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PubMed1978

Sulfide oxidation by spheroplasts of Thiobacillus ferrooxidans.

Thiobacillus ferrooxidans is an acidophilic organism important to metal leaching of low-grade ores. The aforementioned importance is related to the ability of the bacterium to oxidize reduced iron and sulfur, principally found in nature as pyrite (FeS2). The present study dealt with sulfide oxidation at low pH values and the involvement of the cell envelope in the process of the inorganic oxidations. Sulfide oxidation was noted in spheroplasts of T. ferrooxidans prepared by enzymatic and chemical treatments and partially purified by differential centrifugation. No enzyme activities were noted in membrane fractions containing enrichments of lipopolysaccharide symbolic of outer membrane material or in membrane vesicles containing (or associated with) higher levels of proteins. Results to date indicate that in an acid milieu the envelope structure containing both the outer membrane and the intact inner cytoplasmic membrane is required for sulfide oxidation.

Cell Membrane↗
PubMed1978

Development of the hamster retina: a morphologic study.

Hamster retinas were examined by light and electron microscopy during the first 15 postnatal days and at the adult stage. At birth, these retinas were more primitive than those of other rodents, but eventually they became very similar. Initial postnatal growth and development were more rapid than in other rodents of comparable ages. Mitotic figures were present throughout the first 9 days. The primitive inner plexiform layer formed during days 2 to 4; on day 7, several developmental changes became apparent: outer segment disks of the photoreceptor cells formed from flattened, smooth vesicles, horizontal cells became readily identifiable, the outer plexiform layer appeared, and ribbon synapses formed in the receptor cell axon terminals. By day 11, there were numerous conventional synapses in the inner plexiform layer, and ribbon synapses were beginning to appear in the terminal portions of the bipolar axons. The retinal tissue was progressively invaded by developing vasculature. The first photoreceptor outer segment disks formed from smooth membranous vesicles flattening radially around the ends of the connecting cilium. After the outer segment assumed a more mature appearance, with a stacked disk arrangement, further disk formation occurred by plasma membrane infoldings at the outer segment base. The adult hamster retinas were similar to those of other rodents.

Animals↗
PubMed1975

Light dependence of osmium reactivity in mouse photoreceptor cells.

Mouse photoreceptor cells exhibit local accumulations of osmium deposits after prolonged osmic staining at slightly elevated temperatures. Deposits were evident along the membranes of outer segment lamellae, Golgi cisternae and vesicles, nuclear envelopes, and synaptic vesicles. Other membranes within the photoreceptor cells were unreactive. No osmium reactivity was seen in other cells of the retina except for osmiophilic outer segment material which had been phagocytized by the pigment epithelium. In the outer segments, inner segments, and synaptic regions of the photoreceptor cells, the amount of osmium reactivity was increased by light stimulation and decreased following extended dark adaptation. The possible significance of the localized osmium reactivity is discussed.

Animals↗
PubMed1979

The effects of triton X-100 on the transfer of mannose, glucose and n-acetylglusomine phosphate to dolichol monophosphate by preparations of rough and smooth endoplasmic reticulum and of mitochondria of rat liver.

Triton X-100 and exogenous dolichol monophosphate have been used to investigate the nature of enzymes responsible for the transfer of mannose, glucose and N-acetylglucosamine phosphate from nucleotide donors to dolichol monophosphate in vesicles derived from rough and smooth endoplasmic reticulum and mitochondria. Mitochondria were shown to contain the highest specific activities of these enzymes. The responses of the glycosyltransferases to increasing concentrations of Triton X-100 and the effect on these responses of exogenous dolichol monophosphate suggest that the enzymes for mannose and glucose transfer are less hydrophobic, and therefore less intrinsic, in the membrane than the enzyme for N-acetylglucosamine phosphate transfer. In smooth vesicles the results are consistent with mannosyl- and glucosyl-transferases being located at both inner and outer faces of the membrane. In rough vesicles and in mitochondria mannosyl- and glucosyl-transferases were confirmed at the outer face. There is, however, only one site of N-acetylglucosamine phosphate transfer, this being more hydrophobically located in the membrane than the other sites of glycosyl transfer. Mitochondrial enzyme activity closely resembled that of rough endoplasmic reticulum in response to Triton X-100 and exogenous dolichol monophosphate, and is probably associated with the outer membrane.

Acetylglucosamine↗
PubMed1978

Molecular structure of membrane vesicles from Escherichia coli.

The molecular architecture of membrane vesicles prepared from Escherichia coli ML 308-225 has been studied by using crossed immunoelectrophoresis, and a reference pattern of 52 discrete immunoprecipitates has been established. Progressive immunoadsorption experiments conducted with untreated control vesicles and with physically disrupted vesicles demonstrate that the membrane-associated immunogens fall into two categories: (i) those immunogens typified by ATPase (ATP phosphohydrolase, EC 3.6.1.3) and NADH dehydrogenase [NADH: (acceptor) oxidoreductase, EC 1.6.99.3] whose expression is minimal unless the vesicles are disrupted; and (ii) immunogens such as Braun's lipoprotein that are expressed to similar extents in untreated and in disrupted vesicles. A mathematical relationship between the peak area subtended by an immunoprecipitate in the crossed immuno-electrophoresis system and the quantity of vesicles used in the adsorption process has been derived. This relationship allows quantitation of the degree to which specific membrane immunogens partition between exposed and unexposed surfaces of the vesicle membrane. The results demonstrate conclusively that >95% of the membrane in the vesicle preparations is in the form of sealed sacculi with the same polarity as the intact cell. Moreover, the findings provide a strong indication that dislocation of immunogens from the inner to the outer surface of the membrane during vesicle preparation does not occur to an extent exceeding 11%.

Adenosine Triphosphatases↗
PubMed1977

Proton spin-lattice relaxation of retinal rod outer segment membranes and liposomes of extracted phospholipids.

A large fraction of the phospholipid protons of bovine retinal rod outer segment (ROS) disc membrane vesicles yield well-resolved nuclear magnetic resonance lines near physiological temperature. The spin-lattice (T1) relaxation rates of the resolved sharp resonance of ROS disc membranes appear biphasic above 10 degrees C. The rate of the more rapidly relaxing component of each resonance matches closely the relaxation rate of the corresponding resonances of liposomes of purified ROS phospholipids. The slowly relaxing component of each disc membrane resonance is most likely due to phospholipids whose motion is affected by rhodopsin. The primary difference in the relaxation behavior of phospholipids in the ROS membrane vesicles and ROS liposomes appears to be in T1, rather than T2, since the corresponding sharp resonances of both preparations have similar linewidths. These observations suggest that the interaction of rhodopsin with the more fluid membrane phospholipids predominantly affects relatively high frequency segmental motions, which determine T1, while having minimal effects on the lower frequency segmental motions, which influence T2. This conclusion can be rationalized by assuming that a substantial fraction of the interacting phospholipids are relatively fluid with respect to less frequent, larger amplitude segmental motions, but that the more frequent segmental motions (such as beta-coupled trans-gauche isomerizations) are significantly restricted by interaction with protein.

Animals↗
PubMed1978

Relationship between vitamin K-dependent carboxylation and vitamin K epoxidation.

It has been postulated that the liver microsomal conversion of vitamin K hydroquinone to its 2,3-epoxide (epoxidase activity) is coupled in some obligatory fashion to the vitamin K-dependent carboxylation (carboxylase activity) event also occurring in microsomes. This hypothesis is supported by the observations that the requirements for the two reactions are similar and that conditions that promote increased carboxylation increase the epoxidase activity. It has now been shown that both of these reactions are localized in the rough microsomal fraction of a cellular homogenate, and that both activities appear to be located on the luminal rather than the outer surface of microsomal membrane vesicles. The epoxidase activity has been found to be enriched as the microsomal carboxylase activity is fractionated, and a microsomal inhibitor of the carboxylase activity had been shown to also inhibit the epoxidase activity. The enzyme glutathione peroxidase inhibits both of these activities, suggesting that a hydroperoxide of the vitamin might be an intermediate for both reactions. The organic hydroperoxide t-butyl-OOH has also been shown to have weak vitamin K-like activity in an in vitro system. These data strengthen the hypothesis that these two reactions are related, perhaps through a common intermediate, but do not provide a definite molecular role for this interrelationship.

1-Carboxyglutamic Acid↗
PubMed1975

The distribution of anionic sites on the surfaces of mitochondrial membranes. Visual probing with polycationic ferritin.

Polycationic ferritin, a multivalent ligand, was used as a visual probe to determine the distribution and density of anionic sites on the surfaces of rat liver mitochondrial membranes. Both the distribution of bound polycationic ferritin and the topography of the outer surface of the inner mitochondrial membrane were studied in depth by utilizing thin sections and critical-point dried, whole mount preparations for transmission electron microscopy and by scanning electron microscopy. Based on its relative affinity for polycationic ferritin, the surface of the inner membrane contains discrete regions of high density and low density anionic sites. Whereas the surface of the cristal membrane contains a low density of anionic sites, the surface of the inner boundary membrane contains patches of high density anionic sites. The high density anionic sites on the inner boundary membrane were found to persist as stable patches and did not dissociate or randomize freely when the membrane was converted osmotically to a spherical configuration. The observations suggest that the inner mitochondrial membrane is composed of two major regions of anionic macromolecular distinction. It is well-known that an intermembrane space exists between the two membranes of the intact mitochondrion; however, a number of contact sites occur between the two membranes. We determined that the outer membrane, partially disrupted by treatment with digitonin, remains attached to the inner membrane at these contact sites as inverted vesicles. Such attached vesicles show that the inner surface of the outer membrane contains anionic sites, but of decreased density, surrounding the contact sites. Thus, the intermembrane space in the intact mitochondrion may be maintained by electronegative surfaces of the two mitochondrial membranes. The distribution of anionic sites on the outer surface of the outer membrane is random. The nature and function of fixed anionic surface charges and membrane contact sites are discussed with regard to recent reports relating to calcium transport, protein assembly into mitochondrial membranes, and membrane fluidity.

Animals↗
PubMed1977

Structural heterogeneity of the cytoplasmic and outer membranes of Escherichia coli.

The cytoplasmic and outer membranes of gram-negative bacteria can be isolated from spheroplasts, and separated on sucrose density gradients. Lysis of spheroplasts causes extensive membrane fragmentation and since the characteristics of the fragments obtained by different lysis procedures need not be identical, the influence of the disruption method on membrane composition has been examined. Spheroplasts of Escherichia coli J5 were lysed by osmotic shock, which did not significantly separate the cytoplasmic and outer membranes, but resulted in mixed membrane vesicles. Lysis in the French press and by sonication caused extensive membrane fragmentation and separation. Sonication, however, also caused some fusion between fragments of the outer and the cytoplasmic membranes; this intermembrane fusion increased with sonication time. When the cytoplasmic and outer membranes were well separated and intermembrane fusion was minimal or absent, the cytoplasmic and outer membrane fragments were heterogeneous with respect to density and ovarll phospholipid, protein and lipopolysaccharide composition. In addition, cytoplasmic, but not outer, membrane fragments were also heterogeneous with respect to protein composition. It is concluded, therefore, that membrane fragments obtained from the cytoplasmic and outer membranes are heterogeneous independently of the lysis procedures used to obtain these fragments. Possible reasons for this heterogeneity are discussed.

Bacterial Proteins↗
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↗
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↗
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↗
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↗
PubMed1976

Effect of polymyxin on the ultrastructure of the outer membrane of wild-type and polymyxin-resistant strain of Salmonella.

The effect of polymyxin on two sets of Salmonella mutants was studied by thin-section and scanning electron microscopy. Polymyxin (in increasing concentrations, starting just below bactericidal effect) caused the appearance of the previously described rodlike projections on the cell surface of wild-type (smooth, polymyxin-sensitive) bacteria. These projections seemed to involve the outer membrane of the cell wall. In rough mutants, which are deficient in lipopolysaccharide, the projections were much smaller and flat. Higher concentrations of polymyxin were required to produce morphological effects in polyxmin-resistant mutants of both smooth and rough forms. Furthermore, in these mutants polymyxin caused vesicle-like bulging of the total outer membrane quite different in appearance from the rodlike projections of the wild type.

Cell Membrane↗
PubMed1979

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.

Cell Membrane↗
PubMed1978

[Electron microscopic and morphometric investigations of the integument of Acanthocephala (Aschelminthes) (author's transl)].

The integument of the acanthocephalans Echinorhynchus gadi, Acanthocephalus lucii, Polymorphus minutus and Macracanthorhynchus hirudinaceus have been investigated by stereoscan and transmission electron microscopy. The absorptive surface is considerably increased by invaginations of the outer plasma membrane (pore canals and vesicles). The rate of this enlargement has been calculated using morphometric methods. The values range from 20 to 62. The values are compared to values obtained from other gutless parasitic helminths and from the free-living rotifers which are presumably to be regarded as closely related to the acanthocephalans.

Acanthocephala↗
PubMed1978

[Agglutination and acrosome reaction of ram spermatozoa incubated in the bovine folicular fluid].

Fresh-ejaculated sperm of ram was incubated at a temperature of 38 degrees C in the bovine follicular fluid and homologous blood serum (blood plasma). The spermatozoa were studied in native state under a microscope with phase contrast and as ultra-thin sections under a transmission electron microscope. In the follicular fluid and in the blood serum, strong agglutination of spermatozoa occurred, with its maximum after about two hours. Only the heads of spermatozoa agglutinated, the flagella being loose were arranged parallelly. Although the progressive motility of spermatozoa was not observed, the motility of flagella was not affected. The investigation under the electron microscope showed that the agglutination occurred only in acrosomes of intact spermatozoa. It was also found out that the follicular fluid induced the acrosome reaction of spermatozoa. The course of the acrosome reaction is similar to that in the other mammals: first of all plasma membrane becomes undulated, then it fuses with the outer acrosome membrane, giving origin to vesicles within the entire acrosome, except the equatorial segment. The acrosome reaction was found in about 5% spermatozoa, and therefore it may be assumed that secretions of the oviduct and uterus play their role to induce the acrosome reaction.

Acrosome↗