On the Nature of Intramembrane Particles

1977 ◽  
Vol 35 ◽  
pp. 680-683
Author(s):  
J. David Robertson
Keyword(s):  
Chemical Nature ◽  
Membrane Surface ◽  
Freeze Fracture ◽  
Purple Membrane ◽  
Halobacterium Halobium ◽  
Polar Regions ◽  
Hydrophobic Core ◽  
Intramembrane Particles ◽  
Protein Molecules ◽  
Complete Report

The chemical nature of the ubiquitous ∽8-10nm intramembrane particles (IMP's) observed with freeze-fracture-etch (FFE) techniques in biological membranes has not been unequivocally established. Some believe that they represent directly metal coated solid globular protein macromolecules residing within the hydrophobic core of the lipid bilayer. The particles are clearly related to protein molecules in the membrane surface but the extent to which the protein extends into the replicated particles has not been established. The particles at least in some instances could be partly or even entirely lipid bound specifically to the protein with the polypeptide in the polar regions of the membrane. We have used two membranes to study this problem: the purple membrane of Halobacterium halobium and the fusiform vacuole membrane of urothelial epithelial cells (1). A complete report of these studies will be published with collaborators W. Schreil in the case of the purple membrane and J. Vergara on the urothelial membrane. One preliminary report on the purple membrane has already been published with W. Schreil (2).

scholarly journals Oriented adsorption of purple membrane to cationic surfaces

1978 ◽  
Vol 77 (2) ◽  
pp. 611-621 ◽  
Author(s):  
KA Fisher ◽  
K Yanagimoto ◽  
W Stoeckenius
Keyword(s):  
Negative Charge ◽  
Chemical Reactivity ◽  
Membrane Surface ◽  
Freeze Fracture ◽  
Purple Membrane ◽  
Halobacterium Halobium ◽  
Membrane Fragment ◽  
Cytoplasmic Surface ◽  
Selection For ◽  
Cytoplasmic Side

We have investigated the orientation of isolated fragments of Halobacterium halobium purple membrane (PM) adsorbed to poly-L-lysine-treated glass (PL-glass), by quanitative electron microscopy. Three lines of evidence support the conclusion that the cytoplasmic side of the membrane is preferentially absorbed. First, monolayer freeze-fracture reveals nonrandom orientation; more fracture faces (89%) are particulate than smooth. Second, the amount of each membrane surface present can be assayed using polycationic ferritin; 90% of all adsorbed membrane fragments are labeled. Third, it is possible to distinguish two surfaces, "cracked" (the extracellular surface) and "pitted" (the cytoplasmic surface) , in slowly air-dried, platinum-carbon-shadowed membranes. When applied under standard conditions, more than 80% appear cracked. Selection for the cytoplasmic by the cationic substrate suggests that the isolated PM, buffered at pH 7.4 and in the light, has a higher negative charge on its cytoplasmic surface than on its extracellular surface. Nevertheless, cationic ferritin (CF) preferentially adsorbs to the extracellular surface. Orientation provides a striking example of biomembrane surface asymmetry as well as the means to examine the chemical reactivity and physical properties of surfaces of a purified, nonvesicular membrane fragment.

Intramembrane Particles (IMPs) in Halobacterium Halobium

1980 ◽  
Vol 38 ◽  
pp. 792-793
Author(s):  
J.D. Robertson ◽  
W. Schreil ◽  
M. Reedy
Keyword(s):  
Hexagonal Lattice ◽  
Purple Membrane ◽  
Bright Spot ◽  
Halobacterium Halobium ◽  
Fold Axis ◽  
Intramembrane Particles ◽  
Black Arrow ◽  
Dark Shadow ◽  
The One ◽  
Membrane Patch

Whole organisms of Halobacterium halobium were frozen in liquid propane and fractured in a Balzers 360 apparatus at ~−140ÅC at ~10−7 Torr and replicated without etching. Fig. 1 is a reverse print of a micrograph of two fractured organisms. The one above shows two purple patches in the EF face and the one below one in the PF face. The area labelled “1” was used for the filtered image in Fig. 2 and area “2” for the one in Fig. 3. The arrows in Figs. 2-3 indicate the centers of adjacent repeating units of the hexagonal lattice (d ~60Å). The repeating unit in Fig. 2 is a glob of metal representing three bacteriorhodopsin (BR) molecules. Each repeating unit in Fig. 3 consists of a ring of metal surrounding a dark shadow in the center of which there is a bright spot of metal. Each dark shadow ring is the complement of each ~50Å circular glob of metal in Fig. 2. Hence it is the impression of a set of three BR molecules around a three-fold axis of the molecular lattice. Note that there is a slight depression perpendicular to the shadowing direction at the edge of the purple membrane patch (black arrow). The area labelled “3” is another purple membrane patch turned up ~45°. The regular pattern is prominent here because of the shadowing angle. Note the many large intramembrane particles that at first sight appear globular. Many of these cast rectangular shadows (inset enlargements above of particles labelled “4-10)”. Some particles, eg. #7, are linear. The angularity of the shadows shows that these particles represent water crystals (Gross, H., O. Kuebler, P. Bass and H. Moor, 1978, J. Cell Biol. , 79:646). Fig. 4 is an enlargement of a PF face showing triplets of metallic aggregates ~50Å in diameter spaced ~100Å apart. Several are encircled and the spacing indicated by arrows. Fig. 5 is an EF face in which complementary triplets can be seen as sets of three dark circular shadows (encircled to the lower left). Note the very small (<10Å) spots of metal approximately in the center of some triplet components. The center-to-center spacing of ~100Å of three adjacent triplets is indicated to the upper center. The triplet structures in the EF and PF faces are complementary. The features of the filtered image of the EF face in Fig. 3 indicate that the spots of metal in the center of the triplet impressions in Fig. 4 are significant. Fig. 6 is a diagram showing the BR lattice above with three-fold axes separated by 62Å. In the lower part of the diagram the appearance of the PF faces is shown at the same scale. Comparison shows that each triplet consists of three sets of three BR molecules. Each ~50Å metal glob in the triplet is therefore a replica of three bacteriorhodopsin molecules. We interpret the <10Å spots in the dark ring shadows in Fig. 5 and the globs of metal around the ring shadows in the EF face as lipid molecules. They are revealed by decoration and shadowing.

Molecular cytochemistry of freeze-fractured cells

1986 ◽  
Vol 44 ◽  
pp. 894-897
Author(s):  
Pedro Pinto da Silva
Keyword(s):  
Membrane Proteins ◽  
Membrane Surface ◽  
Freeze Fracture ◽  
Biological Membranes ◽  
Bilayer Membrane ◽  
Integral Membrane Proteins ◽  
Distilled Water ◽  
Intramembrane Particles ◽  
Freeze Etching

I will describe four approaches that combine cytochemistry with freeze-fracture: 1) FREEZE-ETCHING; 2) FRACTURE-LABEL; 3) FRACTURE-PERMEATION; and 4) LABEL-FRACTURE. These techniques, in particular fracture-label, involve delicate points of interpretation and numerous validating controls. In the publications listed at the end, these issues have been addressed in detail.1. FREEZE-ETCHING. I developed freeze-etching as a cytochemical approach to prove that membranes were split by freeze-fracture and to show that biological membranes were comprised of a bilayer membrane continuum interrupted by integral membrane proteins.1 - 4 In freeze-etching, the distribution of the marker over the membrane surface exposed by sublimation is compared to that of the intramembrane particles exposed by fracture. It is often required to aggregate the particles into domains larger than the labeling molecules (Fig. 1). This, and the need for freezing in distilled water, severely limits the application of freeze-etching.

Photoreceptor disk membrane substructures by means of the complementary freeze-fracture-replica methods

1978 ◽  
Vol 36 (2) ◽  
pp. 156-157
Author(s):  
A. Tonosaki ◽  
M. Yamasaki ◽  
H. Washioka ◽  
J. Mizoguchi
Keyword(s):  
Cell Membranes ◽  
Freeze Fracture ◽  
Purple Membrane ◽  
Remarkable Case ◽  
Single Face ◽  
Disk Membrane ◽  
Associated Proteins ◽  
Photoreceptor Membranes

A vertebrate disk membrane is composed of 40 % lipids and 60 % proteins. Its fracture faces have been classed into the plasmic (PF) and exoplasmic faces (EF), complementary with each other, like those of most other types of cell membranes. The hypothesis assuming the PF particles as representing membrane-associated proteins has been challenged by serious questions if they in fact emerge from the crystalline formation or decoration effects during freezing and shadowing processes. This problem seems to be yet unanswered, despite the remarkable case of the purple membrane of Halobacterium, partly because most observations have been made on the replicas from a single face of specimen, and partly because, in the case of photoreceptor membranes, the conformation of a rhodopsin and its relatives remains yet uncertain. The former defect seems to be partially fulfilled with complementary replica methods.

Heterogeneity of Size and Distribution of Intramembrane Particles in the Plasma Membrane of Yeast

1977 ◽  
Vol 35 ◽  
pp. 596-597
Author(s):  
E. Keyhani
Keyword(s):  
Plasma Membrane ◽  
Candida Utilis ◽  
Synthetic Medium ◽  
Freeze Fracture ◽  
Translational Diffusion ◽  
Yeast Cells ◽  
Distilled Water ◽  
Intramembrane Particles ◽  
The Matrix ◽  
Water Cell

The matrix of biological membranes consists of a lipid bilayer into which proteins or protein aggregates are intercalated. Freeze-fracture techni- ques permit these proteins, perhaps in association with lipids, to be visualized in the hydrophobic regions of the membrane. Thus, numerous intramembrane particles (IMP) have been found on the fracture faces of membranes from a wide variety of cells (1-3). A recognized property of IMP is their tendency to form aggregates in response to changes in experi- mental conditions (4,5), perhaps as a result of translational diffusion through the viscous plane of the membrane. The purpose of this communica- tion is to describe the distribution and size of IMP in the plasma membrane of yeast (Candida utilis).Yeast cells (ATCC 8205) were grown in synthetic medium (6), and then harvested after 16 hours of culture, and washed twice in distilled water. Cell pellets were suspended in growth medium supplemented with 30% glycerol and incubated for 30 minutes at 0°C, centrifuged, and prepared for freeze-fracture, as described earlier (2,3).

Image Analysis of Intramembrane Particles in Freeze-Fractured Biomembranes Using Computer Simulation Techniques

1975 ◽  
Vol 33 ◽  
pp. 214-215
Author(s):  
D.J. Benefiel ◽  
R.S. Weinstein
Keyword(s):  
Membrane Proteins ◽  
Freeze Fracture ◽  
Integral Membrane Proteins ◽  
Systematic Evaluation ◽  
Intramembrane Particles ◽  
Modeling Methods ◽  
Simulation Techniques ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron ◽  
Computer Simulation Techniques

Intramembrane particles (IMP or MAP) are components of most biomembranes. They are visualized by freeze-fracture electron microscopy, and they probably represent replicas of integral membrane proteins. The presence of MAP in biomembranes has been extensively investigated but their detailed ultrastructure has been largely ignored. In this study, we have attempted to lay groundwork for a systematic evaluation of MAP ultrastructure. Using mathematical modeling methods, we have simulated the electron optical appearances of idealized globular proteins as they might be expected to appear in replicas under defined conditions. By comparing these images with the apearances of MAPs in replicas, we have attempted to evaluate dimensional and shape distortions that may be introduced by the freeze-fracture technique and further to deduce the actual shapes of integral membrane proteins from their freezefracture images.

Freeze-fracture cytochemistry: A goal set by Russell Steere in 1957

1993 ◽  
Vol 51 ◽  
pp. 60-61
Author(s):  
Nicholas J Severs
Keyword(s):  
Chemical Nature ◽  
Biological Systems ◽  
Freeze Fracture ◽  
Structural Components ◽  
Major Goal ◽  
Membrane Biology ◽  
Routine Application ◽  
The Future ◽  
Freeze Etching

In his pioneering demonstration of the potential of freeze-etching in biological systems, Russell Steere assessed the future promise and limitations of the technique with remarkable foresight. Item 2 in his list of inherent difficulties as they then stood stated “The chemical nature of the objects seen in the replica cannot be determined”. This defined a major goal for practitioners of freeze-fracture which, for more than a decade, seemed unattainable. It was not until the introduction of the label-fracture-etch technique in the early 1970s that the mould was broken, and not until the following decade that the full scope of modern freeze-fracture cytochemistry took shape. The culmination of these developments in the 1990s now equips the researcher with a set of effective techniques for routine application in cell and membrane biology.Freeze-fracture cytochemical techniques are all designed to provide information on the chemical nature of structural components revealed by freeze-fracture, but differ in how this is achieved, in precisely what type of information is obtained, and in which types of specimen can be studied.

scholarly journals Spectroscopy and energetics of the purple membrane of Halobacterium halobium

FEBS Letters ◽  
1978 ◽  
Vol 91 (1) ◽  
pp. 131-134 ◽  
Author(s):  
David Cahen ◽  
Haim Garty ◽  
S.Roy Caplan
Keyword(s):  
Purple Membrane ◽  
Halobacterium Halobium

The penetration of Human Defensin 5 (HD5) through bacterial outer membrane: Simulation studies

2021 ◽  
Author(s):  
Tadsanee Awang ◽  
Prapasiri Pongprayoon
Keyword(s):  
Pathogenic Bacteria ◽  
Lipid A ◽  
Dominant Role ◽  
Membrane Surface ◽  
Innate Immune ◽  
Hydrophobic Core ◽  
Gram Negative ◽  
Head Groups ◽  
Bacterial Outer Membrane ◽  
Human Defensin 5

Abstract Human α-defensin 5 (HD5) is one of cationic antimicrobial peptides which plays a crucial role in an innate immune system in human body. HD5 shows the killing activity against a broad spectrum of pathogenic bacteria by making a pore in a bacterial membrane and penetrating into a cytosol. Nonetheless, its pore-forming mechanisms remain unclear. Thus, in this work, the constant-velocity steered molecular dynamics (SMD) simulation was used to simulate the permeation of a dimeric HD5 into a gram-negative LPS membrane model. Arginine-rich HD5 is found to strongly interact with a LPS surface. Upon arrival, arginines on HD5 interact with lipid A head groups and then drag these charged moieties down into a hydrophobic core resulting in the formation of water-filled pore. Although all arginines are found to interact with a membrane, R13 and R32 appear to play a dominant role in the HD5 adsorption on a gram-negative membrane. Furthermore, one chain of a dimeric HD5 is required for HD5 adhesion. The interactions of arginine-Lipid A head groups play a major role in adhering a cationic HD5 on a membrane surface and retarding a HD5 passage in the meantime.

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