Inhibition of Escherichia coli growth and respiration by polymyxin B covalently attached to agarose beads.

Polymyxin B was attached to agarose beads by stable covalent bonds and the antimicrobial activity of the immobilized peptide was examined. Polymyxin-agarose inhibited the growth of Escherichia coli and Pseudomonas aeruginosa, but not Bacillus subtilis. In addition, the respiration of E. coli, E. coli spheroplasts, and B. subtilis protoplasts was inhibited by immobilized polymyxin, whereas the respiration of B. subtilis was unaffected by polymyxin-agarose. The activity of polymyxin-agarose was not due to the release of free peptide from the derivative. These data indicate that polymyxin can inhibit the growth and respiration of gram-negative bacteria by interacting with the outer surface of these cells. It is proposed that perturbation of outer membrane structure by polymyxin-agarose indirectly affected the selective permeability of the inner membrane and inhibited respiration. The results of this study emphasize the importance of outer membrane structural integrity for the normal functions of gram-negative bacteria.     

Relation of Integrity of Outer Membrane with Its Protection from Oxygen in Anabaena Cell

Exposure of Anabaena 7120 cells to membrane perturbant such as EDTA or Tris (pH 8.0, 37℃, for 5–10 min) resulted in the release of outer membrane lipopolysaccharide and proteins from cells. After Tris treatment, the sensitivity of cells to crystal violet and detergents such as SDS and Triton x-100 increased and whole-cell alkaline phosphatase activity enhanced obviously, suggesting that the structure of outer membrane was modified and its permeability increased. At the same time. Tris was found to reduce nitrogen fixation activity of cells considerably in air, but not in anaerobic condition. Reconstitution of Tris-treated cells with released material might recover nitrogen-fixing activity of cells clearly, indicating that the structure of outer membrane is closely related to the protection of nitrogen fixation from oxygen. Although EDTA-treated cells released more lipopolysacharide and proteins than those of Tris-treated cells, the permeability of outer membrane and nitrogen-fixing activity were not influenced significantly. SDS-gel electrophoresis showed that Tris-treated cells released 3–4 specific polypeptides which were not present in the released material from EDTA-treated or water-treated cells. These experiments suggest that membrane perturbants-induced loss of outer membrane function is mediated through the modification of specific position in outer membrane.     

Tris(hydroxymethyl)aminomethane buffer modification of Escherichia coli outer membrane permeability.

The effect of tris(hydroxymethyl)aminomethane (Tris) buffer on outer membrane permeability was examined in a smooth strain (D280) and in a heptose-deficient lipopolysaccharide strain (F515) of Escherichia coli O8. Tris buffer (pH 8.00) was found to increase outer membrane permeability on the basis of an increased Vo of whole-cell alkaline phosphatase activity and on the basis of sensitivity to lysozyme and altered localization pattern of alkaline phosphatase. The Tris buffer-mediated increase in outer membrane permeability was found to be dependent upon the extent of exposure to and concentration of the Tris buffer. The Tris buffer effects were demonstrated not to be due to allosteric activation of cell-associated alkaline phosphatase and were specific for Tris buffer. Exposure of cells to Tris resulted in the release of a limited amount of cell envelope component. Investigators utilizing Tris buffer are cautioned that Tris is not physiologically inert and that it may interact with the system under investigation.     

Effect of ethylenediaminetetraacetic acid-Tris(hydroxymethyl)aminomethane on release of the acid-soluble nucleotide pool and on breakdown of ribosomal ribonucleic acid in Escherichia coli

Brief treatment of Escherichia coli with 2 x 10(-4)m ethylenediaminetetraacetic acid (EDTA)-0.12 m tris(hydroxymethyl)aminomethane (Tris), pH 8.0, or 0.12 m Tris alone resulted in the release of the acid-soluble nucleotide pool at 3 or 23 C. Exposure to EDTA-Tris for up to 90 min at 3 C did not result in the release of increasing amounts of 260-mmu-absorbing material. At 23 and 37 C, EDTA-Tris resulted in a steady increase in acid-soluble 260-mmu-absorbing material. Previous growth environment did not alter the release. There appeared to be degradation of 23S ribonucleic acid (RNA) after 10 min of exposure at 23 C. In addition, there was degradation of nucleotides to nucleosides and bases. This occured either within the cells with altered permeability or in the periplasmic space. This occurred in the presence of EDTA and Tris but was not seen with EDTA-phosphate. The mechanism of this degradation is unclear, since it occurs in ribonuclease I-deficient strains. Exposure to Tris buffer for long periods of time at 23 C resulted in release of the nucleotide pool and in degradation of RNA and nucleotides. These studies point out that the EDTA-Tris effect on E. coli must be divided into two parts, an early (4 to 5 min) change in permeability and a later phase of actual RNA breakdown and nucleotide degradation. Studies utilizing EDTA and Tris as agents altering permeability must thus be viewed with caution. Although the cells are viable, they have lost their acid-soluble nucleotide pool and have undergone degradation of some ribosomal RNA.     

Pseudomonas aeruginosa outer membrane protein OprH: expression from the cloned gene and function in EDTA and gentamicin resistance.

Overexpression of major outer membrane protein OprH of Pseudomonas aeruginosa as a result of mutation (in strain H181) or adaptation to low Mg2+ concentrations (in parent strain H103) is accompanied by increased resistance to polymyxin B, gentamicin, and EDTA. A 2.8-kb EcoRI fragment containing the oprH gene was subcloned into several different expression plasmids in Escherichia coli. These experiments showed that significant levels of OprH could be produced from a promoter on the EcoRI fragment; that the cloned oprH gene was not regulated by Mg2+ deficiency; that there were no differences in the expression of OprH in any construction, regardless of whether the gene from strain H103 or its OprH-overexpressing, polymyxin B-resistant derivative, strain H181, was used; and that overexpression of OprH in E. coli to the level observed in P. aeruginosa H181 did not result in a resistance phenotype. These results favored the conclusion that the mutation in strain H181 was a regulatory rather than a promoter mutation. The oprH gene was cloned behind the benzoate-inducible pm promoter in plasmid pGB25 and transferred to P. aeruginosa H103. Overexpression of OprH from the cloned gene in H103/pGB25 resulted in EDTA resistance but not polymyxin B resistance. This result suggested that another factor, possibly lipopolysaccharide, was affected by the mutation in strain H181. Consistent with this suggestion was the demonstration that mutants of strain H181 with alterations in lipopolysaccharide had reverted to wild-type polymyxin B susceptibility but had unaltered gentamicin and EDTA resistance. These data were consistent with the hypothesis that OprH replaces outer membrane-stabilizing divalent cations.     

Temperature dependence of action of polymyxin B on Escherichia coli

The temperature dependence of the action of polymyxin B on Escherichia coli was studied by using K+, Ca2+, and tetraphenylphosphonium (TPP+) ion-selective electrodes. At room temperature (27 degrees C), Ca2+ was released immediately after addition of polymyxin, while the efflux of K+ occurred after 30 s. The rapid release of Ca2+ was not affected by incubation temperature, while the efflux of K+ was significantly lowered at temperatures below about 25-30 degrees C. The uptake of TPP+ also increased after polymyxin addition. The release of Ca2+ and the uptake of TPP+ supported the disruption of the outer membrane structure reported previously. In experiments with isolated membrane vesicles (the cytoplasmic membrane being exposed), the efflux of K+ was not delayed, but was lowered at temperatures below about 15-20 degrees C. This temperature range differed significantly from that of whole cells, and was interpreted as representing a difference in membrane fluidity between the outer and cytoplasmic membranes. The phase transition temperature of the outer membrane is known to be higher than that of the cytoplasmic membrane; and the temperature dependence of efflux of K+ from membrane vesicles was compatible with the phase transition temperature of liposomes prepared with phospholipids (not containing lipopolysaccharides) extracted from E. coli. Thus, it was speculated that, with whole cells, polymyxin molecules passed through the outer membrane at temperatures above the phase transition and reached the cytoplasmic membrane, increasing its K+ permeability. The mechanism of the permeability change is discussed in terms of deformation of the cytoplasmic membrane structure induced by polymyxin molecules.     

大肠杆菌脑微血管内皮细胞侵袭基因ibeB的编码产物为外膜蛋白前体

为进一步探讨大肠杆菌脑微血管内皮细胞侵袭基因ibeB的生物学特性 ,将ibeB基因克隆到pET2 8a(+)载体 ,以E .coliBL2 1 (DE3)为宿主菌 ,经IPTG诱导后 ,通过Ni2 + NTA树脂提纯IbeB蛋白 .SDS PAGE确定纯化蛋白的分子量 ;应用无蛋白酶的体外转录和翻译系统进一步鉴定ibeB基因表达蛋白的分子量 ;通过 [3 5S]Met标记的体内T7表达体系并结合膜蛋白分离技术定位IbeB蛋白在细菌中的亚细胞分布 ;利用细菌侵袭实验分析IbeB蛋白抗体对E .coliK1侵袭人脑微血管内皮细胞的封闭作用 .结果发现 ,ibeB基因的重组蛋白表达纯化产物呈现出 5 0kD和 34kD两种分子量大小 ,5 0kD存在于表达细菌的可溶性部分 ,而 34kD则存在于包涵体中 ;体外翻译实验也显示出较弱的 5 0kD和较浓的 34kD两个蛋白带 ;体内T7表达体系实验显示 34kD的IbeB成熟蛋白定位于E .coli的外膜 ;抗 34kDIbeB蛋白抗体能封闭E .coli对人脑微血管内皮细胞的侵袭 .这些结果提示 ,大肠杆菌脑微血管内皮细胞侵袭基因ibeB的编码产物为 5 0kD的外膜蛋白前体 ,该前体可通过分子内剪接形成成熟的 34kDIbeB蛋白     

Release of outer membrane fragments from wild-type Escherichia coli and from several E. coli lipopolysaccharide mutants by EDTA and heat shock treatments.

EDTA-induced outer membrane losses from whole cells of wild-type Escherichia coli (O111:B4) and several lipopolysaccharide (LPS) mutants derived from E. coli K-12 D21 were analyzed. EDTA treatment induced losses of LPS (up to 40%), outer membrane proteins OmpA, OmpF/C, and lipoprotein, periplasmic proteins, and phosphatidylethanolamine. The extent of these releases was strain specific. Successively more EDTA was necessary to induce these losses from strains containing LPS with increasing polysaccharide chain length. An additional heat shock immediately following the EDTA treatment had no effect on LPS release, but it decreased the release of outer membrane proteins and reduced the leakage of periplasmic proteins, suggesting that the temporary increase in outer membrane "permeability" caused by Ca2+-EDTA treatment was rapidly reversed by the redistribution of outer membrane components, a process which is favored by a mild heat shock. The fact that the material released from E. coli C600 showed a constant ratio of lipoprotein, OmpA, and phosphatidylethanolamine at all EDTA concentrations tested suggests that the material is lost as specific outer membrane patches. The envelope alterations caused by EDTA did not result in cell lysis.     

Brucella-Salmonella lipopolysaccharide chimeras are less permeable to hydrophobic probes and more sensitive to cationic peptides and EDTA than are their native Brucella sp. counterparts.

A rough (R) Brucella abortus 45/20 mutant was more sensitive to the bactericidal activity of polymyxin B and lactoferricin B than was its smooth (S) counterpart but considerably more resistant than Salmonella montevideo. The outer membrane (OM) and isolated lipopolysaccharide (LPS) of S. montevideo showed a higher affinity for these cationic peptides than did the corresponding B. abortus OM and LPS. We took advantage of the moderate sensitivity of R B. abortus to cationic peptides to construct live R B. abortus-S-LPS chimeras to test the activities of polymyxin B, lactoferricin B, and EDTA. Homogeneous and abundant peripheral distribution of the heterologous S-LPS was observed on the surface of the chimeras, and this coating had no effect on the viability or morphology of the cells. When the heterologous LPS corresponded to the less sensitive bacterium S B. abortus S19, the chimeras were more resistant to cationic peptides; in contrast, when the S-LPS was from the more sensitive bacterium S. montevideo, the chimeras were more susceptible to the action of peptides and EDTA. A direct correlation between the amount of heterologous S-LPS on the surface of chimeric Brucella cells and peptide sensitivity was observed. Whereas the damage produced by polymyxin B in S. montevideo and B. abortus-S. montevideo S-LPS chimeras was manifested mainly as OM blebbing and inner membrane rolling, lactoferricin B caused inner membrane detachment, vacuolization, and the formation of internal electron-dense granules in these cells. Native S and R B. abortus strains were permeable to the hydrophobic probe N-phenyl-1-naphthylamine (NPN). In contrast, only reduced amounts of NPN partitioned into the OMs of the S. montevideo and B. abortus-S. montevideo S-LPS chimeras. Following peptide exposure, accelerated NPN uptake similar to that observed for S. montevideo was detected for the B. abortus-S. montevideo LPS chimeras. The partition of NPN into native or EDTA-, polymyxin B-, or lactoferricin B-treated LPS micelles of S. montevideo or B. abortus mimicked the effects observed with intact cells, and this was confirmed by using micelle hybrids of B. abortus and S. montevideo LPSs. The results showed that LPS is the main cause of B. abortus' resistance to bactericidal cationic peptides, the OM-disturbing action of divalent cationic chelants, and OM permeability to hydrophobic substances. It is proposed that these three features are related to the ability of Brucella bacteria to multiply within phagocytes.     

Comparative analysis of envelope proteomes in Escherichia coli B and K-12 strains

Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane beta-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies.     

Defeat of colicin tolerance in Escherichia coli ompA mutants: evidence for interaction between colicin L-JF246 and the cytoplasmic membrane.

Escherichia coli ompA mutants are tolerant to colicin L-JF246. This tolerance can be overcome by a variety of treatments that have as their target the outer membrane or the peptidoglycan layers of the cell envelope. Thus, increasing the concentration of colicin L, releasing lipopolysaccharide from the outer membrane by treatment of intact cells with ethylenediaminetetracetic acid (EDTA), converting cells to spheroplasts by treatment with lysozyme-EDTA or penicillin, or trypsin, treatment of intact cells will result in an increased colicin sensitivity. These treatments alter the outer membrane of ompA mutants and suggest that the altered outer membrane may allow the penetration of at least a portion of the colicin L molecule to a site of action located within this barrier. To substantiate this, we have demonstrated that membrane vesicles prepared from ompA mutants are sensitive to colicin L and that 14C-labeled colicin L binds rapidly to both the outer and inner membrane fractions of the cell.     

Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [¹⁴C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35 000 is a porin, responsible for the size-dependent permeability of the outer membrane.     

The effect of osmotic shock on the accessibility of the murein layer of exponentially growing Escherichia coli to lysozyme.

The restricted access of lysozyme to the murein layer of exponential phase Escherichia coli is enhanced considerably by osmotic shock. When cells suspended in Tris/EDTA/sucrose are diluted 11-fold in water or 10 mM EDTA in the presence of lysozyme, their susceptibility to lysozyme increases by a factor of 50--100, for both Escherichia coli JC411 and W3110, grown to the early exponential phase in unsuppleneted or supplemented minimal media, and in Brain Heart Infusion. Since an 11-fold dilution causes lysis of lysozyme spheroplasts, the effects of a 2-fold dilution have also been investigated. A 2-fold dilution of cell suspended in TrisEDTA/sucrose still increases their susceptibility to lysozyme by a factor of 10--50, but the resulting spheroplasts remain intact. EDTA is necessary to permit lysozyme access to the murein layer during the dilution, which is ineffective in the presence of 5 mM MgCl2. These results are discussed in terms of the formation of lysozyme spheroplasts from young Escherichia coli.     

Permeability of the membrane of the Escherichia coli envelope for ethidium bromide

The interaction of ethidium bromide, a fluorescent dye, with Escherichia coli cells was studied. The envelope of intact cells was shown to be impermeable for ethidium bromide molecules. The dye penetrated however into E. coli spheroplasts. The barrier properties of the cell envelope against ethidium bromide were ruptured if the cells were treated with EDTA. The results suggest that the outer membrane serves as a principal barrier against penetration of ethidium bromide inside the cells while the cytoplasmic membrane of E. coli is permeable for the dye.     

Mode of action of gramicidin S on Escherichia coli membrane

The action of a cationic antibiotic gramicidin S on the outer and cytoplasmic membranes of Escherichia coli was studied. It was found that gramicidin S disrupted the permeability barrier of the outer membrane, permitting the permeation of an antibiotic ionophore, this being similar to the action of the dimer in compound 48/80 (Katsu, T., Shibata, M. and Fujita, Y. (1985) Biochim. Biophys. Acta 818, 61-66). However, differently from the dimer, gramicidin S further stimulated the efflux of K+ through the cytoplasmic membrane of E. coli. The time course of K+ permeability change accorded well with that of change in the viability of E. coli cells. These changes occurred at temperatures above the phase transition of the cytoplasmic membrane. This temperature range differed greatly from the case of polymyxin B, a polycationic antibiotic acting at temperatures above the phase transition of the outer membrane. We discuss the mode of gramicidin S action on the cytoplasmic membrane of E. coli, in comparison with the results on red blood cells and liposomes.     

Lactoferrin and transferrin damage of the gram-negative outer membrane is modulated by Ca2+ and Mg2+

Lactoferrin and transferrin have antimicrobial activity against selected Gram-negative bacteria, but the mechanism of action has not been defined. We studied the ability of lactoferrin and transferrin to damage the Gram-negative outer membrane. Lipopolysaccharide release by the proteins could be blocked by concurrent addition of Ca2+ and Mg2+. Addition of Ca2+ also blocked the ability of lactoferrin to increase the susceptibility of Escherichia coli to rifampicin. Transferrin, but not lactoferrin, increased susceptibility of Gram-negative bacteria to deoxycholate, with reversal of sensitivity occurring with exposure to Ca2+ or Mg2+. In transmission electron microscopy studies polymyxin B caused finger-like membrane projections, but no morphological alterations were seen in cells exposed to EDTA, lactoferrin or transferrin. These data provide further evidence that lactoferrin and transferrin act as membrane-active agents with the effects modulated by Ca2+ and Mg2+.     

Susceptibility to Polymyxin B of Penicillin G-induced Proteus mirabilis L Forms and Spheroplasts

A polymyxin B-resistant strain of Proteus mirabilis was converted into L forms and spheroplasts in the presence of penicillin G. This treatment caused a 400-fold increase in polymyxin B susceptibility. The acquired susceptibility was in the range of the natural susceptibility reported for susceptible gram-negative bacteria ( approximately 1 mug/ml). The high susceptibility to polymyxin B was lost as soon as the spheroplasts and L forms were allowed to reconvert into the bacillary form in penicillin-free media. This behavior is strong evidence that the natural resistance of Proteus strains to polymyxins is due to the impermeability of the outer cell wall structures to these antibiotic substances.     

Effect of Ethylenediaminetetraacetate upon the Surface of Escherichia coli

The effect of ethylenediaminetetraacetate (EDTA) on the envelope of two strains of Escherichia coli (B and Cla) was studied with freeze-fracturing methods. Untreated cells showed the outer membrane's outer surface with a fine texture of randomly spaced depressions of about 4.5-nm diameter; small areas with symmetrical arrangements of structural surface elements were also observed. The outer membrane's fracture plane revealed a random distribution of particles on its "concave" plane, only occasionally interrupted by particle-free areas. The "convex" aspect of the outer membrane's fracture plane showed only a few scattered particles. The cleavage plane of the inner membrane was often interrupted by many localized elevated plateaus, at which the cleaving process had, for short distances, switched to the outer membrane. The effects of EDTA treatment were mainly seen in the structure of the freeze-etched outer membrane: (i) the pits as well as the symmetrical surface elements of the outer membrane's outer surface had disappeared; (ii) a number of plateaus (about 20 to 50/cell) were seen at which a cleavage plane within the inner membrane had switched to the hydrophobic portion of the outer membrane (outer membrane's fracture plane). These plateaus were also visible in untreated cells; however, EDTA treatment apparently caused an increased exposure of plateaus. Surface areas, exposed by freeze-etching, revealed the underlying plateaus as elevations in the surface contour of the cell, suggesting a slower etching rate in the zones of the plateaus relative to the rest of the outer membrane. Well-defined, particle-free patches in the outer membrane's fracture plane, concave, were more frequent and larger in size after EDTA treatment than in the controls. In the presence of glycerol, the cells often cleaved in the outer membrane's fracture plane, but isolated plateaus were rarely observed. After metabolic poisoning of cells for 15 to 25 min at 37 degrees C, the plateaus had widened. These data suggest that the material of the plateaus has a slow rate of lateral diffusion. Placement of EDTA-treated cells in fresh medium at 37 degrees C caused, after 3 to 5 min, the reoccurrence of the pitted surface structure. We propose that the plateaus represent localized zones, at which newly synthesized lipopolysaccharide has been inserted.     

Evidence for the specific association of the chromosomal origin with outer membrane fractions isolated from Escherichia coli.

DNA-envelope complexes isolated from osmotically lysed spheroplasts of Escherichia coli contained 0.2 to 1% of the total cellular DNA after labeling with [3H]thymidine. Molecular weight determinations indicated that the amount of bound DNA was equivalent in most cases to a maximum of three binding sites per chromosome. Bound DNA from E. coli B/r was distributed approximately equally between inner and outer membrane components when envelopes were fractionated on sucrose equilibrium gradients. Outer membrane-DNA complexes, in particular, fraction H1, with a density of 1.24 g/cm3, were quite stable against shearing and against Sarkosyl NL97. In the case of E. coli B/r, H1-DNA was also relatively resistant to deoxyribonuclease. Inner membrane-DNA complexes, in contrast, were quite labile and readily dissociated to release free DNA. The outer membrane fractions did not appear to contain replication fork DNA, but small amounts may have been present in the inner membrane complexes. A two- to eightfold enrichment for chromosomal origin DNA in the envelope was obtained when cultures of E. coli K-12, synchronized for DNA replication, were pulse labeled at different times in the replication cycle. This enrichment was found invariably in the outer membrane fractions. However, the data do not exclude the possibility that this DNA is bound to regions of adhesion between inner and outer membranes which sediment with a density indistinguishable from that of the outer membrane.     

Ethanol exposure decreases mitochondrial outer membrane permeability in cultured rat hepatocytes

Mitochondrial metabolism depends on movement of hydrophilic metabolites through the mitochondrial outer membrane via the voltage-dependent anion channel (VDAC). Here we assessed VDAC permeability of intracellular mitochondria in cultured hepatocytes after plasma membrane permeabilization with 8 μM digitonin. Blockade of VDAC with Koenig’s polyanion inhibited uncoupled and ADP-stimulated respiration of permeabilized hepatocytes by 33% and 41%, respectively. Tenfold greater digitonin (80 μM) relieved KPA-induced inhibition and also released cytochrome c, signifying mitochondrial outer membrane permeabilization. Acute ethanol exposure also decreased respiration and accessibility of mitochondrial adenylate kinase (AK) of permeabilized hepatocytes membranes by 40% and 32%, respectively. This inhibition was reversed by high digitonin. Outer membrane permeability was independently assessed by confocal microscopy from entrapment of 3 kDa tetramethylrhodamine-conjugated dextran (RhoDex) in mitochondria of mechanically permeabilized hepatocytes. Ethanol decreased RhoDex entrapment in mitochondria by 35% of that observed in control cells. Overall, these results demonstrate that acute ethanol exposure decreases mitochondrial outer membrane permeability most likely by inhibition of VDAC.