Showing papers by "Roland Benz published in 1989"

Pore formation by the Escherichia coli hemolysin: evidence for an association-dissociation equilibrium of the pore-forming aggregates.

Abstract: Lipid bilayer experiments were performed in the presence of hemolysin of Escherichia coli. The toxin had a rather low activity in membranes formed of pure lipids, such as phosphatidylcholine or phosphatidylserine. In membranes from asolectin, a crude lipid mixture from soybean, hemolysin was able to increase the conductance by many orders of magnitude in a steep concentration-dependent fashion, which suggested that several hemolysin molecules could be involved in the conductive unit. Furthermore, the much higher toxin activity in asolectin membranes would be consistent with the assumption that this lipid contains a receptor needed for membrane activity of the toxin. The results of single-channel records showed that the membrane activity of hemolysin is due to the formation of ion-permeable channels with a single-channel conductance of about 500 pS in 0.15 M KCl. The hemolysin channel seemed to be formed by a toxin oligomer which showed an association-dissociation reaction and had a mean lifetime of about 2 s at small transmembrane voltages. The conductance of the hemolysin channels was only moderately dependent on the salt concentration in the aqueous phase. Zero-current membrane potential experiments showed that the hemolysin channel is cation selective. The mobility sequence of the cations in the channel was similar to their mobility sequence in the aqueous phase, which was consistent with the assumption that the hemolysin channel is wide and that the interior field strength is not very high. From the single-channel conductance, a lower limit of about 1.0 nm for the effective channel diameter could be estimated.

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

Abstract: 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 laury(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.

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

Abstract: 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 (dy) was greatly reduced. Starved bacterial cells were less sensitive to subtilin than energized cells. Depolarization of cells by carbonyl cyanide rn-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 trunsnegative 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.

[Identification of human porins. I. Purification of a porin from human B-lymphocytes (Porin 31HL) and the topochemical proof of its expression on the plasmalemma of the progenitor cell].

Abstract: We describe for the first time a porin (Porin 31HL) on the plasmalemm of an eukaryontic cell line, where porins have been found only on the outer mitochondrial membranes. The expression of the porin on the plasmalemm of transformed human B-lymphocytes is demonstrated by cytotoxicity- and indirect immunofluorescence techniques with living and fixed cells. The rabbit xenoantisera used were directed against purified Porin 31HL and free or acetylated synthetic peptides of its nineteen N-terminal amino acids. The three-step purification procedure for Porin 31HL started from a total membrane fraction of the B-cell line, followed by ion-exchange chromatography on CM- and DEAE-cellulose and a final gel filtration in SDS on Sephacryl S-300.

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

Abstract: 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 detergentsolubilized 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. The mitochondrial outer membrane of a variety of eukaryotic cells contains a pore-forming protein termed mitochondrial porin which is responsible for the exchange of water-soluble metabolites across the mitochondrial outer membrane [l - 51. This channel-forming protein was first reconstituted by Schein et al. [6] from crude extracts of Paramecium mitochondria into planar lipid bilayer membranes. Some of the mitochondrial porins have been studied in detail [l, 2, 7-10]. The outer membrane of yeast mitochondria contains a major protein with a molecular mass of 30 kDa, which constitutes at least one fifth of the protein mass of the membrane [1 1 - 131. Reconstitution experiments showed that this protein is the yeast porin [13, 141. It forms channels in lipid bilayer membranes with a single-channel conductance of 4 nS in 1 M KCI [14]. Yeast porin is encoded by a nuclear gene and synthesized on cytoplasmic ribosomes without a leader sequence [12,13]. The 30-kDa porin of yeast was cloned and sequenced [15]. Yeast porin is not particularly hydrophobic, which may indicate that the arrangement of the polypeptide chain in the secondary, tertiary, and quaternary structure is responsible for the role of the protein as an intrinsic membrane protein [15, 161. Disruption of the porin gene resulted in a viable yeast mutant whose growth on glycerolcontaining media is impaired due to a respiratory defect [17]. After some time of adaptation, its growth rate on glycerol recovers to approximately half that of wild-type cells. So far, it was not clear if the mitochondrial outer membrane of this mutant is simply leaky and allows free exchange of hydrophilic

MINI-REVIEW Pores from Mitochondrial Outer Membranes of Yeast and a Porin-Deficient Yeast Mutant: A Comparison

Abstract: 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 KCI. In the open state it behaves as a general diffusion pore with an effective diameter of 1.7nm and possesses properties similar to other mitochondriat 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 singlechannel conductance of 2.0 nS in 1 M KC1 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.

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

Abstract: 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.

[14] Ion carriers in planar bilayers: Relaxation techniques and noise analysis

Abstract: Publisher Summary This chapter describes the relaxation techniques and noise analysis of ion carriers in planar bilayers. Only limited information on the carrier system can be obtained from stationary conductance measurements. Relaxation techniques may be used for the evaluation of the rate constants of the single transport steps. In a relaxation experiment an external parameter, such as pressure, temperature, or voltage, is quickly changed; after the perturbation the approach of the system toward a new stationary state is followed. In the analysis of relaxation experiments with alkali ion carders, it is usually assumed that the association and dissociation reactions in the interface are voltage independent and that the only transport step affected by the electric field in the membrane is the translocation of MS + . Analysis of current noise represents an alternative method for evaluating the rate constants of a carrier system. A straightforward method of extracting information from the fluctuating part of the current δI ( t ) consists in measuring the spectral intensity of current noise, because a function of frequency is the value of δI ( t ) 2 per unit frequency interval.

Topology of Peripheral Kinases: its Importance in Transmission of Mitochondrial Energy

Abstract: All transport systems for anionic mitochondrial metabolites reside in the inner mitochondrial membrane. Therefore, we are entirely accustomed to the idea that the outer membrane is freely permeable for these compounds. However, the permeability for polar metabolites through the outer membrane is restricted to a slightly anion-selective general diffusion pore protein (Colombini, 1979; Benz, 1985) which, at a voltage above 30 mV, adopts a different state, characterized by low conductance and cation selectivity (Ludwig et al., 1988). The latter state of the pore was found to exclude ADP and ATP permeation in intact mitochondria (Benz et al., 1988). Having accepted this fact we turned our curiosity on the question whether a membrane potential across the outer mitochondrial membrane can exist physiologically that results in regulation of anion permeability. The answer we arrived at was that the inner membrane potential might influence the outer membrane where it is in close contact with the inner membrane. The structure and function of these contact sites has been analyzed by electron microscopy in freeze fractured mitochondria. We observed a dynamic regulation of the contacts by the rate of the oxidative phosphorylation and a distance between the two membranes in the sites of 1-2 nm. The regulation of the pore lends support to the concept of a separate compartment of adenine nucleotides in the intermembrane space which would enhance the ATP translocation process because of the following reasons.

Porins from Mitochondrial and Bacterial Outer Membranes: Structural and Functional Aspects

Abstract: The cell envelope of Gram-negative bacteria consists of three different layers, the outer membrane, the murein and the inner membrane (Beveridge, 1981). The inner membrane represents a real diffusion barrier and contains, similar to the mitochondrial inner membrane, the respiration chain and a large number of transport systems. The outer membrane of Gram-negative bacteria plays an important role in the physiology of these organisms. All nutrients or antibiotics either hydrophilic or hydrophobic have to cross this permeability barrier which means that it has special sieving properties. The active components of molecular sieving of the outer membrane are due to presence of a few major proteins called “porins” (Nakae, 1976). The porins are organized as trimers of three identical subunits and form transmembrane channels that have more general properties and sort according to the molecular weight of the solutes (Benz, 1985; Nikaido & Vaara, 1985). On the other hand, also specific porins have been identified in the outer membrane of Gram-negative bacteria which contain binding sites for substrates (Benz, 1988). The porin trimers form basically one channel in the outer membrane with three openings faced to the cell surface and one outlet into the periplasmic space (Engel et al., 1985; Lepault et al., 1988).

Purification of Mammalian Porins

Abstract: The matrix space of mitochondria is surrounded by two unit membranes. Whereas the role of the inner membrane in oxidative phosphorylation was studied in full detail in recent years, the role of the mitochondrial outer membrane in the mitochondrial metabolism has been neglected because of its apparently high permeability for small hydrophilic solutes. The mitochondrial outer membrane contains indeed general diffusion pores which explain its high permeability. The component of the membrane responsible for its molecular filter properties is a protein, called porin or VDAC (Benz, 1985). Mitochondrial porins were identified and characterized from a variety of eukaryotic cells by reconstitution experiments with planar lipid bilayers and liposomes (Colombini, 1979, De Pinto et al., 1987). The mitochondrial pore has a diameter of about 2 nm in the open state and is slightly anion selective for low transmembrane potentials. Voltages larger than 20 mV cause the shift of the pore into closed states with reduced permeability towards substrates and a changed selectivity.

Porin from Thiobacillus versutus.

Abstract: The porin of Thiobacillus versutus IFO 14567 was isolated by extraction of cell-envelopes with sodium dodecyl sulfate. It exhibited strong porin-activity after reconstitution into artificial lipid bilayer membranes. The diameter of the pore was determined as 1.6 nm, with a weak selectivity for cations being observed. The porin migrated as a single band (Mr 35 kDa) on SDS-polyacrylamide gel-electrophoresis after heating (100°C, 5 min). The porin oligomer was not sensitive towards EDTA. Analytical ultracentrifugation studies demonstrated the native oligomer to be a trimer.

PoreFormation bytheEscherichia coli Hemolysin: Evidence foran Association-Dissociation

Abstract: In membranes fromasolectin, acrudelipid mixture fromsoybean, hemolysin wasabletoincrease theconductance bymanyorders ofmagnitude inasteep concentration-dependent fashion, whichsuggested that several hemolysin molecules could beinvolved intheconductive unit. Furthermore, themuchhigher toxin activity inasolectin membranes wouldbeconsistent withtheassumption that this lipid contains areceptor needed formembrane activity ofthetoxin. Theresults ofsingle-channel records showed that themembrane activity ofhemolysin isduetotheformation ofion-permeable channels withasingle-channel conductance of about 500pSin0.15M KCI.Thehemolysin channel seemed tobeformed byatoxin oligomer whichshowed anassociation-dissociation reaction andhadameanlifetime ofabout 2satsmall transmembrane voltages. The conductance ofthehemolysin channels wasonlymoderately dependent onthesalt concentration intheaqueous phase. Zero-current membrane potential experiments showed that thehemolysin channel iscation selective. Themobility sequence ofthecations inthechannel wassimilar totheir mobility sequence intheaqueous phase, which wasconsistent withtheassumption that thehemolysin channel iswideandthat theinterior field strength isnotveryhigh. Fromthesingle-channel conductance, alower limit ofabout 1.0nmfortheeffective channel diameter could beestimated. Themembrane-active cytolysins (hemolysins) areextracellular toxic proteins which areproduced byalarge number ofgram-positive andgram-negative bacteria (12). Cytolysinsecreting bacteria arefrequently pathogenic, andthedirect involvement ofthese extracellular proteins inpathogenesis hasbeendemonstrated insomecases (15). Poreformation in thetarget cell membrane isawell-established mechanism for thealpha-toxin ofStaphylococcus aureus andformembers ofthethiol-activated cytolysins synthesized byvarious gram-positive bacteria (8,9). Thehemolysin ofEscherichia coliisoneofthebeststudied cytolysins ofgram-negative bacteria (11, 16,20). The genetics ofthis toxin havebeenwellcharacterized. Two genes, hlyCandhlyA, arerequired forthesynthesis ofthe active hemolysin (17,22).Oneofthetwogeneproducts, HlyA,isactively excreted withthehelpofa specific transport system consisting oftheproducts oftwoother hly genes, hlyBandhlyD(29). Theexcreted HlyAprotein is hemolytically inactive unless itisactivated inthecytoplasm byHlyC. Theactivation mechanism which converts HlyAto thehemolytically active form, termed HlyA*,isstill unknown. Recent data(28) showed that theactive hemolysin is highly sensitive tophospholipase C andmechanical forces which maydisrupt thestructure ofthetoxin. Ithastherefore beensuggested that hemolytically active HlyA*represents a complex whichconsists ofHlyAandoneormorephospholipid molecules noncovalently linked totheprotein (28). Onlytheactive HlyA*, butnotHlyA, possesses pathogenic potential invivo(J.Hacker, H. Hof,andW. Goebel,