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Biomedical subjects

R Benz

Publications and source records attributed to R Benz.

At least 163 records · Page 9Linked to original sources
PubMed1990

The cationically selective state of the mitochondrial outer membrane pore: a study with intact mitochondria and reconstituted mitochondrial porin.

The outer mitochondrial membrane pore at a voltage above 20 to 30 mV can adopt a state of low conductance which may restrict free permeability of mitochondrial substrates. In order to obtain insight into the physiological meaning of this property we took advantage of the fact that the low conductance pore state could be induced by a polyanion in lipid bilayer membranes as well as in intact mitochondria. Upon reconstitution in artificial bilayers the pore in this substate became exclusively cation selective when the polarity of the applied voltage was negative on the cis-side. This behaviour of the pore would explain why induction of the low conductance pore state in intact mitochondria led to a complete inhibition of mitochondrial intermembranous kinases, such as creatine kinase and adenylate kinase, but not of peripheral kinases, for example hexokinase, when utilizing external ATP. The possibility that the inner membrane potential might be transduced to the outer membrane in the contact sites, suggests the existence of cation selective pores in these sites. This aspect may be important in the regulation of peripheral kinases like creatine kinase, nucleoside diphosphate kinase and adenylate kinase which are located behind the outer mitochondrial membrane.

Adenylate Kinase↗
PubMed1990

LamB (maltoporin) of Salmonella typhimurium: isolation, purification and comparison of sugar binding with LamB of Escherichia coli.

LamB (maltoporin) of Salmonella typhimurium was found to be more strongly associated with the murein than OmpF. It was purified in one step using a hydroxyapatite (HTP) column. Reconstitution of the pure protein with lipid bilayer membrane showed that LamB of S. typhimurium formed small ion-permeable channels with a single channel conductance of about 90 pS in 1 M KCl and some preference for cations over anions. The conductance concentration curve was linear, which suggested that LamB of S. typhimurium does not contain any binding site for ions. Pore conductance was completely inhibited by the addition of 20 mM maltotriose. Titration of the LamB-induced membrane conductance with different sugars, including all members of the maltooligosaccharide series up to seven glucose residues, suggested that the channel contains, like LamB (maltoporin) of Escherichia coli, a binding site for sugars. The binding constant of sugars of the maltooligosaccharide series increased with increasing number of glucose residues up to five (saturated). Small sugars had a higher stability constant for sugar binding relative to LamB of E. coli. The advantage of a binding site inside a specific porin for the permeation of solutes is discussed with respect to the properties of a general diffusion porin.

Bacterial Outer Membrane Proteins↗
PubMed1990

Isolation of Neisseria meningitidis mutants deficient in class 1 (porA) and class 3 (porB) outer membrane proteins.

The class 1 major outer membrane protein of Neisseria meningitidis is a serious candidate for a meningococcal vaccine. To facilitate studies on the function of this protein, mutants were isolated that lacked this protein or the structurally related class 3 protein. These mutants were obtained by using the antibody-dependent bactericidal action of the complement system. The class 1 protein-deficient strain grew normally in vitro, whereas growth of the class 3 protein-deficient strain was slightly retarded. The class 3 protein-deficient strain displayed increased resistance to the antibiotics tetracycline and cefsulodin, which is consistent with the proposed role of the protein as a pore-forming protein. The class 1 protein was purified to homogeneity from the class 3 protein-deficient strain. Lipid bilayer experiments revealed that this protein also formed pores. The class 1 protein pores were cation selective.

Bacterial Outer Membrane Proteins↗
PubMed1990

Aerolysin of Aeromonas sobria: evidence for formation of ion-permeable channels and comparison with alpha-toxin of Staphylococcus aureus.

Aerolysin from Aeromonas sobria AB3 was isolated and purified. The pure toxin formed sodium dodecyl sulfate-insoluble oligomers in a lipidic environment. The addition of aerolysin to the aqueous phase bathing lipid bilayer membranes resulted in the formation of ion-permeable channels which had a single-channel conductance of about 70 pS in 0.1 M KCl. This defines the toxin as a channel-forming component similar to other toxins but without any indication for an association-dissociation reaction, since the channels had a long lifetime at low voltages. At voltages higher than 50 mV, the aerolysin channel switched into a closed state with a low residual conductance. The single-channel conductance was a linear function of the total aqueous conductance, which suggested that the toxin oligomers formed aqueous channels with an estimated minimal diameter of about 0.7 nm. The aerolysin pores were found to be slightly anion selective. The pore-forming properties of aerolysin were compared with those of alpha-toxin of Staphylococcus aureus. Both aerolysin and alpha-toxin share secondary structure features, must oligomerize to form pores in lipid bilayer membranes, and form channels with similar properties.

Aeromonas↗
PubMed1990

Studies on human porin. III. Does the voltage-dependent anion channel "Porin 31HL" form part of the chloride channel complex, which is observed in different cells and thought to be affected in cystic fibrosis?

"Porin 31HL", of known primary structure, is an integral protein of the plasmalemma of human B cells (Thinnes, F.P. et al. (1989) This Journal 370, 1253-1264; Kayser, H. et al. (1989) This Journal 370, 1265-1278). Purified "Porin 31HL" from human B lymphocytes was reconstituted into lipid bilayer membranes, where it formed defined voltage-dependent channels. Five minutes preincubation with 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, potent inhibitor of chloride transport, altered the channel-forming properties of the protein, so that it now showed small irregular channels instead of distinct steps. In addition, the voltage-dependence of the channel was abolished by the action of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate. Functional and structural similarities between "Porin 31HL" and porin preparations from other human tissues and from other species suggest that this protein may be part of the chloride channel complex, which is defective in cystic fibrosis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1990

Porin of Rhodobacter capsulatus: biochemical and functional characterization.

The major outer membrane protein of Rhodobacter capsulatus 37b4 (capsule-free) was isolated. Strong porin-activity was observed after reconstitution into artificial lipid bilayer membranes with a single channel conductance of 3.15 nS in 1 M KCl. The porin migrated as a broad, single band (Mr above 90,000) on sodium dodecyl sulfate polyacrylamide gel electrophoresis and dissociated into a single species of polypeptides (Mr 36,000) on treatment with EDTA (10 mM at 30 degrees C, 20 min) or by heating (100 degrees C, 5 min). Analytical ultracentrifugation studies demonstrated the native porin to be a trimer. The monomers chromatofocused as a single, sharp peak on fast performance liquid chromatography and only one band, corresponding to an isoelectric point of about 4.0, was obtained on isoelectric focusing. Gas-phase sequencing of the 23 N-terminal residues revealed Glu-Val-Lys-Leu-Ser-Gly-Asp-Ala-Arg-Met-Gly-Val-Met-Tyr-Asn-Gly-Asp-Asp- X-Asn- Phe-Ser-Ser.

Amino Acid Sequence↗
PubMed1989

Characterization of the mitochondrial porin from Drosophila melanogaster.

Mitochondrial porin was isolated from the fruit fly Drosophila melanogaster at different developmental stages, starting from whole mitochondria. The porin from adults' mitochondria was fully characterized. The protein had a molecular mass of 31 kDa as judged from sodium dodecylsulfate electrophoretograms. It was very resistive against digestion with V8 proteinase of Staphylococcus aureus and a larger number of fragments were only obtained after digestion with papain. Drosophila porin showed little interaction with antibodies raised against mitochondrial porins from mammalia and Neurospora crassa, but a strong reactivity with antibodies raised against yeast porin. Reconstitution experiments with planar lipid bilayer membranes showed that the protein was able to form ion-permeable pores with a single-channel conductance of 0.41 nS in 0.1 M KCl. At low transmembrane voltages Drosophila porin had the properties of a general diffusion pore with an estimated effective diameter of about 1.7 nm and a small selectivity for anions over cations. Voltages larger than 20 to 30 mV resulted in a closure of the pore. The closed states of the pore were found to be cation-selective. The addition of a synthetic polyanion to the aqueous phase on one side of the membrane resulted in an asymmetric shift of the voltage dependence and the pore became already closed at very small voltages negative at the cis-side (the side of the addition of the polyanion).

Animals↗
PubMed1989

One single lysine residue is responsible for the special interaction between polyphosphate and the outer membrane porin PhoE of Escherichia coli.

Site-directed mutagenesis was performed with the phosphate starvation-inducible outer membrane porin PhoE of Escherichia coli K-12 to study the molecular basis of its anion selectivity. Lysines 18, 29, 64, and 125 were replaced by glutamic acids, and the properties of the mutant porins were investigated in in vivo and in vitro experiments. Lipid bilayer experiments showed that all these mutations had no influence on the pore structure because PhoE and the mutants had the same single channel conductance in KCl solution. Selectivity measurements revealed that the mutations changed the ionic selectivity of PhoE, but the change was dependent on the location of the lysine. Replacement of Lys18 and Lys29 by glutamic acid had a relatively small influence. The effect of the Lys64 substitution was somewhat larger, and the effect of the replacement of Lys125 resulted in the most drastic change in selectivity and in the loss of the interaction of PhoE with polyphosphate, whereas the replacement of the other lysines had no effect on the polyphosphate interaction behavior. The results are consistent with the assumption that the charge spot in PhoE consists of only 1 lysine per monomer, located in position 125 of the primary sequence and probably close to the pore interior.

Bacterial Outer Membrane Proteins↗
PubMed1989

Interaction of non-classical detergents with the mitochondrial porin. A new purification procedure and characterization of the pore-forming unit.

The effect of different families of detergents on the solubilization and purification of the pore-forming protein (porin) of the mitochondrial outer membrane of bovine heart was investigated in detail. With Tritons, dimethylamine oxides and zwittergents, porin solubilization with respect to total mitochondrial membrane protein was more efficient with the more hydrophobic members of each series. With most detergents the protein eluted as protein-detergent micelles in the void volume of hydroxyapatite/celite columns. In contrast, the protein was bound to the column material and was eluted after the addition of salt to the elution buffer when the detergents octylglucoside, zwittergent Z-314 and lauryl(dimethyl)-amine oxide were used. The protein purified in the presence of the latter detergent had a higher pore-forming activity in lipid bilayer membranes compared to porin isolated in the presence of Triton X-100. The binding of porin to the hydroxyapatite/celite column was used to study the lipid content of the active pore-forming complex. The analysis revealed that the complex contained no phospholipid but rather five molecules of cholesterol/polypeptide chain.

Animals↗
PubMed1989

The peptide antibiotic subtilin acts by formation of voltage-dependent multi-state pores in bacterial and artificial membranes.

The peptide antibiotic subtilin was shown to induce a rapid efflux of amino acids from intact bacterial cells and cytoplasmic membrane vesicles, and to prevent amino acid uptake by cells preincubated with the peptide. Upon addition of subtilin the trans-membrane potential (delta psi) was greatly reduced. Starved bacterial cells were less sensitive to subtilin than energized cells. Depolarization of cells by carbonyl cyanide m-chlorophenylhydrazone prevented subtilin action, but its activity could be restored by a valinomycin-induced potassium diffusion potential. Using this technique, we deduced a threshold potential of about -90 to -100 mV to be essential for subtilin action on intact cells. A similar value was obtained in macroscopic conductance measurements with black lipid membranes. The current-voltage characteristic was symmetric, i.e. subtilin induced membrane currents with trans-negative and trans-positive voltages. Single-channel experiments revealed short-lived multi-state pores of the alamethicin type. The pores had lifetimes of several hundred milliseconds and pore diameters of up to approximately 2 nm.

Amino Acids↗
PubMed1989

Molecular basis of porin selectivity: membrane experiments with OmpC-PhoE and OmpF-PhoE hybrid proteins of Escherichia coli K-12.

Lipid bilayer experiments were performed with one OmpF-PhoE and several OmpC-PhoE hybrid porins of Escherichia coli K-12. All hybrid pores had approximately the same pore-forming activity, which indicated that the structure of the pores remained essentially unchanged by the genetic manipulation. This result was supported by single-channel experiments because all pores had similar single-channel conductances in potassium chloride. Measurements with other salts indicated a drastic change in the ionic selectivity when the fusion site in the ompC-phoE hybrid genes passed along the sequence of the porins from the N-terminal to the C-terminal end. Selectivity measurements using zero-current membrane potentials showed that the selectivity suddenly changed from anion to cation selectivity when a relatively short portion from the N-terminal end of PhoE was replaced by the corresponding part of OmpC. The replacement of increasing portions led to an increase in the cation selectivity until that of OmpC was reached. The change in the anion to cation selectivity is correlated with exchange of lysine-18 and serine-28 by aspartic acids. The anion selectivity of the phosphate starvation-inducible PhoE porin is closely related to the presence of several lysines spread along the primary sequence of the polypeptide chain.

Amino Acid Sequence↗
PubMed1989

Identification of a new pore in the mitochondrial outer membrane of a porin-deficient yeast mutant.

Reconstitution experiments were performed on lipid bilayer membranes in the presence of detergent-solubilized mitochondrial outer membranes of a porin-free yeast mutant and of its parent strain. The addition of the detergent-solubilized material resulted in a strong increase in the membrane conductance which was not observed if only the detergent was added to the aqueous phase. Surprisingly, the membrane conductance induced by the detergent extracts of the mutant membrane was only a factor of 20 less than that caused by the outer membrane of the parent strain under otherwise identical conditions. Single-channel recordings of lipid bilayer membranes in the presence of mitochondrial outer membranes of the yeast mutant suggested the presence of a transient pore. The reconstituted pores had a single-channel conductance of 0.21 nS in 0.1 M KCl and the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. The pores present in the mitochondrial outer membranes of the yeast mutant shared some similarities with the pores formed by mitochondrial and bacterial porins although their effective diameter is much smaller than those of the 'normal' mitochondrial porins which have a single-channel conductance of about 0.4 nS in 0.1 M KCl, corresponding to an effective diameter of 1.7 nm. Zero-current membrane-potential measurements suggested that the second mitochondrial porin is slightly cation-selective. Its possible role in the metabolism of mitochondria is discussed.

Bacterial Outer Membrane Proteins↗
PubMed1989

Porin of Paramecium mitochondria isolation, characterization and ion selectivity of the closed state.

Porin was isolated and purified from mitochondria of Paramecium tetraurelia. The protein showed a single band of apparent Mr 37,000 on sodium dodecyl sulfate polyacrylamide electrophoretograms. The reconstitution of the protein into artificial lipid bilayer membranes revealed it to be a porin giving pores with an average single-channel conductance of 0.26 nS in 0.1 M KCl. This conductance is about half of that of other eukaryotic porins studied to date. The pore formed by the mitochondrial porin of Paramecium was found to be voltage-dependent and switched to a defined substrate at membrane voltages larger than 20 mV. In the open state the pore exhibited the characteristics of a general diffusion pore because the mobility sequence of the ions inside the pore was similar to that in the bulk aqueous phase. The effective diameter was estimated to be about 1.3 nm. The properties of the low conductance state of the pore were studied in detail. In this state the pore favored the passage of cations, in contrast to the open state which favored anions slightly. The possible role of the low-conductance state in the regulation of transport processes across the outer mitochondrial membrane and in mitochondrial metabolism is discussed.

Animals↗
PubMed1989

Pores from mitochondrial outer membranes of yeast and a porin-deficient yeast mutant: a comparison.

Reconstitution experiments were performed on lipid bilayer membranes in the presence of purified mitochondrial porin from yeast and of detergent-solubilized mitochondrial outer membranes of a porin-free yeast mutant. The addition of the porin resulted in a strong increase of the membrane conductance, which was caused by the formation of ion-permeable channels in the membranes. Yeast porin has a single-channel conductance of 4.2 nS in 1 M KCl. In the open state it behaves as a general diffusion pore with an effective diameter of 1.7 nm and possesses properties similar to other mitochondrial porins. Surprisingly, the membrane conductance also increased in the presence of detergent extracts of the mitochondrial outer membrane of the mutant. Single-channel recordings of lipid bilayer membranes in the presence of small concentration of the mutant membranes suggested that this membrane also contained a pore. The reconstituted pores had a single-channel conductance of 2.0 nS in 1 M KCl and the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. This means that the pores present in the mitochondrial outer membranes of the yeast mutant have a much smaller effective diameter than "normal" mitochondrial porins. Zero-current membrane potential measurements suggested that the second mitochondrial porin is slightly cation-selective, while yeast porin is slightly anion-selective in the open state but highly cation-selective in the closed state. The possible role of these pores in the metabolism of mitochondria is discussed.

Electric Conductivity↗
PubMed1989

[LH episodes in high performance sports].

For the elucidation of hypothalamic factors, which predominantly cause exercise-amenorrhea 10 untrained and 6 trained volunteers were experimentally tested during 6 hours of rest (R) and 2 hours long distance run (E). The exercise exerted several physical and metabolic demands, leading to increment of LH pulse frequently mainly in trained women (interpulse-interval IPI, R: 104 +/- 50, E: 78.8 +/- 33), due to unaltered beta-endorphin (R: 6.4 +/- 2, E: 6.5 +/- 1.5) and augmented norepinephrine (R: 462 +/- 275, E: 1267 +/- 773 pg/ml). In untrained subjects LH pulses decreased in frequency, as a consequence of increased beta-E and NE (R: 6 +/- 3, E: 12.2 +/- 7.4 pg/ml) (R: 432 +/- 208, E: 947 +/- 473).

Adult↗
PubMed1989

Organ-specific antibodies against ovary in patients with systemic lupus erythematosus.

We studied whether patients with systemic lupus erythematosus can have organ-specific antibodies directed against ovaries. With a recently developed enzyme-linked immunosorbent assay system that uses both a soluble and an extractable corpus luteum antigen fraction, we detected both IgG and IgM antibodies in the sera of patients with systemic lupus erythematosus (16 of 19, 84%). The highest levels of IgG antibodies were seen when the extractable organ fraction (ST80) was used as the antigen.

Antibodies↗