the membrane. From the average value of the single-channel conductance a channel diameter of about 0.9 nm is estimated. This channel size is consistent with the sugar permeability which has been reported for lipid vesicles reconstituted in the presence of the protein.

Cations, Monovalent↗
PubMed1978

Charge-pulse relaxation studies with lipid bilayer membranes modified by alamethicin.

Charge-pulse relaxation studies with the alamethicin-lipid membrane system reveal a triphasic decay of membrane voltage. At short times (resolution time 2 microseconds), where a voltage decay due to the orientation of alamethicin dipoles from the interface into the membranes interior ("gating current") could possibly be expected, only a slow decrease with a time constant determined by the bare membrane conductance occurs. After approximately 1 ms (depending on the experimental conditions) the formation of alamethicin pores starts, leading to an increase in the voltage decay rate. When the characteristic voltage Vcpc is approached, pores close and after passing Vcpc the voltage decreases slowly again according to the bare membrane conductance. Vcpc is determined as a function of the initially applied voltage Vo, alamethicin and KCl concentration. Since the membrane voltage decreases continuously, the system does not reach the equilibrium states obtained at constant voltages. Taking the presented experimental results into account the estimate of the electrical potential at the functional membrane of photosynthesis induced by a saturating single turnover flash of deltaphio approximately 105-135 mV (Zickler, Witt and Boheim (1976) FEBS Lett. 66, 142-148) is changed to deltaphio approximately 200 mV.

Alamethicin↗
PubMed1978

Porin activity in the osmotic shock fluid of Escherichia coli.

Osmotic shock fluid of Escherichia coli exhibited pore-forming activity. This activity could be followed by an in vitro assay based on the conductivity increase for ions due to the presence of pores in black lipid membranes. The histogram (the distribution of conductivity increments in a single pore experiment) obtained with osmotic shock fluid from E. coli was identical to the histogram obtained by detergent-solubilized porin isolated from the outer membrane. The osmotic shock fluid from porin-negative mutants also exhibited pore activity, although the histogram and ion specificity were different from those of porin. Antibodies raised against detergent-solubilized porin were able to form precipitin lines by the Ouchterlony immunodiffusion technique when shock fluids, but not detergent-solubilized porin, were used. These antibodies prevented the formation of pores when shock fluids contained porin but not when shock fluids obtained from porin-negative mutants were used. Macroscopic membrane conductivity of shock fluids due to porin exhibited a concentration dependence, in contrast to detergent-solubilized porin. These results indicate that the hydrodynamic properties of periplasmic or "soluble" porin are different from those of the detergent-solubilized porin of the outer membrane. Periplasmic porin comprises about 0.7% of total protein in the osmotic shock fluid.

Antibodies, Bacterial↗
PubMed1977

Properties of lipid bilayer membranes made from lipids containing phytanic acid.

Besides the preparation of phytanic acid (3,7,11,15-tetramethylhexadecylic acid) according to the Dumas-Stass reaction, the synthesis of four different lipids containing phytanic acid residues is described. Diphytanoyl phosphatidylcholine was synthesized beginning from glycerylphosphorylcholine, whereas the other lipids, diphytanoyl phosphatidylethanolamine, diphytanoyl phosphatidylserine and monophytanoyl glyceride were prepared by total synthesis. Some properties of lipid bilayer membranes made from the lipids containing phytanic acid were investigated. The specific capacity of these membranes was measured. Its value of approximately 400 nF cm-2 was found to be similar to the value of membranes from lipids with unbranched fatty acid residues. Charge pulse experiments were performed using dipicrylamine as a molecular probe of membrane structure. The results were discussed on the basis of a higher viscosity of the membranes from lipids containing phytanic acid residues compared with unbranched fatty acid residues.

Amines↗
PubMed1977

Properties of bilayer membranes in the presence of dipicrylamine. A comparative study by optical absorption and electrical relaxation measurements.

In order to test the question if a pool of lipophilic ions may exist in black lipid membranes which cannot be detected by electrical relaxation measurements we have performed simultaneously measurements of the optical absorption of a lipophilic ion. The absorbance of membrane-bound dipicrylamine at 410 nm was measured with a sensitive spectrophotometer which can detect absorbance changes larger than or equal to 4-10(-5). A minimal concentration of about 6-10(11) dipicrylamine ions per cm2 of the membrane could be detected with this instrument. The dipicrylamine concentration in the membrane obtained with the optical method Ntopt is compared with the concentrations Ntel obtained from simultaneous electrical relaxation meausurements. Ntopt and Ntel agreed at low dipicrylamine concentrations (10(-8)--10(-7) M in the aqueous phase) and showed saturation at higher concentrations (up to 5-10(-6) M). In the saturation range Ntopt was maximally four times higher than Ntel. The significance of this difference is discussed together with general aspects of the saturation phenomenon.

Aniline Compounds↗
PubMed1977

Influence of membrane structure on the kinetics of carrier-mediated ion transport through lipid bilayers.

Charge-pulse relaxation experiments of valinomycin-mediated Rb+ transport have been carried out in order to study the influence of membrane structure on carrier kinetics. From the experiment data the rate constants of association (kR) and dissociation (kD) of the ion-carrier complex as well as the rate constants of translocation of the complex (kMS) and of the free carrier (kS) could be obtained. The composition of the planar bilayer membrane was varied in a wide range. In a first series of experiments, membranes made from glycerolmonooleate dissolved in different n-alkanes (n-decane to n-hexadecane), as well as solvent-free membranes made from the same lipid by the Montal-Mueller technique were studied. The translocation rate constants kS and kMS were found to differ by less than a factor of two in the membranes of different solvent content. Much larger changes of the rate constants were observed if the structure of the fatty acid residue was varied. For instance, an increase in the number of double bonds in the C20 fatty acid from one to four resulted in an increase of kS by a factor of seven and in an increase of kMS by a factor of twenty-four. The stability constant K = kR/kD of the ion-carrier complex as well as the translocation rate constants kS and kMS were found to depend strongly on the polar headgroup of the lipid. The incorporation of cholesterol into glycerolmonooleate membranes reduced kR, kMS and kS up to seven-fold.

Alkanes↗