Ultrastructural analysis of black lipid membranes

SummaryTriton X-100 (in concentrations which did not cause a significant solubilization of membrane material) caused aggregation of the intramembrane particles of human erythrocyte ghosts.Ghosts from which the extrinsic proteins had been removed by alkali treatment showed a temperature-induced aggregation of the particles. With virtually no spectrin present, the particles in these stripped ghosts could still be aggregated by manipulations with ionic strength and pH, or by the addition of calcium.Recombinant vesicles were made from a Triton X-100 extract and a mixture of phospholipids with a composition which resembled that of the inner monolayer of erythrocyte membrane. In these recombinants the same manipulations with ionic strength and pH and the addition of calcium caused a rearrangement of the particles, resulting in the appearance of particle-free areas. In recombinants prepared from a Triton X-100 extract and egg phosphatidylcholine the lateral distribution of the particles was not altered by these manipulations.It is concluded that in the erythrocyte membrane the intramembrane particles can be aggregated by effects of external agents on lipid components. In this light the role of spectrin in stabilizing the membrane by interactions with lipids in the inner monolayer is discussed.

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“…Freeze-fracture electron microscopy was performed as outlined previously [33]. Glycerol was added to the samples to prevent freeze damage.…”

Section: Methodsmentioning

confidence: 99%

The lateral distribution of intramembrane particles in the erythrocyte membrane and recombinant vesicles

Gerritsen

Verkleij

Deenen

1979

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…It seems that both the acid p H and the uranyl ions contribute to 'surface staining' by UA. The interaction of uranyl ions with phospholipids has been described by several authors (Shah, 1969;Ververgaert & Elbers, 1971;Silver et aZ., 1971) and it seems that lipids are 'stabilized' or 'fixed' so that the deleterious effect of drying on the virus particles is in fact neutralized. This is not the case with PTA which, on the contrary, causes damage to membranes as shown in mitochondria and cell membranes (Muscatello & Horne, 1968;Munn, 1968;Muscatello & Guarriero-Bobyleva, 1970).…”

Section: Discussionmentioning

confidence: 95%

Negative staining of viruses

Nermut

1972

Journal of Microscopy

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SUMMARY Several techniques and procedures of negative staining are described and compared from the point of view of both ‘drying’ and ‘specific staining’ artefacts. These are shown on examples from several virus species. The ‘drying artefacts’ can be prevented using the freeze‐drying technique or reduced using ‘paper‐filtration’, ‘pseudoreplication’ or the Kleinschmidt techniques. The ‘specific staining’ artefacts can be avoided by using another stain which does not affect the structure under study. Different ways are shown to stain the surface or the interior of virus particles.

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“…Cells were washed with 0.9% NaC1 and resuspended in 0.9% NaC1 to which 30% glycerol was added as a cryoprotectant. Samples were quenched from 0°C in a mixture of liquid and solid nitrogen and fractured in a Denton freeze-etch apparatus as described before [32]. Electron micrographs were made with a Philips 301 electron microscope.…”

Section: Methodsmentioning

confidence: 99%

Architecture of the outer membrane of Escherichia coli K12 IV. Relationship between outer membrane particles and aqueous pores

Alphen

Verkleij

et al. 1979

Biochimica et Biophysica Acta (BBA) - Biomembranes

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SummaryThe hypothesis that intramembraneous particles, observed in the outer membrane of Escherichia coli by freeze-fracture electron microscopy, are the morphological representation of aqueous pores, was tested. A mutant which is deficient in five major outer membrane proteins, b, c, d, e and the phage receptor protein, contains a largely decreased number of intramembraneous particles and also shows a greatly decreased rate of uptake of several solutes. In derivatives of this strain which contain only one of these proteins in large amounts a strong decrease of the number of intramembraneous particles is observed, which is accompanied by a complete restoration of the rate of uptake of those solutes which use pores in which the protein in question is involved. The results provide strong evidence for the notion that an individual pore contains only one protein species, a property which has been found earlier for individual particles. The observed correlation between particles and aqueous pores strongly supports the hypothesis that the particles are the morphological representation of pores. Implications of this hypothesis for the structure of the particles are discussed.

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Ultrastructural analysis of black lipid membranes

Cited by 8 publications

References 23 publications

The lateral distribution of intramembrane particles in the erythrocyte membrane and recombinant vesicles

The lateral distribution of intramembrane particles in the erythrocyte membrane and recombinant vesicles

Negative staining of viruses

Architecture of the outer membrane of Escherichia coli K12 IV. Relationship between outer membrane particles and aqueous pores

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