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Results for “outer membrane vesicles”

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At least 667 records · Page 37Linked to original sources
PubMed1989

Arylsulphatase and acid phosphatase activity associated with developing and ripe spermatozoa of the mussel Mytilus edulis.

Arylsulphatase and acid phosphatase activity were demonstrated cytochemically in spermatozoa of the marine mussel Mytilus edulis. Reaction product resulting from arylsulphatase activity was measured using an integrating microdensitometer and found to increase with incubation time and to be variable according to the pH of the incubation medium. Two peaks in activity, at pH 4.5 and 6.0 were evident for some experimental protocols suggesting the possibility of two isoenzymes; however, studies on the ultrastructural localization of the enzyme showed no difference between sites of activity for the two pH values. Ultrastructural localization of arylsulphatase showed activity associated with the Golgi body of developing spermatids and in particular within the proacrosomal vesicles but limited to the periphery of the acrosomal vesicle which is formed with the fusion of the proacrosomal vesicles. In spawned spermatozoa arylsulphatase activity was localized in association with the axial rod and subacrosomal material; activity also occurred along the outer acrosomal membrane and within the acrosomal vesicle and also associated with the acrosomal process following the acrosome reaction. Sulphate groups were demonstrated cytochemically within the vitelline coat of oocytes in the mantle tissue. These findings suggest that arylsulphatase could be one of the lysins previously demonstrated in M. edulis spermatozoa. Acid phosphatase activity was demonstrated in spawned spermatozoa around the nuclear envelope and along the outer acrosomal membrane.

Acid Phosphatase↗
PubMed1988

Distribution and properties of the glycylsarcosine-transport system in rabbit renal proximal tubule. Studies with isolated brush-border-membrane vesicles.

The distribution and properties of the peptide-transport system in rabbit renal proximal tubule was examined with glycylsarcosine as the substrate and using brush-border-membrane vesicles derived from pars convoluta (outer cortex) and pars recta (outer medulla). The dipeptide was transported into these vesicles against a concentration gradient in the presence of an inward-directed H+ gradient, demonstrating the presence of a H+-coupled peptide-transport system in outer-cortical as well as outer-medullary brush-border membranes. Even though the transport was electrogenic and was energized by a H+ gradient in both membranes, the system was more active in outer medullary membranes than in outer cortical membranes. Kinetic analysis showed that, although the affinity of the transport system for glycylsarcosine was similar in both membrane preparations, the capacity of the system was significantly greater in outer medulla than in outer cortex. In addition, the pH profiles of the peptide-transport systems in these membrane preparations also showed dissimilarities. The greater dipeptide uptake in one membrane vis-à-vis the other may probably be due to the difference in the affinity of the transport system for H+ and/or the difference in peptide/H+ stoichiometry.

Animals↗
PubMed1985

Relation between thyroid peroxidase, H2O2 generating system and NADPH-dependent reductase activities in thyroid particulate fractions.

In thyroid gland, iodination takes place on the apical plasma membrane and requires the presence of the thyroid peroxidase and H2O2 generating system. H2O2 generation and NBT (nitro blue tetrazolium) reductase activity (both of which are NADPH-dependent) as well as peroxidase activity were compared for their respective orientations in membrane vesicles. The possible role of NADPH-NBT reductase activity in H2O2 generation was also examined. Results favor the conclusion that thyroid peroxidase is oriented towards the luminal side of the vesicles, whereas the NADPH site of NADPH oxidase-dependent H2O2 generation is located on the external side of the same or of different vesicles. Furthermore, it is shown that different NADPH-NBT reductase activities are present on both the outer and inner surfaces of the membrane vesicles, and that none of these activities is able to produce either H2O2 or O-2. The idea that a multi-component complex is involved in H2O2 generation is discussed, and a model is proposed which takes into account the possible spatial separation of the thyroid peroxidase site from the NADPH site of this H2O2 generation system on the apical membrane of the thyrocyte.

Animals↗
PubMed1979

Antigenic architecture of membrane vesicles from Escherichia coli.

The antigenic architecture of membrane vesicles prepared from Escherichia coli ML 308--225 has been studied using crossed immunoelectrophoresis. Progressive immunoadsorption experiments conducted with control vesicles and with physically disrupted vesicles were used to monitor and quantitate the expression of 14 different immunogens. Eleven immunogens, including NADH dehydrogenase (EC 1.6.33.3), D-lactate dehydrogenase (EC 1.1.1.27), dihydro-orotate dehydrogenase (EC 1.3.3.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.43), polynucleotide phosphorylase (EC 2.3.7.8), and beta-galactosidase (EC 3.2.1.23), exhibit minimal expression (10% or less) unless the vesicles are disrupted. Three unidentified antigens are expressed to a similar extent in untreated and disrupted vesicles. Consideration of these and other results [Owen, P., & Kaback, H. R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3148] in terms of membrane polarity, dislocation of antigens, and possible transmembrane orientation of some immunogens reveals that over 95% of the membrane in the vesicle preparations is in the form of sealed sacculi with the same orientation as the intact cell. Furthermore, antigens are distributed across the membrane in a highly asymmetric manner, indicating that dislocation of components from the inner to the outer surface of the membrane during vesicle preparation does not occur to an extent exceeding 10%.

Antigen-Antibody Reactions↗
PubMed2003

Reconstituted TOM core complex and Tim9/Tim10 complex of mitochondria are sufficient for translocation of the ADP/ATP carrier across membranes.

Precursor proteins of the solute carrier family and of channel forming Tim components are imported into mitochondria in two main steps. First, they are translocated through the TOM complex in the outer membrane, a process assisted by the Tim9/Tim10 complex. They are passed on to the TIM22 complex, which facilitates their insertion into the inner membrane. In the present study, we have analyzed the function of the Tim9/Tim10 complex in the translocation of substrates across the outer membrane of mitochondria. The purified TOM core complex was reconstituted into lipid vesicles in which purified Tim9/Tim10 complex was entrapped. The precursor of the ADP/ATP carrier (AAC) was found to be translocated across the membrane of such lipid vesicles. Thus, these components are sufficient for translocation of AAC precursor across the outer membrane. Peptide libraries covering various substrate proteins were used to identify segments that are bound by Tim9/Tim10 complex upon translocation through the TOM complex. The patterns of binding sites on the substrate proteins suggest a mechanism by which portions of membrane-spanning segments together with flanking hydrophilic segments are recognized and bound by the Tim9/Tim10 complex as they emerge from the TOM complex into the intermembrane space.

Amino Acid Sequence↗
PubMed1983

Chloride transport in the thick ascending limb of Henle's loop: potassium dependence and stoichiometry of the NaCl cotransport system in plasma membrane vesicles.

Cells were isolated from the thick ascending limb of Henle's loop of rabbit kidney outer medulla and a plasma membrane fraction was prepared by differential centrifugation. Sodium and rubidium uptake into the plasma membrane vesicles were determined by a rapid filtration technique. In the presence of a 100 mM KCl gradient and 0.5 mM sodium the vesicles took up 252 +/- 82 pmoles sodium/mg protein X 15s; 52% of the uptake was dependent on the presence of chloride or inhibited by 10(-3) M bumetanide. If KCl was stepwise replaced by choline chloride, sodium uptake decreased in the absence of bumetanide but was only insignificantly altered in the presence of bumetanide. Potassium exerted a halfmaximum stimulation at 22.3 +/- 9.7 mM. In tracer exchange experiments under zero salt gradient conditions, sodium uptake was also strongly reduced in the absence of potassium. Rubidium uptake into the same membrane fraction was highest in the presence of a NaCl gradient, and decreased 41% when sodium was replaced by choline or when 10(-3) M bumetanide was present. Replacement of sodium chloride by sodium nitrate also inhibited rubidium uptake. When the sodium, chloride and potassium dependence of the bumetanide sensitive sodium uptake was investigated in more detail, Hill coefficients for sodium of 1.0 +/- 0.03, for chloride of 1.8 +/- 0.2 and for potassium of 0.98 +/- 0.03 were obtained. These results are consistent with the presence of a Na-Cl-K cotransport system in the medullary thick ascending limb of Henle's loop which may operate with a stoichiometry of 1/2/1.

Animals↗
PubMed1976

Disruption of Escherichia coli outer membranes by EM 49. A new membrane active peptide.

A new peptide antibiotic, EM 49, is shown to disrupt the structure of Escherichia coli outer membranes and release outer membrane fragments into the surrounding media. Evidence supporting this conclusion indludes EM 49 stimulated release of outer membrane phospholipids, lipopolysaccharide, and membrane fragments having a phospholipid and polypeptide composition similar to outer membranes. The density of the membrane fragments released by EM 49 was 1.22 g/cm3, which was identical to isolated outer membranes. Approximately 10 to 15% of the E. coli lipopolysaccharide was released upon treatment with EM 49. Both scanning and transmission electron microscopy revealed that the antibiotic caused the formation of numerous protrusions or blebs on the surface of E. coli with apparent release of membrane vesicles from the cells. Direct interaction between EM 49 and outer membranes was demonstrated using outer membranes labeled with the fluorescent dye diphenylhexatriene. Treatment of the fluorescent-labeled outer membranes with EM 49 increased fluorescence intensity and decreased polarization, indicating that the peptide perturbed outer-membrane structure. In addition, strong interactions between EM 49 and purified E. coli phospholipids were detected using the Hummel and Dreyer technique. Association constants between the peptide and phospholipids were approximately 10(5) M-1. A model for the disruptive effect of EM 49 on outer-membrane structure is proposed in which the fatty acid chain of the antibiotic is inserted into the hydrophobic core of the membrane. This orientation would allow the polycationic, peptide portion of the antibiotic to disrupt the antibiotic to disrupt the normal electrostatic interactions between divalent cations and components of the outer membrane. Evidence supporting this conclusion includes specific protection of E. coli from EM 49 by Mg2+ and Ca2+ and inhibition of EM 49 stimulated phospholipid release by these cations. Disruption of the antibiotic to penetrate to the inner membrane, which is probably the primary killing site of EM 49.

Anti-Bacterial Agents↗
PubMed2000

Novel toluene elimination system in a toluene-tolerant microorganism.

In studies of Pseudomonas putida IH-2000, a toluene-tolerant microorganism, membrane vesicles (MVs) were found to be released from the outer membrane when toluene was added to the culture. These MVs were found to be composed of phospholipids, lipopolysaccharides (LPS), and very low amounts of outer membrane proteins. The MVs also contained a higher concentration of toluene molecules (0.172 +/- 0. 012 mol/mol of lipid) than that found in the cell membrane. In contrast to the wild-type strain, the toluene-sensitive mutant strain 32, which differs from the parent strain in LPS and outer membrane proteins, did not release MVs from the outer membrane. The toluene molecules adhering to the outer membrane are eliminated by the shedding of MVs, and this system appears to serve as an important part of the toluene tolerance system of IH-2000.

Bacterial Outer Membrane Proteins↗
PubMed1987

Axial heterogeneity of organic cation transport along the rabbit renal proximal tubule: studies with brush-border membrane vesicles.

Brush-border membrane vesicles prepared from rabbit kidney outer cortex (rich in S1 and S2) and outer medulla (rich in S3) were used to evaluate the axial heterogeneity of tetraethylammonium transport in the proximal tubule. The vesicle preparations had similar Km values but the Vmax values differed, suggesting that axial heterogeneity of tetraethylammonium secretion may be due to differences in transport across the brush-border membrane.

Animals↗
PubMed1999

Export of virulence genes and Shiga toxin by membrane vesicles of Escherichia coli O157:H7.

Membrane vesicles released by Escherichia coli O157:H7 into culture medium were purified and analyzed for protein and DNA content. Electron micrographs revealed vesicles that are spherical, range in size from 20 to 100 nm, and have a complete bilayer. Analysis of vesicle protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates vesicles that contain many proteins with molecular sizes similar to outer membrane proteins and a number of cellular proteins. Immunoblot (Western) analysis of vesicles suggests the presence of cell antigens. Treatment of vesicles with exogenous DNase hydrolyzed surface-associated DNA; PCR demonstrated that vesicles contain DNA encoding the virulence genes eae, stx1 and stx2, and uidA, which encodes for beta-galactosidase. Immunoblot analysis of intact and lysed, proteinase K-treated vesicles demonstrate that Shiga toxins 1 and 2 are contained within vesicles. These results suggest that vesicles contain toxic material and transfer experiments demonstrate that vesicles can deliver genetic material to other gram-negative organisms.

Bacterial Outer Membrane Proteins↗
PubMed1994

Intra-acrosomal organization of a 90-kilodalton antigen during spermiogenesis in the rat.

The localization of an acrosomal protein was studied using a monoclonal antibody MN7 raised against mouse spermatozoa. MN7 specifically recognized the anterior acrosome of several mammalian (mouse, rat, hamster) spermatozoa fixed with paraformaldehyde. An immunoblot study with periodate treatment showed that MN7 recognized a carbohydrate region of a 90 kDa protein in an extract of mouse and rat cauda epididymal spermatozoa. The change in distribution of the MN7 antigen during acrosome development was investigated in the rat testis using the pre-embedding immunoperoxidase technique. The antigen first appeared in the proacrosomic granules of spermatids in steps 1-2. Small vesicles adjacent to the outer acrosomal membrane and the developing acrosomic system were immunoreactive during steps 4-7. The majority of the antigen was then redistributed to the head-cap portion during steps 8-18, and finally restricted to the anterior acrosome in the step 19-spermatid. These results suggest that the antigen is transported to the acrosome by way of the vesicles that originate from the Golgi apparatus during early spermiogenesis, and are then delivered to the final destination within the acrosome by the intra-acrosomal migration during late spermiogenesis.

Acrosome↗
PubMed1979

Aggregations of synaptic vesicles on the exposed inner membrane of presynaptic mitochondria in brain.

Fragments of rat cerebral cortex have been incubated under various conditions. When divalent cations are present, patches of the external membrane of some mitochondria are disassembled leaving the inner mitochondrial membrane exposed to the cytoplasm. Sometimes the entire external membrane is missing. In presynaptic bulbs the synaptic vesicles are attracted and adhere to the exposed outer face of the inner mitochondrial membrane. The mode of attraction and adhesion of the vesicles is discussed. Possibly this could serve as a model for further investigation of the attraction of vesicles to the active zone of the presynaptic membrane.

Animals↗
PubMed1982

Heat-induced blebbing and vesiculation of the outer membrane of Escherichia coli.

Thermal damage to the outer membrane of Escherichia coli W3110 was studied. When E. coli cells were heated at 55 degrees C in 50 mM Tris-hydrochloride buffer at pH 8.0, surface blebs were formed on the cell envelope, mainly at the septa of dividing cells. Membrane lipids were released from the cells during the heating period, and part of the released lipids formed vesicle-like structures from the membrane. This vesicle fraction had a lipopolysaccharide to phospholipid ratio similar to that of the outer membrane of intact cells, whereas it had a lower content of protein than the isolated outer membrane. After heating bacterial cells at 55 degrees C for 30 min, the resulting leakage from the cells of a periplasmic enzyme, alkaline phosphatase, amounted to 52% of the total activity, whereas no release of a cytoplasmic enzyme, glucose-6-phosphate dehydrogenase, was detected. The results obtained suggest that surface blebs formed by heat treatment almost completely consist of the outer membrane and that the blebs may be gradually released from the cell surface into the heating menstruum to partially form vesicles.

Alkaline Phosphatase↗
PubMed1987

Channels in the mitochondrial outer membrane: evidence from patch clamp studies.

Patch clamp techniques were applied to outer mitochondrial membranes of giant mitochondria from mice kept on a cuprizone diet or to vesicles produced by fusing membranes derived from the outer membrane of Neurospora mitochondria. In the negative range of potentials the conductances decreased with increases in the magnitude of voltage, suggesting the closing of channels. Experiments in which mitochondria were treated with the polyanion polymethacrylate maleate styrene (1:2:3) or succinic anhydride suggest that the channels correspond to VDAC. Although sometimes conductance also decreased with increasing potential over a narrow range of positive potentials, more commonly the conductances increased. Although this phenomenon may represent a detachment of the patch, the changes in conductance are reversible, suggesting that they correspond to the formation or the opening of channels.

Electric Conductivity↗
PubMed1978

Asymmetric permeability of the membrane of egg phosphatidylcholine-cholesterol vesicles to 2,2,6,6 tetramethyl piperidinyl-l-oxycholine.

The rates of uptake and release of 2,2,6,6 tetramethyl piperidinyl-l-oxycholine (Tempo-choline) for vesicles made of dipalmitoyl phosphatidylcholine (DPPC) and of egg phosphatidylcholine-cholesterol mixtures were measured by ESR and found to have interesting temperature-dependences. In the former case, both rates exhibit a sharp maximum at the critical temperature of phase transition of the bilayer membrane. In the latter case, the permeability of the membrane to Tempo-choline is asymmetric with respect to uptake and release: uptake is appreciable at temperatures higher than 66 degrees C, while release is observable only at temperatures higher than 80 degrees C. The asymmetric permeability is explained in terms of the asymmetric distribution of cholesterol between the outer and inner membranes of the vesicle.

Cholesterol↗
PubMed1987

Free diffusion to and from the inner nuclear membrane of newly synthesized plasma membrane glycoproteins.

Sindbis virus-infected baby hamster kidney (BHK) cells were analyzed by thin section fracture-label. Specific immunolabel with antiviral glycoprotein antibodies was used in conjunction with colloidal gold-conjugated protein A. As we previously reported (Torrisi, M. R., and S. Bonatti, 1985, J. Cell Biol., 101:1300-1306), Sindbis transmembrane glycoproteins are present in the inner nuclear membrane as well as in the outer nuclear membrane, endoplasmic reticulum, Golgi stacks and vesicles, and plasma membranes. Viral glycoproteins located on the inner nuclear membrane resemble those present on the outer membrane in terms of amount, distribution, and preferential partition after fracture. We show in this paper that Sindbis glycoproteins after treatment with cycloheximide are removed from the inner nuclear membrane with the same kinetics as their counterparts present on the outer membrane. This finding strongly suggests that newly synthesized transmembrane glycoproteins may freely diffuse to and from the inner nuclear membrane before entering into the intracellular transport pathway to the plasma membrane.

Animals↗
PubMed1982

Generation and release of DNA-binding vesicles by Haemophilus influenzae during induction and loss of competence.

Genetic transformation of bacterial cells required the induction of a state of competence to bind and absorb free DNA molecules. Induction of competence in Haemophilus influenzae was accompanied by the generation on the cell surface of membrane extensions ("blebs") 80 to 100 nm in diameter. When competent cells were returned to normal growth conditions, they shed these structures as free vesicles with a concomitant loss of cellular DNA-binding activity. Purified vesicle preparations retained the ability to bind double-stranded DNA in a nuclease-resistant, salt-stable form. Binding was specific for DNA molecules containing the 11-base pair Haemophilus uptake sequence, required Na+ and divalent cations (Mg2+, Ca2+, or Mn2+), and was inhibited by the presence of EDTA or high concentrations of salt (greater than 0.5 M NaCl). Binding was not stimulated by nucleotide triphosphates and was insensitive to the uncoupling agents dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Vesicles contained the major Haemophilus outer membrane proteins and were enriched in several minor proteins.

Cations, Divalent↗