Article

Membrane Structure: Some General Principles

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Abstract

The arrangement of lipids and some proteins in the erythrocyte membrane has been discussed. The conclusions from this are listed here as a set of general guidelines for the structure of membranes of higher organisms: some of these rules may be wrong. But at this stage it seems useful to sharpen our thoughts in this way and thereby focus attention on various specific points. 1) The basis of a membrane is a lipid bilayer with (i) choline phospholipids and glycolipids in the external half and (ii) amino (and possibly some choline) phospholipids in the cytoplasmic half. There is effectively no lipid exchange across the bilayer (unless enzymatically catalyzed) (68). 2) Some proteins extend across the bilayer. Where this is so, they will in general have carbohydrate on their surface remote from the cytoplasm. This carbohydrate may prevent the protein diffusing out of the membrane into the cytoplasm; it acts as a lock on the protein. 3) Just as lipids do not flip-flop, proteins do not rotate across the membrane. Lateral motion or rotation of lipids and proteins in the plane of the bilayer may be expected. 4) Most membrane protein is associated with the inner, cytoplasmic, urface of the membrane. Proteins are not usually associated exclusively with the outer half of the lipid bilayer. 5) Membrane proteins are a special class of cytoplasmic proteins, not of secreted proteins.

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... Shortly after his initial discovery of lipid asymmetry, Bretscher realized, however, that the asymmetry could not be maintained purely by a slow rate of lipid flip-flop. Based on the assumption that in all cells capable of protein synthesis (this excludes red blood cells) all phospholipid synthesis is likely to occur on the cytoplasmic side of a membrane, he concluded [15,16] that the transfer of choline-containing phospholipids from the cytoplasmic leaflet of the membrane to the outer leaflet must be enzymatically catalysed. He coined the word flippase to describe the class of enzymes which catalyse this transfer. ...
... He coined the word flippase to describe the class of enzymes which catalyse this transfer. Because he was of the opinion, however, that movement of lipids between the two leaflets could not occur at any physiologically relevant rate unless enzymatically catalysed, Bretscher [15,16] did not mention any energy requirement for flippase activity. It appears that he was envisaging a facilitated diffusion process. ...
... Bretscher's prediction [15,16] of enzyme-mediated lipid facilitated diffusion across a membrane was first demonstrated in 1985 by Bishop and Bell [28,29] in the case of phosphatidylcholine transport from the cytoplasmic side of the endoplasmic reticulum to the luminal side. They found that the transport of dibutyroylphosphatidylcholine across the membrane of microsomal vesicles derived from the endoplasmic reticulum was saturable and inhibited by structural lipid analogues and Fig. 3. Distribution of phospholipids on each side of the red blood cell plasma membrane (adapted from [11]). ...
Article
The plasma membrane phospholipid distribution of animal cells is markedly asymmetric. Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are concentrated in the inner leaflet, whereas phosphatidylcholine (PC) and sphingomyelin (SM) are concentrated in the outer leaflet. This non-equilibrium situation is maintained by lipid pumps (flippases or floppases), which utilize energy in the form of ATP to translocate lipids from one leaflet to the other. Scramblases, which are activated when physiologically required, transport lipids in both directions across the membrane and can abolish lipid asymmetry. Lipid asymmetry also causes imbalances in the areas occupied by lipid in the two membrane leaflets, contributing to membrane curvature. The asymmetry of PS across the plasma membrane plays a crucial signalling role in numerous physiological processes. Exposure of PS on the external surface of blood platelets stimulates blood coagulation. PS exposure by other cells during apoptosis provides an “eat me” signal to surrounding macrophages. Many peripheral and integral membrane proteins have polybasic PS-binding domains on their cytoplasmic surfaces which either provide a membrane anchor or affect activity. These domains can also determine trafficking within the cell and control regulation via an electrostatic switch mechanism, as well as potentially acting as “death sensors” when cytoplasmic PS is transferred to the extracellular leaflet during apoptosis. Apart from these physiological roles, external PS exposure by microorganisms, viruses and cancer cells allows them to mimic the immunosuppressive anti-inflammatory action of apoptotic cells and proliferate, thus providing a strong medical motivation for future research in the field of lipid asymmetry in membranes.
... IP 3 diffuses in the cytosol where it binds IP 3receptors, to mediate calcium (Ca 2+ ) responses and, in some cases, trigger secretion (Di Paolo and De Camilli, 2006). (Fairn et al., 2011;Bretscher, 1973). This different distribution is actively maintained by energy-dependent transport, in fact exposure of PS on the outer leaflet is a marker of apoptosis (Fadok et al., 1992). ...
... 2) Transbilayer exchange is relatively quick for lipid with apolar headgroups such as DAG and cholesterol (with t 1/2 of seconds to minutes), but it is very slow for lipids with polar headgroups such as glycerophospholipids (t 1/2 of hours to days for PC) or glycosylated lipids (Holthuis and Levine, 2005). This movement can be energetically eased by protein flippases that mask the hydrophilic head from the hydrophobic core of the bilayer (Bretscher, 1973;Sanyal and Menon, 2009). An example is the family of the P4-ATPases, transmembrane proteins implicated in translocation of phospholipids between membrane leaflets of different organelles. ...
Conference Paper
Non-vesicular intracellular lipid traffic is mediated by lipid transfer proteins (LTPs), which contain domains with an internal cavity that can solubilise and transfer lipids. One of the most widespread LTP folds is the Steroidogenic Acute Regulatory Transfer (StART) domain, which forms a hydrophobic pocket, and appears in proteins with different localisations and lipid specificities. The aim of this study was to characterise a new StART-like domain family, which we identified by a bioinformatics approach. I studied aspects of the localisations, functions and structural properties of six StART-like proteins in S. cerevisiae. The yeast StART-like proteins were endoplasmic reticulum (ER)-integral membrane proteins with transmembrane domains, and they localised at membrane contact sites: Lam1p/Lam3p, and Lam2p/Lam4p at junctions between ER and plasma membrane (PM); Lam5p/Lam6p at junctions between the ER and the vacuolar membrane, at nucleus-vacuole junction (NVJ) and at ER-mitochondria contacts. To study their functions, I purified the second StART-like domain of Lam4p, and I identified sterol as its lipid ligand from in vitro binding assays and in a spectroscopy approach with fluorescent ergosterol. We named the whole family LAM for Lipid transfer proteins Anchored at Membrane contact sites. The sterol binding property of the domains was related to a phenotype shared by LAM1, LAM2 and LAM3 delete strains, which showed an increased sensitivity to the sterol-sequestering polyene antifungal drug Amphotericin B (AmB). The two most sensitive strains (lam1∆ and lam3∆), displayed low sphingolipid levels, which is as yet unexplained. All AmB phenotypes were rescued by StART-like domains from the human LAMa, Lam2/4p and Lam5/6p, suggesting that these domains bind sterol. Simultaneous deletion of LAM1, LAM2, and LAM3 significantly reduced the extent of cortical ER-PM contacts, implying that they create the structure of the particularly punctate contact site they target. Finally, I started structural analysis of Lam4S2 to study the mechanism of sterol binding and to confirm our structural model.
... According to that model, integral transmembrane proteins are arranged in the plasma membrane of living cells such that the polar regions are facing the aqueous phase and the hydrophobic regions are embedded on a viscous phospholipid bilayer and those proteins are able to move freely on that two-dimensional, approximately homoge- neous luid "sea" of phospholipids [18]. One year later, in 1973, Bretscher published a Science paper in which he discusses overall membrane organization based on evidences collected from experiments performed in red blood cells [19]. According to that paper, the plasma membrane of mammalian cells was not as simple as depicted by the luid mosaic model. ...
... Some of the integral proteins span the membrane and their glycosylation is responsible for locking them at the membrane impeding their migration to the cytoplasm. Another important contribution from this paper is that proteins not only interact with the outer layer of the plasma membrane but also with the inner layer, and membrane proteins are a subtype of cytoplasmic proteins that are not secreted [19]. In the same year, Yu and collaborators, also performing experiments in red blood cells, showed that when those cells are incubated with the nonionic detergent Triton X-100, there are some fractions of the cellular proteins that are resistant to the detergent extraction and seem to form oligomeric complexes with some of the lipid components, which were preferentially composed by nonglycosylated proteins and sphingolipids [20]. ...
Chapter
Full-text available
Atherosclerosis is a chronic inlammatory process that initiates with accumulation of apolipoprotein B containing lipoproteins (LPs) in the subendothelium (intima), especially in areas where the laminar low is disturbed. LP retention triggers an inlammatory response leading to activation of endothelial and vascular smooth muscle cells that culminates with recruitment of leukocytes. Atherosclerosis is the leading cause of vascular disease worldwide being its major clinical manifestations ischemic heart disease, isch-emic stroke, and peripheral arterial disease. Even though a lot has been done to unravel the role of turbulent low and mechanotransduction for atherosclerosis development, litle is known about the role of plasma membrane (PM) cholesterol in this process. This chapter is going to be focused on exploring what has been done so far to decipher the role of PM cholesterol in regulating actin architecture, cellular mechanical properties, and cellular contractility in muscle and nonmuscle cells.
... The plasma membrane is composed of phosphatidylcholines, phosphatidylserines, phosphatidylethanolamines, sphingolipids, phosphoinositides and cholesterol [Keren, 2011]. The early models of the plasma membrane comprise a lipid bilayer with an asymmetric distribution of the phospholipids in the inner and outer membrane leaflets [Bretscher, 1973] In this model, major proteins and glycoproteins were placed in their specific orientation across the membrane, and other proteins were associated with the inner surface of the bilayer [Bretscher, 1973]. This picture was accompanied by other models which assumed that the positions of membrane lipids and proteins are stationary [Benson, 1966, Danielli and Davson, 1935, Gorter and Grendel, 1924, Stoeckenius and Engelman, 1969. ...
... The plasma membrane is composed of phosphatidylcholines, phosphatidylserines, phosphatidylethanolamines, sphingolipids, phosphoinositides and cholesterol [Keren, 2011]. The early models of the plasma membrane comprise a lipid bilayer with an asymmetric distribution of the phospholipids in the inner and outer membrane leaflets [Bretscher, 1973] In this model, major proteins and glycoproteins were placed in their specific orientation across the membrane, and other proteins were associated with the inner surface of the bilayer [Bretscher, 1973]. This picture was accompanied by other models which assumed that the positions of membrane lipids and proteins are stationary [Benson, 1966, Danielli and Davson, 1935, Gorter and Grendel, 1924, Stoeckenius and Engelman, 1969. ...
Article
Deformability of the plasma membrane, the outermost surface of metazoan cells, allows cells to be dynamic, mobile and flexible. Factors that affect this deformability, such as tension on the membrane, can regulate a myriad of cellular functions, including membrane resealing, cell motility, polarization, shape maintenance, membrane area control and endocytic vesicle trafficking. This review focuses on mechanoregulation of clathrin‐mediated endocytosis. We first delineate the origins of cell membrane tension and the factors that yield to its spatial and temporal fluctuations within cells. We then review the recent literature demonstrating that tension on the membrane is a fast‐acting and reversible regulator of clathrin‐mediated endocytosis. Finally, we discuss tension‐based regulation of endocytic clathrin coat formation during physiological processes. This article is protected by copyright. All rights reserved
... It is well known that biological membranes are of great importance to maintain cell homeostasis since they encompass and compartmentalize cells and organelles. The major components of all cell membranes are lipids and proteins with small amounts of carbohydrates [30,31]. Most membranes exhibit a lipid-protein ratio of ~1, but the proportion between lipids and proteins may vary considerably depending on the membrane functions [31][32][33]. ...
... The major components of all cell membranes are lipids and proteins with small amounts of carbohydrates [30,31]. Most membranes exhibit a lipid-protein ratio of ~1, but the proportion between lipids and proteins may vary considerably depending on the membrane functions [31][32][33]. Choosing a class of biomolecules, such as lipids or proteins, as the best substrates to optimize the photodynamic effects is not a straightforward task and cannot be generalized. In this way, it is essential to address detailed studies on specific targets. ...
Article
In this review, we describe how photooxidation changes membrane properties that can ultimately lead to permanent membrane damage. Lipid photooxidation occurs in the presence of reactive oxygen species such as singlet oxygen and by direct reactions of lipids with a photosensitizer in the excited state. Indeed, lipid oxidation triggers chemical transformations that can alter lipid packing; change the membrane surface area, thickness and elastic modulus; and induce pore formation and phase separation. Here, we highlight how lipid hydroperoxides promote membrane remodelling and phase separation. Further, we emphasize the alterations caused by truncated oxidized lipids that lead to increased membrane permeability. Finally, the consequences of lipid photooxidation on cell functions are also discussed.
... A liposome is a spherical chamber/vesicle, bounded by bilayer of an amphiphilic lipids or a mixture of such lipids, containing aqueous solution inside the chamber [97]. Liposomes are used as mimic membrane of living cell to study biological systems, physical and chemical properties [98]. ...
Thesis
Irreversible electroporation (IRE) is a minimally invasive and non-thermal technique in which an ultra-short pulse with high electric field has been found to be successful for ablation of certain tumor and cancer cells. As a mimic of biomembranes of cells, lipid membranes of giant unilamellar vesicles (GUVs), composed of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC), with diameters equal or greater than 10 μm is currently used. In this regard, at first a microcontroller-based IRE technique is developed indigenously where a special purpose power supply is used to construct high voltage generator and a MOSFET (N-Channel Power MOSFET, SCILLC) based switching circuit is designed for generating square pulses with very high energy as required for electroporation. Then investigated the electro- deformation and pore formation of GUVs at constant tension induced by IRE signal. It is also observed the membrane fusion of GUVs induced by the IRE signal. The constant tension is induced on the GUVs at electric field strength 340V/cm with pulse width 200 µs. The pore formation in GUVs is increased with the increase of constant tension which supported the results of the mechanical tension-induced pore formation. The pore formation in the lipid membranes is explained by the classical theory of pore formation. These results provide important information for the mechanism of IRE-induced pore formation in biomembranes and lipid membranes. Therefore, optimization of various parameters of IRE technique is necessary for the study of pore formation in lipid membranes of GUVs.
... In other words, the disorganization of a tiny part of a membrane may be enough to destabilize the homeostasis of an organelle and consequently of the whole-cell [10,11]. The proportion between lipids and proteins varies considerably depending on the type and function of a biological membrane, but most membranes exhibit lipid-protein mass ratio ~1 [55][56][57]. Even though the photo-oxidation probably affects both, their distinct effects may display a singular response that could trigger cell death and other signaling processes [58]. ...
... The red cell membrane is composed of approximately 45% lipid, 45% protein and 10% carbohydrate (Anstee, 1986). Phospholipid makes up about 65% of the membrane lipid and is distributed asymmetrically in the lipid bilayer, sphingomyelin and phosphatidylcholine are in the outer leaflet and phosphotidyl ethanolamine and phosphatidyl serine in the inner leaflet (Bretscher, 1973). A further 30% of the lipid is cholesterol which is located in the hydrophobic core of the membrane. ...
Thesis
This thesis describes the production of a monoclonal antibody to the low incidence blood group antigen Wra which is thought to be allelic to Wrb (a high frequency antigen known to be associated with glycophorin A and band 3). The main approach was to immunise mice with Wr(a+) red cells and select appropriate antibodies by screening them against a panel of red cells by haemagglutination. One anti-Wra antibody (BGU1-WR) was found from the 10 74 hybridomas screened.BGU1-WR belongs to immunoglobulin subclass IgG1 and has an affinity constant of 1.82x10 7. This antibody and a previously described anti-Wrb monoclonalantibody was used to investigate the nature of the antigens. Haemagglutination studies showed that both antigens are resistant to proteinase treatment, probably do not involve sialic acid and do not require intact disulphide bonds on native red cells. The site number of the Wra and Wrb antigens was determined by direct binding of 125I-labelled antibody to red cells. Wr(a+b+) cells were shown to have approximately 70,000 Wra and 150,000 to 350,000 Wrb sites per cell. The possible expression of these antigens on other cells was investigated using flow cytometry. The Wra antigen was not found on leucocytes from a Wr(a+) donor or on 19 cell lines of different cell and tissue origin of unknown Wra phenotype, including 2 erythroid-like lines. Wrb was only found on 2 myeloid-like cell lines. SDS-PAGE, immunoblotting and immunoprecipitation were used to investigate the molecular structure of the antigens. Neither antibody recognises the denatured, SDS- treated antigens under the wide variety of conditions used. Numerous immunoprecipitation experiments showed that anti-Wrb immunoprecipitates glycophorin A and band 3. Under identical conditions anti-Wra did not immunoprecipitate any detectable component of the red cell membrane. Thus the Wra antigen appears to differ from the Wrb antigen casting doubt on the antithetical relationship of these antigens.
... Galectin-8 serves as a danger receptor and an ''eat-me'' signal detected by the autophagy cargo receptor NDP52, which directs selective autophagy against damaged BCVs and the bacteria contained therein [14][15][16][17]. Similar to glycans, certain lipids are also asymmetrically distributed in biological membranes [18]: sphingomyelin is enriched in the outer leaflet of the plasma membrane while phosphatidylserine and phosphatidylinositol are located primarily in the inner, cytoplasmic leaflet. The translocation of phosphatidylserine to the outer leaflet during apoptosis marks dying cells and leads to their timely removal [19]. ...
Article
Full-text available
Pathogenic bacteria enter the cytosol of host cells through uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the vacuolar membrane [1]. Bacterial invaders are sensed either directly, through cytosolic pattern-recognition receptors specific for bacterial ligands, or indirectly, through danger receptors that bind host molecules displayed in an abnormal context, for example, glycans on damaged BCVs [2, 3, 4]. In contrast to damage caused by Listeria monocytogenes, a Gram-positive bacterium, BCV rupture by Gram-negative pathogens such as Shigella flexneri or Salmonella Typhimurium remains incompletely understood [5, 6]. The latter may cause membrane damage directly, when inserting their Type Three Secretion needles into host membranes, or indirectly through translocated bacterial effector proteins [7, 8, 9]. Here, we report that sphingomyelin, an abundant lipid of the luminal leaflet of BCV membranes, and normally absent from the cytosol, becomes exposed to the cytosol as an early predictive marker of BCV rupture by Gram-negative bacteria. To monitor subcellular sphingomyelin distribution, we generated a live sphingomyelin reporter from Lysenin, a sphingomyelin-specific toxin from the earthworm Eisenia fetida [10, 11]. Using super resolution live imaging and correlative light and electron microscopy (CLEM), we discovered that BCV rupture proceeds through two distinct successive stages: first, sphingomyelin is gradually translocated into the cytosolic leaflet of the BCV, invariably followed by cytosolic exposure of glycans, which recruit galectin-8, indicating bacterial entry into the cytosol. Exposure of sphingomyelin on BCVs may therefore act as an early danger signal alerting the cell to imminent bacterial invasion.
... While glycans reside exclusively on the non-cytosolic surface of lysosomes and the plasma membrane, also certain lipids display strict asymmetric distributions across organellar bilayers. For instance, sphingomyelin (SM) is highly enriched in the exoplasmic leaflet of the plasma membrane whereas phosphatidylserine (PS) is primarily located in the cytosolic leaflet [12][13][14] . Translocation of PS to the outer leaflet during apoptosis marks dying cells and leads to their timely removal 15 . ...
Article
Full-text available
Lysosomes are vital organelles vulnerable to injuries from diverse materials. Failure to repair or sequester damaged lysosomes poses a threat to cell viability. Here we report that cells exploit a sphingomyelin-based lysosomal repair pathway that operates independently of ESCRT to reverse potentially lethal membrane damage. Various conditions perturbing organelle integrity trigger a rapid calcium-activated scrambling and cytosolic exposure of sphingomyelin. Subsequent metabolic conversion of sphingomyelin by neutral sphingomyelinases on the cytosolic surface of injured lysosomes promotes their repair, also when ESCRT function is compromised. Conversely, blocking turnover of cytosolic sphingomyelin renders cells more sensitive to lysosome-damaging drugs. Our data indicate that calcium-activated scramblases, sphingomyelin, and neutral sphingomyelinases are core components of a previously unrecognized membrane restoration pathway by which cells preserve the functional integrity of lysosomes. Activation of ESCRT prevents potentially lethal outcomes of minor perturbations in lysosomal integrity. Here authors show that Ca2 + -activated scrambling of sphingomyelin and its cytosolic turnover drives lysosomal repair independently of ESCRT.
... The study also highlighted the presence of PtdSer in, and its ability to recruit charge-based protein probes to, endosomal compartments, while not being detectable in the cytoplasmic-facing cis-Golgi, ER or mitochondria. While it is possible the LactC2 probe does not have high enough sensitivity to detect the relatively low levels of PtdSer present in these organelles [9,10], it is also possible that, like in the PM, PtdSer leaflet distribution in intracellular organelle membranes is asymmetrical [17]. Indeed, there existed significant evidence prior to the development of the LactC2 probe suggesting this is the case, at least in the ER [18][19][20][21]. ...
Article
Full-text available
Phosphatidylserine (PtdSer), an essential constituent of eukaryotic membranes, is the most abundant anionic phospholipid in the eukaryotic cell accounting for up to 10% of the total cellular lipid. Much of what is known about PtdSer is the role exofacial PtdSer plays in apoptosis and blood clotting. However, PtdSer is generally not externally exposed in healthy cells and plays a vital role in several intracellular signaling pathways, though relatively little is known about the precise subcellular localization, transmembrane topology and intracellular dynamics of PtdSer within the cell. The recent development of new, genetically-encoded probes able to detect phosphatidylserine is leading to a more in-depth understanding of the biology of this phospholipid. This review aims to give an overview of recent developments in our understanding of the role of PtdSer in intracellular signaling events derived from the use of these recently developed methods of phosphatidylserine detection.
... With molecular models, this complex has dimensions of -1.2 by 0.6 by 2.6 nm; i.e., addition of the fatty acyl group increases the width of the complex but not its length or depth. Such a complex could be inserted into one layer of a bilayer bacterial cell membrane assuming a fluid-mosaic type of membrane with a lipid bilayer ranging from 3.5 to 4.5 mm in thickness (16,96,136). This is in agreement with and extends preliminary theories on the integration of hopanoids into bilayer membranes (100,109,112,119). ...
... This is called bilayer asymmetry (see following chapter for more details). The sphingolipids (except glucosylceramide) are synthesized on the luminal surface of the Golgi, whereas the phospholipid PS and PE are actively concentrated in the cytosolic leaflet (Bretscher, 1973;D'Angelo et al., 2007;Simons and Van Meer, 1988). ...
Thesis
Oxysterol binding protein (OSBP) is a lipid transfer protein that regulates cholesterol distribution in cell membranes. OSBP consists of a pleckstrin homology (PH) domain, two coiled-coils, a “two phenylalanines in acidic tract” (FFAT) motif and a C-terminal lipid binding OSBP-Related Domain (ORD). The PH domain recognizes PI(4)P and small G protein Arf1-GTP at the Golgi, whereas the FFAT motif interacts with the ER-resident protein VAP-A. By binding all these determinants simultaneously, OSBP creates membrane contact sites between ER and Golgi, allowing the counter-transport of cholesterol and PI(4)P by the ORD. OSBP also contains an intrinsically disordered ~80 aa long N-terminal sequence, composed mostly of glycine, proline and alanine. We demonstrate that the presence of disordered N-terminus increases the Stoke’s radius of OSBP truncated proteins and limits their density and saturation level on PI(4)P-containing membrane. The N-terminus also prevents the two PH domains of OSBP dimer to symmetrically tether two PI(4)P-containing (Golgi-like) liposomes, whereas protein lacking the disordered sequence promotes symmetrical liposome aggregation. Similarly, we observe a difference in OSBP membrane distribution on tethered giant unilamellar vesicles (GUVs), based on the presence/absence of N-terminus. Protein with disordered sequence is homogeneously distributed all over the GUV surface, whereas protein without N-terminus tends to accumulate at the interface between two PI(4)P-containing GUVs. This protein accumulation leads to local overcrowding, which is reflected by slow in-plane diffusion. The effect of N-terminus is also manifested in monomeric OSBPderived proteins that tether ER-like and Golgi-like membranes in the presence of VAP-A. Findings from our in vitro experiments are confirmed in living cells, where N-terminus controls the recruitment of OSBP on Golgi membranes, its motility and the on-and-off dynamics during lipid transfer cycles. Most OSBP-related proteins contain low complexity N-terminal sequences, suggesting a general effect.
... Typically β integrin-like proteins consist of three different domains, which include the highly conserved extracellular domain. The presence of 56 conserved cysteine residues in the extracellular domain of all tested β integrin-like proteins (Hynes 2012; our data) confirms the well-known fact that disulfide bonds in polypeptide chains of different proteins are mainly located on the outside of the membrane, providing the native structure of polypeptides and being critical for interactions between polypeptide chains (Bretscher 1973). β-A transcripts corresponding predicted β integrin-like proteins appear to be the products of alternative splicing of the same gene β-A, whereas β-B transcripts resembling the oyster β3-integrin seem to be a result of the allelic diversity of the gene β-B. ...
Article
Full-text available
This study is based on the Illumina RNA-sequencing data obtained for a de novo assembly of the transcriptome from early developmental stages and some tissues and cells of the adult mussel Mytilus trossulus (Mytilidae, Mollusca) using the Trinity program. A total of 200,079 contigs were obtained, and compared to the NCBI database using BLAST to search for sequence similarity. The number of annotated contigs under the GO term 3 categories was estimated to reach 19.96%. The BUSCO analysis determined a level of 99.2% completeness for the assembled transcriptome. The main findings include evidence that the mussel β integrin-like protein sequences are very similar to the β integrin-like proteins so far sequenced for all classes of Mollusca, while the highest similarity is observed between mussel and oyster (Crasostrea gigas) β integrin-like proteins. Our transcriptome dataset contributes to the genetic databases of non-model animals such as bivalves and represents the first characterization of expressed sequences during early development of the mollusc M. trossulus from the Sea of Japan including the identification of candidate genes involved in cell adhesion.
... Mammalian cells, however, do not have a cell wall. The outermost layer of a mammalian cell is only a delicate, twolayered structure of phospholipids with proteins embedded 6 . While the internal cytoskeleton mechanically helps mammalian cells maintain their shapes, mammalian cells can be easily damaged or destroyed when exposed to physical or biological assaults. ...
Article
Full-text available
Mammalian cells are different from plant and microbial cells, having no exterior cell walls for protection. Environmental assaults can easily damage or destroy mammalian cells. Thus, the ability to develop a biomimetic cell wall (BCW) on their plasma membrane as a shield can advance various applications. Here we demonstrate the synthesis of BCW with a framing template and a crosslinked matrix for shielding live mammalian cells. The framing template is a supramolecular DNA structure. The crosslinked matrix is a polyelectrolyte complex made of alginate and polylysine. As the entire procedure of BCW synthesis is strictly operated under physiological conditions, BCW-covered mammalian cells can maintain high bioactivity. More importantly, the data show that BCW can shield live mammalian cells from not only physical assaults but also biological assaults. Thus, this study has successfully demonstrated the synthesis of BCW on live mammalian cells with great potential of shielding them from environmental assaults.
... Besides imaging by endogenous SHG, exogenous staining enables visualization of the plasma membranes and monitoring the change in the membrane potential under the SHG Biophys Rev microscope (Campagnola et al. 1999;Moreaux et al. 2000Moreaux et al. , 2003Dombeck et al. 2004;Nuriya et al. 2006Nuriya et al. , 2016Vanzi et al. 2013). The distribution of lipid molecules between the outer and inner leaflet of the membrane bilayer is asymmetric (Bretscher 1973;Murate et al. 2015). This implies that the cellular membranes are non-centrosymmetric, i.e., SHG-active. ...
Article
Optical second harmonic generation (SHG) is a nonlinear optical process which is sensitive to the symmetry of media. SHG microscopy allows for selective probing of a non-centrosymmetric area of sample. This type of nonlinear optical microscope was first used to observe ferroelectric domains and has been applied to various specimens including the biological samples to date. Imaging of the endogenous SHG of biological tissue has been utilized for the selective observation of filament systems in tissues such as collagen, myosin, and microtubules, which exhibit a polar structure. The cellular membrane can be selectively observed by the SHG microscope through membrane staining with amphiphilic polar dye molecules. It has been reported that, by imaging exogenous SHG of the membrane, sensitive detection of membrane damage could be realized using the SHG microscope. Because the staining dye is fluorescent, both SHG and two-photon excited fluorescence (TPF) images can be obtained simultaneously. How the SHG intensity depends on the molecular alignment of the polar dye molecules that reflects the ordering of lipid molecules in the plasma membrane and the necessity of the normalization of the SHG intensity by the TPF intensity is discussed. Furthermore, the assessment of the membrane damage induced by exposing polycation to HeLa cells has been compared with the conventional cytotoxicity and cell viability tests to demonstrate the higher sensitivity of the present SHG-based assay.
... In other words, the disorganization of a tiny part of a membrane may be enough to destabilize the homeostasis of an organelle and consequently of the whole-cell [10,11]. The proportion between lipids and proteins varies considerably depending on the type and function of a biological membrane, but most membranes exhibit lipid-protein mass ratio ~1 [55][56][57]. Even though the photo-oxidation probably affects both, their distinct effects may display a singular response that could trigger cell death and other signaling processes [58]. ...
Article
Abstract The lipid composition impacts directly on the structure and function of the cytoplasmic as well as organelle membranes. Depending on the type of membrane, specific lipids are required to accommodate, intercalate, or pack membrane proteins to the proper functioning of the cells/organelles. Rather than being only a physical barrier that separates the inner from the outer spaces, membranes are responsible for many biochemical events such as cell-to-cell communication, protein-lipid interaction, intracellular signaling, and energy storage. Photochemical reactions occur naturally in many biological membranes and are responsible for diverse processes such as photosynthesis and vision/phototaxis. However, excessive exposure to light in the presence of absorbing molecules produces excited states and other oxidant species that may cause cell aging/death, mutations and innumerable diseases including cancer. At the same time, targeting key compartments of diseased cells with light can be a promising strategy to treat many diseases in a clinical procedure called Photodynamic Therapy. Here we analyze the relationships between membrane alterations induced by photo-oxidation and the biochemical responses in mammalian cells. We specifically address the impact of photosensitization reactions in membranes of different organelles such as mitochondria, lysosome, endoplasmic reticulum, and plasma membrane, and the subsequent responses of eukaryotic cells. Keywords OrganellesPhotodynamic therapyPhotosensitizerPhotoagingCancerLipids
... Or, le transfert spontané des glycérolipides est trop lent pour équilibrer la quantité de lipides entre les deux feuillets ( Figure 4). Ainsi, dès 1973, l'hypothèse que ces mouvements puissent être dus à des facilitateurs a été émise (Bretscher, 1973). Ces facilitateurs ATP indépendants, nommés à l'origine flippases, ont été renommés flip-floppases/scramblases pour plus de clarté, ces mouvements étant bidirectionnel et le terme flippase ayant été depuis attribué aux ATPases de type P4 (voir paragraphe suivant). ...
Thesis
Dans les cellules eucaryotes, chaque membrane a une composition lipidique qui lui est propre. La composition des membranes est finement régulée en fonction des conditions environnementales et physiologiques de la plante. Cette homéostasie lipidique est le résultat des processus de synthèse, conversion, dégradation et de trafic des lipides. Si la majorité des enzymes impliqués dans le métabolisme des lipides est identifiée, la majorité des mécanismes de transferts intermembranaires des lipides reste à caractériser. Nous nous concentrons sur l'homéostasie lipidique des chloroplastes et plus particulièrement celle des galactolipides, lipides essentiels des membranes photosynthétiques. Les galactolipides sont synthétisés au niveau de l'enveloppe des chloroplastes. Cependant, une grande partie des galactolipides proviennent de la phosphatidylcholine, elle-même synthétisée dans le réticulum endoplasmique. Cette délocalisation de la voie de synthèse sur deux compartiments souligne l'importance de l’étape de transfert lipidique associé.Des études transcriptomiques ont montré qu'ALA10, une ATPase de type P4, flippase de phospholipides, est surexprimée dans des conditions faisant varier la synthèse des galactolipides, telles que l'inhibition chimique des MGDG synthases par la galvestine-1 ou la carence de phosphate.Le but de cette thèse est de caractériser ALA10 et d'analyser son rôle dans ce trafic lipidique et dans l’homéostasie des galactolipides chloroplastiques.Pour comprendre le rôle d'ALA10, nous avons d'abord étudié sa localisation subcellulaire à l'aide d'une fusion traductionnelle avec la GFP et effectué des analyses lipidiques de différentes lignées exprimant ALA10 à différents niveaux. L’analyse de la composition lipidique indique qu'ALA10 stimule la synthèse des galactolipides et limite la désaturation de la phosphatidylcholine dans le réticulum endoplasmique. Nous avons recherché les partenaires protéiques potentiels d'ALA10 permettant d'expliquer ces effets et utilisé une approche de complémentation de fluorescence bimoléculaire afin d'étudier ces interactions. Nous avons pu déterminer qu'ALA10 interagit avec ALIS1 et ALIS5, deux sous-unités beta potentiellement nécessaires à la localisation et à la fonction d'ALA10, et confirmer leurs colocalisation avec ALA10 à l'aide de fusions GFP/CFP. ALA10 peut être localisée dans le réticulum endoplasmique à proximité des chloroplastes avec ALIS5 ou au niveau de la membrane plasmique avec ALIS1. Nous avons aussi pu déterminer qu'ALA10 interagit avec l’acide gras désaturase, FAD2, et une E3-ubiquitine ligase, PUB11. L''interaction avec FAD2 confirme un lien entre ALA10 et la désaturation de la phosphatidylcholine.Nous avons ensuite étudié l'effet d'ALIS1 et d'ALIS5 sur la fonction d'ALA10 en utilisant des lignées n’exprimant pas ces protéines ou les surexprimant avec ALA10. L'observation en microscopie électronique a révélé que la forme des chloroplastes et leurs relations avec le système endomembranaire sont modifiées en fonction de l'ALIS coexprimée avec ALA10. Les analyses lipidiques effectuées sur les plantes mutantes confirment un effet d’ALA10 sur l’homéostasie des galactolipides et la désaturation de la phosphatidylcholine. Les résultats suggèrent plusieurs fonctions d'ALA10, dépendantes de l’ALIS. Cet effet apparait variable en fonction de la photopériode ou du rythme circadien et indiquent une régulation post traductionnelle d'ALA10. Le rôle de PUB11 dans cette régulation a été exploré.Au final, cette étude révèle que, dans les cellules chlorophylliennes, ALA10, une flippase de phospholipides du réticulum endoplasmique, est impliquée dans la dynamique de désaturation de la phosphatidylcholine. Son activité stimule la synthèse des galactolipides et active la biogénèse des membranes photosynthétiques, probablement, en favorisant les échanges de lipides entre le chloroplaste et le réticulum endoplasmique.
... Biomembrane integral globular proteins interact with membrane lipids, mainly their acyl tails, due to hydrophobic forces and much less to hydrophilic interactions between the lipid head groups and protein hydrophilic groups [15], [39], [40]. As originally proposed [1], [14], the basic nano-scale structural organization of biomembranes has remained relatively consistent with current evidence [9], [10], [15], [18], [21]- [24], [40]- [45], with some modifications [26], [27], [32], [34], [35], [40], [45]. This will be briefly discussed. ...
Article
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The original Fluid-Mosaic Membrane Model of cellular and intracellular biomembrane structure was based primarily on thermodynamic principals and the available data on biomembrane dynamics at the time of its presentation [Singer S.J. and Nicolson G.L.,"The Fluid Mosaic Model of the structure of cell membranes", Science, 175 (1972), 720-731]. Approximately 50 years later this basic model remains relevant for describing the fundamental, simplified structural characteristics of biomembranes and some of their primary dynamic properties. However, plasma and intracellular membranes are certainly more complex than the original scheme that was proposed, and concepts of membrane hierarchy, fluidity, intra-and extracellular membrane connections, specialized membrane domains and different aspects of membrane dynamics and electrical properties have evolved over the intervening years. For example, the importance of specialized membrane domains, such as lipid rafts and phospholipid-protein complexes, peripheral, cytoskeletal and extracellular membrane connections and channels and their importance in determining the macrostructure and especially the dynamics of biomembranes and their roles in signaling and homeostasis have become much more complicated. In addition, the roles of membrane-associated cytoskeletal structures and extracellular matrix in limiting the mobility and range of motion of membrane components have added new layers of complexity and hierarchy to the original model. These dynamic structures lend themselves to new concepts in biomembrane medicine, and one of these, Membrane Lipid Replacement, has been developed to naturally modify membrane glycerolphospholipid composition and membrane protein-lipid interactions, and in the process repair and restore disease-and aging-associated damage to intracellular and plasma membranes. The use of Membrane Lipid Replacement with specific oral glycerolphospholipids has resulted in significant improvements in clinically important symptoms, such as fatigue, pain, and neurological symptoms.
... Membranes of eukaryotic cells are important, e.g., for protecting the cells and acting as transporters of biomolecules into the cell interior [1]. Nowadays, it is well established that lipid membranes can be separated into nano-or microdomains, usually referred to as "lipid rafts" [2,3], which play a role in many biological processes. ...
Article
It is well established that lipid aldehydes (LAs) are able to increase the permeability of cell membranes and induce their rupture. However, it is not yet clear how LAs are distributed in phase-separated membranes (PSMs), which are responsible for the transport of selected molecules and intracellular signaling. Thus, we investigate here the distribution of LAs in a PSM by coarse-grained molecular dynamics simulations. Our results reveal that LAs derived from mono-unsaturated lipids tend to accumulate at the interface between the liquid-ordered/liquid�disordered domains, whereas those derived from poly-unsaturated lipids remain in the liquid-disordered domain. These results are important for understanding the effects caused by oxidized lipids in membrane structure, properties and organization.
... Today, however, based on varied experimental and simulation data, it has been proposed to be a membrane with heterogeneity in the distribution of its components (1,14). Experiments with mammalian erythrocytes demonstrated that some lipids were identified within the outer leaflet of the membrane (phosphatidylcholine, sphingomyelin, and GM1 ganglioside) that were different from those localized in the inner leaflet (phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine) (15,16). Related to lateral membrane heterogeneities, in giant plasma membrane vesicles, it was observed that glycosylphosphatidylinositol-anchored proteins preferentially partitioned into liquid ordered domains, whereas the inner-leaflet-anchored protein flotillin-2 was located in liquid disordered domains (17). ...
Article
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Fluorescence Resonance Energy Transfer (FRET) is a high resolution technique that allows the characterization of spatial and temporal properties of biological structures and mechanisms. In the present work, we developed an in silico single-molecule FRET methodology to study the dynamics of fluorophores inside lipid rafts. We monitored the fluorescence of a single acceptor molecule in the presence of several donor molecules. By looking at the average fluorescence, we selected situations with a single acceptor and donor molecules, and we used them to determine the raft size in the range of 5 to 16 nm. We conclude that our method is robust and insensitive to variations in the diffusion coefficient, donor density or selected fluorescence threshold.
... The cell membrane functions as more than just mechanical support and protection for cells. 1 The cell membrane is also involved in the communications between different cells as well as the communications between cells and the extracellular environment. 2 Such communications mainly rely on interactions between receptors and ligands expressed on the cell surface. ...
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The cell surface is the forward position in cancer immunotherapy, with surface ligand and receptor interactions between various cells for determining immune privilege or recognition. Therefore, cell surface engineering (CSE) that manipulates the surface interactions between the immune effector cells (IECs) and tumor cells represents a promising means for eliciting effective anticancer immunity. Specifically, taking advantage of the development in biomaterials and nanotechnology, the use of functional bionanomaterials for CSE is attracting more and more attention in recent years. Rationally designed functional biomaterials have been applied to construct artificial functional modules on the surface of cells through genetic engineering, metabolic labeling, chemical conjugation, hydrophobic insertion, and many other means, and the CSE process can be performed both ex vivo and in vivo, on either IECs or tumor cells, and results in enhanced anticancer immunity and various new cancer immunity paradigms. In this review, we will summarize the recent exciting progresses made in the application of functional bionanomaterials for CSE especially in establishing effective recognition and interaction between IECs and tumor cells.
... Of particular interest in paleolimnological reconstructions are sterols and stanols. Sterols are natural, unsaturated steroid alcohols that represent an important group of compounds for the functioning and structure of biological membranes (Bretscher, 1973). Stanols are the reduction products of sterols by the process of hydrogenation, which is typically mediated by microbes in animal gastrointestinal tracts or in the environment (Reeves and Patton, 2001;Bull et al., 2002). ...
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The lack of long-term monitoring data for many wildlife populations is a limiting factor in establishing meaningful and achievable conservation goals. Even for well-monitored species, time series are often very short relative to the timescales required to understand a population's baseline conditions before the contemporary period of increased human impacts. To fill in this critical information gap, techniques have been developed to use sedimentary archives to provide insights into long-term population dynamics over timescales of decades to millennia. Lake and pond sediments receiving animal inputs (e.g., feces, feathers) typically preserve a record of ecological and environmental information that reflects past changes in population size and dynamics. With a focus on bird-related studies, we review the development and use of several paleolimnological proxies to reconstruct past colony sizes, including trace metals, isotopes, lipid biomolecules, diatoms, pollen and non-pollen palynomorphs, invertebrate sub-fossils, pigments, and others. We summarize how animal-influenced sediments, cored from around the world, have been successfully used in addressing some of the most challenging questions in conservation biology, namely: How dynamic are populations on long-term timescales? How may populations respond to climate change? How have populations responded to human intrusion? Finally, we conclude with an assessment of the current state of the field, challenges to overcome, and future potential for research.
Technical Report
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Volume 9 (2021) of the Universal Journal of Fluid Mechanics by the EU Academy of Sciences (EUAS)
Chapter
Membranes provide an essential physical boundary to the cell, allowing separation of a living cell from its environment, as well as subcellular compartmentalization of functional organelles in eukaryotic cells. Each membrane has a unique lipid and protein composition, which is optimized for its function as the interface between the extracellular and intracellular environments. As the primary structural component of this interface, phospholipids form the membrane bilayer backbone, and provide the major barrier function and other functions such as signal transduction and molecular recognition. One crucial feature of membrane phospholipids is their asymmetrical distribution between the two leaflets in various cell membranes, which appears to be a common theme from yeast to human cells. For example, in the plasma membrane of red blood cells, the extracellular leaflet is predominantly occupied by phosphatidylcholine (PtdCho) and sphingomyelin (SM), while the cytosolic leaflet is enriched in phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn). The asymmetric distribution of membrane lipids and their regulated transbilayer movement plays important roles in many cellular processes and functions, including apoptosis, blood coagulation and cell membrane integrity. But how is membrane lipid asymmetry generated and maintained? More specifically, how do phospholipids traverse the membrane bilayer, known as flip-flop, in a living cell? Since phospholipids are amphipathic molecules with large polar groups, their movement across the hydrophobic membrane interior is thermodynamically unfavorable. Experimental evidence demonstrates that in biological membranes, this process is mediated and facilitated by a number of membrane proteins, which function as phospholipid translocases that include P4-ATPases, ATP-binding cassette (ABC) transporters, TMEM16 family members, and others. Furthermore, recent advancement of structural studies on some of these proteins starts to shed light on the molecular mechanisms of phospholipid translocation by lipid translocases. In this chapter, we review our knowledge on phospholipid translocases in eukaryotic cells, and discuss our current understanding toward their functions and mechanisms.
Chapter
Transport of materials through cell membranes is of significant interest. We consider specifically the transport of gold nanoparticles that are in current use for delivery of pharmaceuticals, photothermal therapy, as contrast agents for imaging, and for targeted cancer therapy. We use coarse-grained molecular dynamics simulations to “observe” details of interactions between nanoparticles and a lipid bilayer model membrane during the permeation process. The nanoparticles are characterized at the molecular level (distributions of ligand configurations, their dependence on ligand length and surface coverage). Observation of membrane properties that agree with experimental values validates the simulations. We investigate the mechanisms of permeation of a gold nanoparticle, with either hydrophobic (alkane-thiols) or hydrophilic (PEG (polyethyleneglycol)) ligands attached via a sulfur covalent linkage to spherical (or nanorod) gold cores, and their dependence on surface coverage, ligand length, core diameter, and core shape. Lipid response such as lipid flip-flops, lipid extraction, changes in order parameter of the lipid tails are examined in detail. The mechanism of permeation of a PEGylated nanorod is shown to occur by tilting, lying down, rotating, and straightening up. Information provided by molecular dynamics simulations helps to understand why some systems work better than others, and aids design of new ones.
Chapter
Cellular membranes are highly complex liquid-crystalline entities, which makes it difficult for researchers to connect specific components and their effects on overall membrane structure, function, biochemical and biophysical properties. To circumvent this issue, model membranes with controlled compositions have since become a staple of biomembrane research, helping researchers better understand the inner mechanisms of cell membranes. These simplified lipid systems have predominately been composed of symmetric lipid bilayers--where both leaflets are composed of the same constituents. Only recently has there been a shift towards the use of bilayer systems with asymmetric distributions of lipids across the two monolayers. This is because most (if not all) biological membranes possess lipid asymmetry which has sparked an intense desire to study its effects on membrane structure, dynamics and membrane-associated molecules. In recent years, many have sought out to develop asymmetric model construction methods to facilitate these studies. In this chapter, we aim to describe novel and relevant asymmetric preparation methods, as well as their pros and cons to paint an image of the current state of biomembrane research and the challenges the field faces. Ultimately, these techniques are at the forefront of an exciting biomembrane renaissance.
Article
Intracellular organelles are subsystems within a cell, whose activity and chemical composition reflect the metabolic state of live cells. Alterations in cellular homeostasis occurring in disease, ageing and development are also reflected at the level of organelles. By targeting organelles with pharmacological agents and genetic tools, the aim is to improve disease diagnosis and to restore cell function. In this Review, we discuss biological pathways that can be exploited to target the delivery of exogenous cargo with organelle-level precision. We investigate how these pathways can be leveraged for imaging, diagnosis and therapy at the organelle level, and highlight the potential of nucleic acids as delivery systems to target specific organelles in vivo, including the nucleus, lysosomes, secretory organelles and mitochondria. The programmability, modularity and biocompatibility of nucleic acid-based scaffolds make them well suited to accomplishing next-generation targeting with organelle-level resolution in living organisms.
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The plasma membrane (PM) is often described as a wall, a physical barrier separating the cell cytoplasm from the extracellular matrix (ECM). Yet, this wall is a highly dynamic structure that can stretch, bend, and bud, allowing cells to respond and adapt to their surrounding environment. Inspired by shapes and geometries found in the biological world and exploiting the intrinsic properties of conductive polymers (CPs), several biomimetic strategies based on substrate dimensionality have been tailored in order to optimize the cell–chip coupling. Furthermore, device biofunctionalization through the use of ECM proteins or lipid bilayers have proven successful approaches to further maximize interfacial interactions. As the bio-electronic field aims at narrowing the gap between the electronic and the biological world, the possibility of effectively disguising conductive materials to “trick” cells to recognize artificial devices as part of their biological environment is a promising approach on the road to the seamless platform integration with cells.
Article
The overlapping of the electric double layer (EDL) in a nanochannel yields many interesting and significant electrokinetic phenomena such as ionic current rectification (ICR), which occurs only at a relatively low bulk salt concentration (∼1 mM) where the EDL thickness is comparable to the nanochannel size. In an attempt to raise this concentration to higher levels and the ICR performance improved appreciably, a branched nanochannel filled with polyelectrolytes (PEs) is proposed in this study. We show that these objectives can be achieved by choosing appropriate PE. For example, if the stem side of an anodic aluminun oxide nanochannel is filled with polystyrene sulfonate (PSS) an ICR ratio up to 850 can be obtained at 1 mM, which was not reported in previous studies. Taking account of the effect of electroosmotic flow, the underlying mechanisms of the ICR phenomena observed are discussed and the influences of the solution pH, the bulk salt concentration, and how the region(s) of a nanochannel is filled with PE examined. We show that the ICR behavior of a branched nanochannel can be modulated satisfactorily by filling highly charged PE and the solution pH.
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The membrane-impermeable, nitrene precursor reagent, N-(4-azido-2-nitrophenyl)-2-aminoethyl sulfonate (NAP-taurine), has been employed to study the comparative topology of resealed ghosts and their parent erythrocytes. We have found significant differences in the labeling pattern obtained from resealed ghosts as compared with that obtained from intact erythrocytes. Bands 2.3, 2.4, and 4 (nomenclature adapted from Fairbanks et al. (Fairbanks, G., Steck, T. L., and Wallach, D. F. H. (1971) Biochemistry 10, 2606)) show marked changes in labeling depending on the volume of solution in which the lysis and resealing is carried out. From this result we infer that the positions of certain protein components in the direction normal to the plane of the bilayer is measurably affected by the environment of the membrane.
Article
The cellular site of initial glycosylation of proteins from Saccharomyces cerevisiae has been studied. Short pulses of [U-14C]mannose label the ribosomal fraction of the yeast. Most of the label was associated with polysomes; monosomes contained only a small amount of radioactivity. All of the radioactivity present in the polysomal fraction was accounted by mannose and smaller amounts of glucose and glucosamine. Puromycin treatment detached more than 50% of the radioactivity from the polysomes; treatment of polysomes at pH 10.0 also caused the release of radioactivity. These results indicate that initial sugar binding occurs while the nascent polypeptide chains are still growing on the ribosomes. When the cells were preincubated with 2-deoxy-D-glucose, incorporation of [U-14C]mannose into the polysomes and the cell wall was inhibited, whereas its incorporation into membrane fractions was unimpaired. It was concluded that 2-deoxy-D-glucose inhibited the synthesis of glycoproteins by interference with the initial glycosylation steps at the ribosomal level.
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In experiments on isolated human erythrocyte membranes, cross-links between protein components have been produced both by the catalyzed oxidation of intrinsic sulfhydryl groups with the o-phenanthroline cupric ion complex and by reaction with dimethyl dithiobispropionimidate, a bisalkylimidate containing a disulfide linkage between the two reactive groups. The mixture of molecular species has been analyzed by a two-dimensional gel electrophoresis procedure in which the cross-links are split by reductive cleavage during an initial phase of the separation in the second dimension. Cross-linked complexes are recognized by the positions of the off-diagonal elements in the final pattern. Complexes apparently derived from stable oligomers of peptides of the same molecular weight have been found corresponding to many of the bands in the standard sodium dodecyl sulfate gel pattern. Heterocomplexes formed from peptides of different molecular weight have been tentatively identified in a few instances but are less common than oligomers from single bands. In addition cross-linking yields very high molecular weight complexes of spectrin and many of the lower molecular weight protein components of the membrane. The observed cross-linking patterns are consistent with the picture of transmembrane and cytoplasmic surface components anchored to the submembrane network of spectrin fibers. The cross-linking patterns of broken ghosts and intact cells treated with these membrane-permeable reagents are almost identical except for the addition of hemoglobin complexes in the latter.
Article
Accurately evaluating interfacial behavior is key and of exceptional meaning to characterize the mechanical behavior of layered structures. Different from various sensors in macroscopic regime, it is still a challenge to realize monitoring of interfacial behavior at nanoscale. Inspired by the prayer wheel, i.e., a Buddhist ritual apparatus, a novel nanoscale system based on double-walled carbon nanotubes is proposed and its rotating dynamics performance is investigated by molecular dynamics simulation. A theoretical model is also established to explain the underlying mechanism. It is found that the proposed nanoscale system functions well to predict the crack propagation speed of a layered structure. What is more, the dynamics response affected by various interfacial and dimension parameters is evaluated. The results not only provide insights into the design the small-scale interfacial sensors of layered nanostructures, but also would be promising for health monitoring of laminate structures.
Article
The increasing amount of knowledge about the macromolecular composition of biological membranes has led to the elaboration of newer models of membrane ultrastructure which have not yet been definitively evaluated by means of direct or indirect observations. The studies of model systems by Baumeister and Hahn emphasize that it might be possible to directly determine the placement of heavy metal atomic staining agents after these are specifically attached to a membrane. High resolution bright field imaging of very thin sections of embedded membranes has shown that the pair of dense lines which traditionally defines a unit membrane in positively stained specimens is formed by the superimposed projection of many individual stain “granules” having diameters down to 5-10Å.
Article
The ability to measure the passive membrane permeation of drug-like molecules is of fundamental biological and pharmaceutical importance. Of significance, passive diffusion across the cellular membranes plays an effective role in the delivery of many pharmaceutical agents to intracellular targets. Hence, approaches for quantitative measurement of membrane permeability have been the topics of research for decades, resulting in sophisticated biomimetic systems coupled with advanced techniques. In this review, recent developments in experimental approaches along with theoretical models for quantitative and real-time analysis of membrane transport of drug-like molecules through mimetic and living cell membranes are discussed. The focus is on time-resolved fluorescence-based, surface plasmon resonance, and second-harmonic light scattering approaches. The current understanding of how properties of the membrane and permeant affect the permeation process is discussed.
Article
Outer and inner leaflets of plasma cell membranes have different lipid compositions, and the membrane properties of each leaflet can differ from each other significantly due to these composition differences. However, because of the experimental difficulty in measuring the membrane properties for each leaflet separately, the differences are not well understood at a molecular level. In this study, we constructed two lipid bilayer systems, modeling outer and inner leaflets of plasma membranes of mouse hepatocytes based on experimental composition data. The ion concentration in the interlamellar water phase was also set to match the concentration in extra- and intracellular fluids. The differences in physical properties between the outer and inner leaflets of mouse hepatocyte cell membrane models were investigated by performing 1.2 μs-long all-atomistic molecular dynamics calculations under physiological temperature and pressure conditions (310.15 K and 1 atm). The calculated electron density profiles along the bilayer normal for each model bilayer system captured well the asymmetric feature of the experimental electron density profile across actual cell plasma membranes, indicating that our procedure of modeling the outer and inner leaflets of the cell plasma membranes was satisfactory. We found that compared to the outer leaflet model, the inner leaflet model had a very bulky and soft structure in the lateral direction. To confirm the differences, membrane fluidity was measured from the lateral diffusivity and relaxation times. The fluidity was significantly higher in the inner leaflet model than in the outer leaflet model. We also discuss two topics that are of wide interest in biology, i.e., the interdigitation of acyl tails of lipid molecules between two monolayers and the lateral concentration fluctuation of lipid molecules in the bilayers.
Article
Stratum corneum intercellular lipids play an important role in skin barrier function and are therefore considered useful in pharmaceutical and cosmetics products. In this study, we formulated cholesterol (Cho), the main component of intercellular lipids, into bicelles using a semi-spontaneous method, with the ultimate aim of extending the utility of bicelles in cosmetics. Bicelles are disk-shaped structures in which the planar part comprises long-chain phospholipids and the rims comprise short-chain phospholipids. We used soybean lecithin (SL) and nonionic surfactants (NS) to formulate Cho into bicelles using a semi-spontaneous method. Cho was successfully formulated into bicelles using poly(oxyethylene) cholesteryl ether (with an average of 10 ethylene oxide units, ChEO10) as the NS. We identified the boundaries of the bicelle region in the SL-Cho-ChEO10 system by measuring particle sizes. A bicelle region was formed up to around a maximum mixing ratio of 10wt% Cho, representing a mole fraction (mol%) of approximately 33. Phase changes in the system were observed. Comparison of poly(oxyethylene) cholesteryl ethers containing an average of 10 or 20 (ChEO20) ethylene oxide units revealed that the size of the hydrophilic group affects both the bicelle formation region and the mixing ratio of Cho. We analyzed the state of the SL-Cho-ChEO10 bicelles by differential scanning calorimetry and found that samples containing only phospholipid (SL/Ch/ChEO10 = 1/0/0) provided an endothermic peak between 50 and 55 °C, corresponding to the gel-liquid crystal transition point of the phospholipid. In contrast, the bicelle regions (SL/Cho/ChEO10 = 0.4/0/0.6 and 0.37/0.05/0.58) lacked a gel liquid crystal transition point and a liquid-ordered state was induced by Cho or ChEO10. This indicates that these bicelles are highly stable towards thermal changes.
Thesis
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La thèse a pour objectif l'implémentation d'approches computationnelles dédiées à l'étude des propriétés optiques non linéaires (ONL) du second ordre de molécules organiques π-conjuguées de type "push-pull" dans leurs états solvaté et agrégé. Dans une première partie, la première hyperpolarisabilité de quatre séries de mérocyanines fortement dipolaires est calculée en utilisant la théorie de la fonctionnelle de la densité (DFT) avec différentes fonctionnelles d'échange-corrélation (FXCs). En particulier, les performances de FXCs hybrides appartenant à la famille "Minnesota 2006", ainsi que les FXCs à séparation de portée LC-BLYP, ωB97X-D et CAM-B3LYP, sont analysées par rapport à des valeurs de référence obtenues à partir de calculs Møller−Plesset du second ordre, et à des données expérimentales provenant de mesures de diffusion Hyper-Rayleigh (HRS). Cette étude se concentre principalement sur deux effets : l'influence du taux d'échange Hartree-Fock exact inclus dans la FXC sur l'intensité des réponses HRS statiques, et l'impact de l'optimisation du paramètre de séparation de portée dans les FXCs à séparation de portée, effectuée selon une procédure non-empirique et spécifique au système considéré. Les effets de dispersion en fréquence sont également étudiés, ainsi que leur rôle crucial dans la comparaison entre données théoriques et expérimentales. La deuxième partie de la thèse présente une étude de l'organisation structurelle et les propriétés ONL du second ordre de nanoparticules organiques fluorescentes (FONs) formées de chromophores dipolaires π-conjuguées. Des simulations de dynamique moléculaire sont couplées à des calculs DFT afin d'étudier le processus d'agrégation moléculaire, l'orientation moléculaire des chromophores dipolaires au sein des nanoparticules, ainsi que l'effet des fluctuations dynamiques sur leurs propriétés ONL. Ces calculs permettent de rationaliser la forte augmentation de la première hyperpolarisabilité induite par l'agrégation, et mettent en évidence l'impact des effets de polarisation mutuelle et des couplages intermoléculaires sur les réponses ONL.
Article
The lipid composition impacts directly on the structure and function of the cytoplasmic as well as organelle membranes. Depending on the type of membrane, specific lipids are required to accommodate, intercalate, or pack membrane proteins to the proper functioning of the cells/organelles. Rather than being only a physical barrier that separates the inner from the outer spaces, membranes are responsible for many biochemical events such as cell-to-cell communication, protein-lipid interaction, intracellular signaling, and energy storage. Photochemical reactions occur naturally in many biological membranes and are responsible for diverse processes such as photosynthesis and vision/phototaxis. However, excessive exposure to light in the presence of absorbing molecules produces excited states and other oxidant species that may cause cell aging/death, mutations and innumerable diseases including cancer. At the same time, targeting key compartments of diseased cells with light can be a promising strategy to treat many diseases in a clinical procedure called Photodynamic Therapy. Here we analyze the relationships between membrane alterations induced by photo-oxidation and the biochemical responses in mammalian cells. We specifically address the impact of photosensitization reactions in membranes of different organelles such as mitochondria, lysosome, endoplasmic reticulum, and plasma membrane, and the subsequent responses of eukaryotic cells.
Article
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The 95,000-dalton polypeptide in human red blood cell membranes constitutes about 25% of the membrane protein. Previous labeling studies have shown that different regions of this polypeptide are exposed to the inside and outside of the cell and have suggested a role for the protein in anion exchange across the membrane. This polypeptide has been fragmented by chymotrypsin digestion of intact red cells and by treatment of purified polypeptide with 2-nitro-5-thiocyanobenzoic acid, hydroxylamine, and N-bromosuccinimide. The sites of cleavage by each of these reagents have been located relative to the NH-2 and COOH-terminals of the intact 95,000-dalton polypeptide. Polypeptide obtained from cells labeled with 1-isothiocyanate-4-benzene [35S]sulfonic acid (an inhibitor of anion transport), 125I and lactoperoxidase, or 32P has been similarly fragmented and these labels have been assigned to specific regions of the polypeptide. There are at least two sites of phosphorylation of the polypeptide; the major sites lies within 10,000 daltons of the NH2-terminal requiring that this portion of the polypeptide lie inside the cell. Sites of chymotrypsin cleavage and 125I and lactoperoxidase labeling are in a 7,000-dalton region toward the COOH-terminal of the polypeptide; this region must lie outside the cell. Between these two regions the polypeptide must traverse the lipid bilayer an odd number of times. 1-Isothiocyanate-4-benzenesulfonic acid also labels the protein near the site of chymotrypsin cleavage.
Article
State‐of‐the‐art methods for using DNA to engineer cell surfaces on both a monovalent and polyvalent scale are introduced in this Minireview. The application of these methods for either the promotion or inhibition of cell–environment communication in different settings is also described. Abstract The cell membrane is not only a physical barrier, but also a functional organelle that regulates the communication between a cell and its environment. The ability to functionalize the cell membrane with synthetic molecules or nanostructures would advance cellular functions beyond what evolution has provided. The aim of this Minireview is to introduce recent progress in using synthetic DNA and DNA‐based nanostructures for cell‐surface engineering. We first introduce chemical conjugation and physical binding methods for monovalent and polyvalent surface engineering. We then introduce the application of these methods for either the promotion or inhibition of cell–environment communication in numerous applications, including the promotion of cell–cell recognition, regulation of intracellular pathways, protection of therapeutic cells, and sensing of the intracellular and extracellular microenvironments. Lastly, we summarize current challenges existing in this area and potential solutions to solve these challenges.
Article
The cell membrane is not only a physical barrier, but also a functional organelle regulating the communication between a cell and its environment. An ability to functionalize the cell membrane with synthetic molecules or nanostructures would advance cellular functions beyond what the evolution of nature has provided. The purpose of this minireview is to introduce recent progress in using synthetic DNA and DNA‐based nanostructures for cell surface engineering. We first introduce chemical conjugation and physical binding methods for monovalent and polyvalent surface engineering. We further introduce the application of these methods for either promotion or inhibition of cell‐environment communication in numerous applications including promotion of cell‐cell recognition, regulation of intracellular pathways, protection of therapeutic cells and sensing of intracellular and extracellular microenvironment. Lastly, we summarize current challenges existing in this area and potential solutions to solve these challenges.
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Spectrin, a major protein constituent of mammalian red blood cell membrane preparations, has been localized on the inner surface of human red blood cell membranes by techniques that utilized specific ferritin-conjugated antibodies and fixation of membranes shortly after hemolysis so as to allow penetration of the ferritin-antibody labels. The labeling of spectrin was shown to be specific by the following criteria. (a) Nonhomologous ferritin-conjugated antibodies did not specifically bind to either membrane surface. (b) Blocking the membrane-bound spectrin with excess unconjugated antispectrin antibodies prevented ferritin-antibody labeling. (c) Removal of spectrin by treating the membrane preparation with a low ionic strength buffer containing ethylenediaminetetraacetate and ß-mercaptoethanol prevented labeling by specific ferritin-conjugated antibodies.
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An electron microscopic stain for specific saccharides was prepared by the conjugation of ferritin to concanavalin A, a plant agglutinin that specifically binds to oligosaccharides containing terminal d-glucose, d-mannose, or sterically related sugar residues. A technique was developed to allow topological visualization of erythrocyte and other membranes by means of transmission electron microscopy, and the distribution of the binding sites for ferritin-concanavalin A on such membrane preparations was determined. The conjugate was found to bind specifically to the outer, but not the inner, surface of erythrocyte membranes. The number of conjugate molecules bound per unit area of the membrane was larger for rabbit than for human erythrocytes.
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Ferritin conjugates of two plant agglutinins, concanavalin A and ricin, have been used as specific electron microscopic stains for covalently-bound saccharide residues on membrane fragments from a myeloma-cell homogenate. The results indicate that different saccharide residues are uniformly localized to a single surface of each membrane fragment. In particular, the ferritin-concanavalin A conjugate binds exclusively to the cisternal side of membrane fragments of the rough endoplasmic reticulum. If it is postulated that the biogenesis of eukaryotic plasma membranes involves an assembly-line process from precursor intracellular membranes, these observed asymmetric distributions of saccharides on cell membranes can be explained.
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The polypeptides of the human erythrocyte membrane were analyzed by polyacrylamide gel electrophoresis in 1% sodium dodecyl sulfate. Six major bands (I-VI) together make up over two-thirds of the protein staining profile. Component III (mol wt 89,000) predominates in the ghost membrane; it constitutes 30% of the protein and numbers over 106 chains/ghost. Components I and II form a slow-moving doublet (approximate mol wt 250,000) containing 25% of the protein. The molar amounts of I + II, IV (mol wt 77,500), V (mol wt 41,300), and VI (mol wt 36,200) are similar, falling in the range 3.4-4.6 × 105 chains/ghost. Four bands were recognized in gels stained by the periodic acid-Schiff procedure. A broad Schiff-positive zone just behind the tracking dye corresponds to membrane lipids. Three bands of lower mobility are sialoglycoproteins. The most prominent of these has an apparent molecular weight of 83,500 and contains at least 57% of the sialic acid of ghosts. The Schiff-positive bands were not colored by protein stains. Sialidase treatment of ghosts selectively increased the mobilities of the sialoglycoproteins without affecting the protein-staining profile. Attempts to produce subunits from the large polypeptides by treatment with various denaturing agents were unsuccessful. Normally, no polypeptides of size less than 15,000 were seen in ghost electrophorograms. However, heating ghosts with low levels of sodium dodecyl sulfate and high levels of salt produced diffuse bands of low average molecular weight. This highly variable effect is attributed to degradation by proteinases. Components I, II, and V were solubilized by incubating ghosts at low ionic strength. Component VI was released by washing with buffered saline at concentrations above 0.1 M. Both elution procedures were rapid (15 min), complete, and selective; they were also conservative in that new bands were not created and the electrophorograms of released and retained material were complementary. The eluted material contained negligible sialic acid and no Schiff-positive lipids. Two classes of membrane protein were distinguished by their response to the elution procedures. Components I, II, V, and VI compose one class. They make up 30-35 % of the protein and are tenuously related to the membrane, possibly by predominantly ionic bonds. The second class, which includes components III, IV, and the sialoglycoproteins, together with various minor components, constitutes 65-70% of the protein. These polypeptides are tightly bound; their properties may reflect participation in hydrophobic protein-protein and protein-lipid interactions.
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A fluid mosaic model is presented for the gross organization and structure of the proteins and lipids of biological membranes. The model is consistent with the restrictions imposed by thermodynamics. In this model, the proteins that are integral to the membrane are a heterogeneous set of globular molecules, each arranged in an amphipathic structure, that is, with the ionic and highly polar groups protruding from the membrane into the aqueous phase, and the nonpolar groups largely buried in the hydrophobic interior of the membrane. These globular molecules are partially embedded in a matrix of phospholipid. The bulk of the phospholipid is organized as a discontinuous, fluid bilayer, although a small fraction of the lipid may interact specifically with the membrane proteins. The fluid mosaic structure is therefore formally analogous to a two-dimensional oriented solution of integral proteins (or lipoproteins) in the viscous phospholipid bilayer solvent. Recent experiments with a wide variety of techniqes and several different membrane systems are described, all of which abet consistent with, and add much detail to, the fluid mosaic model. It therefore seems appropriate to suggest possible mechanisms for various membrane functions and membrane-mediated phenomena in the light of the model. As examples, experimentally testable mechanisms are suggested for cell surface changes in malignant transformation, and for cooperative effects exhibited in the interactions of membranes with some specific ligands.
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The major glycoprotein of the human erythrocyte membrane has been isolated by treatment with lithium di-iodosalicylate and found to be a single polypeptide chain with a molecular weight of about 50,000. This molecule, which is 60% carbohydrate and 40% protein, carries multiple blood-group antigens, the receptors for influenza viruses, and various plant agglutinins. Four unique carbohydrate-containing peptides (α-1, α-2, α-3, and β) are produced by tryptic digestion of the isolated glycoprotein; their order in the molecule has been determined by sequential tryptic digestion of intact erythrocyte membranes and partially digested glycoprotein fragments. Cleavage of the native protein with cyanogen bromide produces five fragments; two of these (C-5 and C-1) contain most of the carbohydrate in the molecule and are derived from the N-terminal half of the polypeptide chain. The nonpolar amino acids of this glycoprotein are located predominantly in the C-terminal fragment (C-2). Phytohemagglutinin conjugated to ferritin has been used to map the distribution of glycoprotein receptors over the surfaces of intact erythrocytes by freeze-etching and electron microscopy. This label localizes to sites on the membrane that overlie the intramembranous particles. These findings suggest that the glycoprotein is oriented at the cell surface with its oligosaccharide-rich N-terminal end exposed to the exterior, while its C-terminal segment interacts with other components in the interior of the membrane to form intramembranous particles.
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Phospholipid spin labels incorporated in the sarcoplasmic reticulum from rabbit-skeletal muscle undergo rapid lateral diffusion within the plane of the membrane. The diffusion constant, D, is 6x10(-8) cm(2)/sec at 37 degrees . With this diffusion constant, a phospholipid molecule can diffuse a distance of the order of 5000 nm in 1 sec.
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The proteins located on the exterior of the membrane of influenza virus can be preferentially labeled by incorporation of tritium. This incorporation is achieved by sodium borotritide reduction of the Schiff's base formed between pyridoxal phosphate and protein amino groups of purified virions. The preferential labeling is due to the inability of phosphate esters, such as pyridoxal phosphate, to penetrate cell membranes. Labeled virions remain fully infectious and retain hemagglutinating activity.
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The freeze-etch technique was used to observe red blood cell ghosts labeled on both surfaces with covalently bound ferritin. Ferritin molecules were never observed on fracture faces, thus indicating that fracture does not show membrane-surface detail. Subliming away the surrounding ice did expose the ferritin on the membrane surface. These results were consistent with the concept that membranes split during the fracture process of freeze-etching.
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Human red blood cell membranes contain at least 17 polypeptides of differing molecular weight ranging from 27,000 to 220,000. Previous publications have reported higher values for the largest molecular weight species, but are in error owing to incorrect calibration of sodium dodecylsulfate-polyacrylamide gels with unreduced proteins. The two highest molecular weight species which constitute approximately 40% of the total membrane protein have molecular weights of 200,000 and 220,000. It is shown that these polypeptides are not aggregates of a lower molecular weight species, and analysis of the entire membrane by gel filtration in 6 m guanidine hydrochloride reveals no significant amount of protein with molecular weight less than that of hemoglobin. Solubilization of a fraction of the membrane protein in EDTA according to the method of Marchesi et al. (Marchesi, S. L., Steers, E., Marchesi, V. T., and Tillack, T. W., Biochemistry, 9, 50 (1970)) produced a heterogeneous mixture of polypeptide chains. Prolonged exposure to this solvent leads to dissolution of over 90% of the membrane protein.
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Treatment of erythrocytes or their isolated membranes with radioactive iodoacetate results in the specific modification of one of the membrane proteins, as determined by acrylamide gel electrophoresis in sodium dodecyl sulfate. The reactive groups of this protein are almost completely blocked before any of the other major membrane proteins react.The reaction with N-ethylmaleimide does not show a similar specificity of labeling, indicating a considerable degree of reagent specificity for the reaction. The specifically labeled protein was extracted from the membrane with sodium chloride and further purified by chromatography on Sephadex G-200 in sodium dodecyl sulfate. This preparation shows a single band on acrylamide gel electrophoresis in sodium dodecyl sulfate with a molecular weight of 35,000. The labeled amino acid was identified as cysteine from its elution position on ion exchange chromatography.
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Small and medium lymphocytes from the peripheral blood and lymphoid tissues of the rabbit react in suspension with antibodies directed against different immunoglobulin determinants. Through immunofluorescence, it was possible to show that numerous discrete spots on the surface of the positive lymphocytes carry immunoglobulin molecules. The positive lymphocytes are about one-half of all lymphocytes in the different preparations; thymus lymphocytes are all negative. With antisera specific for rabbit IgM as well as with antisera directed against allotypic determinants specific for IgM or IgG, it was possible to show that about nine-tenths of the immunoglobulin-positive lymphocytes carry IgM molecules on their surface. With antisera directed against a- and b-locus determinants, it was also possible to demonstrate that both heavy and light chains were present in the surface immunoglobulins. Furthermore, in animals which were heterozygous at the a or the b locus, it was found that each lymphocyte had immunoglobulins synthesized under the influence of only one of two alleles. A very small proportion of lymphocytes could be shown to have a specific surface reaction with one antigen (horse ferritin); the proportion of these cells increased very much after immunization.
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Excerpt In order to understand the mechanism of immunological stimulation, it is worth starting with a minimum theory of antigen reception. We assume first that nothing except antibody recognizes antigen, and we must therefore assume that the receptor for antigen is antibody already present at a site, in or on the cell, prior to exposure to antigen. Presumably the receptor antibody is made by the cell, and represents an accurate sample of the antibodies that the progeny of the cell will produce if stimulation is successfully accomplished. The relationship between receptor and product may well be even simpler and more direct than this: an individual plasma cell makes a single amino acid sequence, and this sequence is present in the parent lymphocyte. The arguments in favor of this hypothesis can be found in a recent review (Lennox and Cohn, 1967), from which the present account is derived. It would be premature...
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Four different amino-reactive reagents, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (SITS),¹ 1-fluoro-2,4-dinitrobenzene (FDNB), 2,4,6-trinitrobenzene sulfonic acid (TNBS), and 2-methoxy-5-nitrotropone (MNT) decrease the anion permeability of the human red blood cell, as measured by sulfate fluxes, whereas the sulfhydryl agent, parachloromercuriphenyl sulfonic acid (PCMBS), does not. In contrast, PCMBS increases the cation permeability as measured by K⁺ leakage, whereas SITS does not. Of the other agents, FDNB increases the cation permeability to the same extent as PCMBS but MNT and TNBS produce smaller increases. PCMBS does not protect against FDNB as it does against other sulfhydryl agents (X-irradiation) and the FDNB effect on cations is attributed to amino groups. Studies of the binding of SITS indicate that it does not penetrate into the membrane and its failure to influence cation permeability is attributed to its inability to reach an internal population of amino groups. It is concluded that two ion permeability barriers, both involving proteins, are present in the red blood cell. The more superficial barrier contains amino groups and controls anion flow; the more internal barrier contains sulfhydryl and amino groups and controls cation flow. The amino groups contribute to the control of permeability by virtue of their positive charges, but the role of sulfhydryl groups is not clear. Only a small fraction of the membrane protein amino and sulfhydryl is involved in the barriers.
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A glycoprotein has been extracted in water-soluble form from human red cell membranes with lithium diiodosalicylate. After extraction of the membranes and phosphocellulose chromatography a homogeneous preparation is obtained which was 60 percent carbohydrate and 40 percent protein (by weight). The preparation contains AB and MN blood group antigens, receptors for influenza virus, and various phytohemagglutinins.
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The phosphatides of the erythrocytes of several species of mammals have been analysed chromatographically. The cells of the true ruminants contained substantial amounts of cephalin and sphingomyelin but usually no lecithin. The cells of dog and guinea-pig contained lecithin but relatively small amounts of sphingomyelin. The biological implications of the lack of lecithin in ruminant cells have been discussed.
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1.A study has been made of the action of heated cobra venom, a partially purified human pancreatic phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4) and crude sea-snake venom on the phospholipids of human red cell ghosts and intact, washed erythrocytes.2.Marked variations were found between the action of the phospholipase A from these different sources on red cell ghosts and their actions on intact erythrocytes.3.Differences were also demonstrated between these three phospholipases in their ability to attack different phospholipids in the intact erythrocyte membrane.4.The pffect of plasma on the actions of these enzyme preparations on erythrocytes has been studied.5.Heparin has been shown to inhibit the phospholipase A activity of cobra venom and human pancreas.6.The relationship of these findings to the problem of the mechanism of haemolysis is discussed.
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We have examined the blood of man and of the rabbit, dog, guinea pig, sheep, and goat. There exists a great difference in the size of the red blood cells of these animals, but the total surfaces of the chromocytes See PDF for Structure from 0.1 cc. blood do not show a similarly great divergence, because animals having very small cells (goat and sheep) have much greater quantities of these cells in their blood than animals with blood cells of larger dimensions (dog and rabbit). We give all the results of our experiments, omitting only those in which we were unable to avoid losses in the procedure of evaporation of the acetone. It is clear that all our results fit in well with the supposition that the chromocytes are covered by a layer of fatty substances that is two molecules thick.
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High-rate antibody-forming cells and immunological memory cells can be selectively retained if filtered through a column coated with relevant antigen. This trapping can be blocked if the cells are incubated with an anti-immunoglobulin serum prior to column passage. A similar blocking is not observed when cells are treated with an anti-lymphocyte serum, thereby excluding the possibility that any antibodies combining with surface structures could cause this effect. By the use of antisera specific for heavy or light chain antigens, it was possible to locate such antigens in the antigen-binding receptor areas of immune cells. Criss-cross studies using antisera specific for gamma 1 or gamma 2a heavy chains showed that the membrane receptor has the same heavy chain as will be present in the eventual product of that cell, the humoral antibody.
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1.1. The diazonium salt of [35S]sulfanilic acid can be used as a label for outer components of the human erythrocyte membrane; the reagent does not penetrate intact cells.2.2. Modified cells become permeable to Na+ and K+ but not to water-soluble nonelectrolytes. They eventually lyse in isotonic buffer.3.3. About 20% of the label bound to intact cells can be recovered in an ethanol-ether membrane extract. Phospholipase D (cabbage) changes the way in which it partitions between ether and water.4.4. If the residue left after ethanol-ether extraction is dissolved in 3% sodium dodecyl sulfate and sized by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate, a complex but reproducible pattern is observed on staining with Coomassie brilliant blue. The most intensely labeled material has a molecular weight of about 140 000. Some peaks are free of label.5.5. Similar patterns are obtained if intact membranes are dissolved in 3% sodium dodecyl sulfate; sodium dodecyl sulfate dissociates the protein and lipid as effectively as ethanol-ether.6.6. If the residue left after ethanol-ether extraction is exposed first to 0.8 M NaCl, a medium in which it is largely insoluble, much of the protein shifts from high to low molecular weight. The most intensely labeled material does not. The membrane contains protein complexes which can be dissociated by sodium dodecyl sulfate after exposure to salt. The dissociation is less extensive when intact membranes are exposed to salt.
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THERE is increasing evidence that lymphocytes have antigen-specific receptors on their surface1-3 and it seems reasonable to suppose that the receptors are immunoglobulins (Ig)1. The existence of Ig on the surface of lymphocytes has also been inferred from the transforming effect of anti-Ig sera4 and the opsonic adherence to macrophages of lymphocytes treated with anti-Ig5. We report here the demonstration of Ig determinants on the surface of living mouse lymphocytes by the use of immunofluorescence and immunoautoradiography.
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1) Induction of humoral antibody formation involves the obligatory recognition of two determinants on an antigen, one by the receptor antibody of the antigen-sensitive cell and the other by carrier antibody (associative interaction). 2) Paralysis of antibody formation involves the obligatory recognition of only one determinant by the receptor antibody of the antigen-sensitive cell; that is, a nonimmunogenic molecule (a hapten) can paralyze antigen-sensitive cells. 3) There is competition between paralysis and induction at the level of the antigen-sensitive cell. 4) The mechanisms of low- and high-zone paralysis, and maintenance of the unresponsive state, are identical. 5) High-zone paralysis occurs when both the carrier antibody and the receptor antibody are saturated, so that associated interactions cannot take place. 6) The mechanisms of paralysis and induction for the carrier-antigen-sensitive cell are identical to those for the humoral-antigen-sensitive cell. 7) The formation of carrier-antigen-sensitive cells is thymus-dependent, whereas humoral-antigen-sensitive cells are derived from bone marrow. Since carrier antibody is required for induction, all antigens are thymus-dependent. 8) The interaction of antigen with the receptor antibody on an antigen-sensitive cell results in a conformational change in an invariant region of the receptor and consequently paralyzes the cell. As the receptor is probably identical to the induced antibody, all antibody molecules are expected to be able to undergo a conformational change on binding a hapten. The obligatory associated recognition by way of carrier antibody (inductive signal) involves a conformational change in the carrier antibody, leading to a second signal to the antigen-sensitive cell. 9) The foregoing requirements provide an explanation for self-nonself discrimination. Tolerance to self-antigens involves a specific deletion in the activity of both the humoral- and the carrier-antigen-sensitive cells.
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The membranes of a cell have the principal function of setting the boundaries between the cell and the environment and between compartments within the cell. These boundaries prevent the movement of all polar solutes from one compartment to another, unless such movement is required for biological activity; under these circumstances, special transport systems are required. Thus, membranes can be considered as structures which are selectively permeable. The barrier to movement of polar solutes across the membrane is provided by one of the two major components of the membrane: the lipids. The other major component of the membrane, the proteins, provides the permeability function. Membrane proteins also determine most of the other properties of a membrane: They carry the determinants of specificity which distinguish one cell from another and allow for recognition between cells; they determine the shape and architecture of the membrane; they are the receptors for information about the environment and relay that information to other parts of the cell; and they are enzymes with a precise compartmental localization.
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The paramagnetic resonance spectra of N-oxyl-4','-dimethyloxazolidine derivatives of 12-ketostearic acid, 5-ketostearic acid, 5-ketotricosanoic acid, and 5alpha-androstan-3-one-17beta-ol have been observed in sonicated phospholipid dispersions, the walking-leg nerve fibers of Homarus americanus, and in erythrocytes oriented by hydrodynamic shear. The preferred orientation of these spin labels in the nerve and erythrocyte membranes is one in which the long amphiphilic axis is perpendicular to the membrane surface. The resonance spectra indicate that these labels in the nerve fiber are bound to a lipid bilayer very similar to that found in the sonicated phospholipid dispersion, whereas any lipid bilayer in the erythrocyte must be much more rigid.
Article
Spin-labeled phosphatidylcholine is a paramagnetic analog of phosphatidylcholine. The vesicle which results from prolonged sonication of egg phosphatidylcholine and spin-labeled phosphatidylcholine in salt solution has an aqueous compartment and a bilayer membrane. Sodium ascorbate at 0° abolishes the paramagnetism of spin-labeled molecules in the external monolayer of the vesicle membrane (65 % of the total paramagnetism of a vesicle) without affecting the paramagnetism of internal molecules. The consequent asymmetry in the distribution of paramagnetic molecules between the two monolayers of the vesicle membrane decays with a half-time of 6.5 hr at 30°. It follows that the probability of a spin-labeled phosphatidylcholine molecule passing from the internal monolayer of the vesicle membrane to the external monolayer is 0.07/hr at 30°, and the probability of a spinlabeled phosphatidylcholine molecule passing from the external monolayer of the vesicle membrane to the internal monolayer is 0.04/hr at 30°.
Article
Nuclear-resonance spectra of phosphatidylcholine (PC) vesicles are broadened by spin-labeled PC. The broadening of the N-methyl proton line is proportional to spin-labeled PC concentration; the broadening by 1 molecule in 100 of spin-labeled PC is 9.7 Hz at 35 degrees C; the broadening is not due to collisions between vesicles, exchange of spin label between vesicles, or fusion of vesicles. These findings are proof of rapid diffusion in the plane of the PC bilayer (lateral diffusion). A further study of the broadening reveals the molecular frequency of the translation step for lateral diffusion to be much greater than 3 x 10(3) sec(-1) at 0 degrees C.
Article
Molecular weights for the human erythrocyte membrane glycoprotein and other glycoproteins calculated relative to protein standards on SDS acrylamide gel electrophoresis depend upon the percent crosslinking of gels. This anomaly is due to a decreased binding of SDS to the oligosaccharide side chains relative to the polypeptide backbone; removal of sialic acid fails to correct the anomaly. Previous molecular weights proposed for the human erythrocyte membrane glycoprotein utilizing SDS gel electrophoresis are probably incorrect and a new value of 55,000 is proposed based on corrected SDS-gel data.
Article
The paramagnetic resonance spectrum of a highly concentrated region of spin-labeled phosphatidylcholine included in oriented bilayers of phosphatidylcholine (PC) changes dramatically in time. This time dependence of the spectra is due to the lateral diffusion of the oriented labeled molecules spreading in the planes of the corresponding monolayers. The resonance spectra can be analyzed in terms of a time-dependent superposition of spectra corresponding to the different concentrations of spin label. It is possible to estimate the diffusion constant: D ≃ 1.8 ± 0.6 × 10-8 cm2/sec at room temperature (25°). If lateral diffusion is assumed to be due to pairwise exchange of neighboring molecules, then this diffusion coefficient corresponds to an exchange frequency which is of the order of 107 sec-1. This rate is high enough to suggest that the lateral translation of molecules bound to membranes may sometimes have biological significance.
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Dichroism can be photoinduced in a frog retina once it has been fixed with glutaraldehyde. This dichroism is absent in the normal retina because rhodopsin is free to undergo rotational Brownian motion.
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The determination of the electron density distribution of the membrane cross sections of nerve myelin and the discs of the outer segments of retinal rods by a new X-ray evaluation method indicates, as in the case of the cross-sectional structure of the photosynthetic membrane, an “asymmetric” mass distribution. In connection with the detection of planar protein lattices in the membranes of photosynthetic bacteria, the chloroplasts of higher plants, erythrocytes, and retinal discs as well as the detection of a new category of lipid characteristics, comprising isothermal confromational and phase changes by specific ligand binding, a basis is established for the development of a general concept of biomembrane structure.
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The specificity of RNA from free and bound polyribosomes for free and bound ribosomes was examined using the system for cell-free protein synthesis dependent on RNA. RNA from free polyribosomes can stimulate amino acid incorporation in the system with free ribosomes in the presence of KCl extract from free polyribosomes, which was prepared by treatment of polyribosomes with 0.8M KCl. Whereas RNA from bound polyribosomes can stimulate amino acid incorporation in system with bound ribosomes. This suggests that in course of protein synthesis a distinct specificity is present between messenger RNA and ribosomes.
Article
Human erythrocyte glycophorin has been purified and partially sequenced. The portion of the sequence reported here (51 residues) represents a unique region of glycophorin that, on the basis of labeling experiments reported elsewhere, is probably associated with the hydrophobic interior of the red cell membrane. This sequence has the anticipated hydrophobic characteristics for interaction with the hydrocarbon interior of a membrane and, as far as we are aware, represents the first reported sequence from a hydrophobic region of an integral membrane protein. There is a distribution of charged and hydrophobic residues topographically similar to a cross-section of a phospholipid bilayer, a structure many now accept as the basic feature of a biological membrane. We propose that the hydrophobic sequence of approximately 23 residues comprises the intramembranous domain of human erythrocyte glycophorin and is intimately and possibly specifically associated with lipids of the membrane.
Article
Two classes of membrane-bound ribosomes were shown in rat liver cells. A small portion of them (loose ribosomes) was dissociated from the rough endoplasmic reticulum by RNase treatment, whereas the remainder still attached tightly to the membrane (tight ribosomes). Pulse-labeling experiment showed that they are both active in protein synthesis, but serum albumin is exclusively synthesized on tight ribosomes.
Article
Much more phosphatidyl-ethanolamine is labelled when erythrocyte ghosts, rather than intact cells, are exposed to the reagent [35S]FMMP‡ The ability to label phosphatidyl-ethanolamine from the cell exterior is not increased by prior treatment of the cells with pronase. This suggests that phosphatidyl-ethanolamine may exist in an unreactive state in the outer half of the lipid bilayer, or that most of it resides in the inner half of the bilayer of the erythrocyte membrane.
Article
Experiments with transferred stearate layers were performed to determine the location of fracture planes in frozen ice-lipid systems. Bilayers and multilayers of carbon-14-labeled stearate were frozen in contact with an aqueous phase and then fractured. The distribution of radioactivity on both sides of the fracture showed that the stearate layers were cleaved apart predominantly in the plane of their hydrocarbon tails. Because bilayers split in this manner, it was possible to measure time-dependent exchange of label between the layers. Exchange occurred with a half-time of 50 minutes in the presence of calcium and 25 minutes in the absence of calcium. Since stearate bilayers and multilayers are models of hydrophobically stabilized structures, the strong influence of their hydrophobic region on the fracture plane provides an explanation of how the freeze-etch technique of electron microscopy can expose inner, hydrophobic faces of cell membranes.
Article
Acetic anhydride has been used as a reagent to investigate the relative reactivities of various components within the human red blood cell membrane. The reagent penetrates the membrane rapidly and reacts with intracellular and membrane proteins as well as lipid amino groups. To determine the relative reactivities of the various components in intact and disaggregated membranes, a double labeling procedure with 14C- and 3H-labeled reagents was performed. Either whole red cells or intact ghosts are initially reacted with reagent containing one radioisotope. After hemolysis and/or appropriate washings, the singly labeled membranes are treated with reagent containing the second isotope in the presence of sodium dodecyl sulfate. Comparisons of the relative reactivities of the membrane components in the intact and disaggregated states can then be made by fractionation of the membranes by acrylamide gel electrophoresis and analysis of the gels for the two radioisotopes. This procedure separates the membrane into a lipid fraction plus a number of protein components. The results of the double labeling indicate that there is little difference in the relative reactivities of the membrane protein components that can be attributed to their incorporation into the membrane structure. The lipid fraction does show a change in its relative reactivity toward acetic anhydride when the membrane is disaggregated which is dependent on whether whole red cells or intact ghosts are initially labeled. These results suggest that the structure of the membrane of the intact cell may be different from that of the isolated ghost and that caution must be exercised in deducing membrane structure solely from studies of isolated membranes.
Article
1. The temperature dependence of the steady-state self-exchange of chloride between human red cells and a plasma-like electrolyte medium has been studied by measuring the rate of (36)Cl(-) efflux from radioactively labelled cells. Between 0 and 10 degrees C the rate increased by a factor of eight corresponding to an Arrhenius activation energy of 33 kcal/mole.2. The rate of chloride exchange decreased significantly in experiments where 95% of the chloride ions in cells and medium were replaced by other monovalent anions of a lyotropic series. The rate of chloride self-exchange was increasingly reduced by bromide, bicarbonate, nitrate, iodide, thiocyanate, and salicylate. The latter aromatic anion was by far the most potent inhibitor, reducing the rate of chloride self-exchange to 0.2% of the value found in a chloride medium.3. The temperature sensitivity of the chloride self-exchange was not affected significantly by the anionic inhibitors. The Arrhenius activation energies of chloride exchange were between 30 and 40 kcal/mole in the presence of the six inhibitory anions mentioned above.4. The rate of self-exchange of bromide, thiocyanate, and iodide between human red cells and media was determined after washing and labelling cells in media containing 120 mM bromide, thiocyanate, or iodide respectively. The rate of self-exchange of the three anions were 12, 3, and 0.4% of the rate of chloride self-exchange found in the chloride medium.5. The Arrhenius activation energies of the self-exchange of bromide, iodide, and thiocyanate were all between 29 and 37 kcal/mole, the same magnitude as found for the self-exchange of chloride.6. Although approximately 40% of the intracellular iodide and salicylate ions appeared to be adsorbed to intracellular proteins, the rate of tracer anion efflux followed first order kinetics until at least 98% of the intracellular anions had been exchanged.7. The self-exchange of salicylate across the human red cell membrane occurred by a different mechanism than the one utilized by the inorganic monovalent anions. The activation energy of salicylate exchange (13.2 kcal/mole) was significantly lower than that of inorganic anion exchange. Salicylate exchange increased with decreasing pH in contrast to the exchange of chloride, which decreases when pH is lowered.
Article
An enzymatic iodination procedure utilizing lactoperoxidase (LPO), radioactive iodide, and hydrogen peroxide generated by a glucose oxidase-glucose system has been described and utilized for a study of the red cell membrane. 97% of the incorporated isotope is in the erythrocyte ghost and 3% is associated with hemoglobin. No significant labeling of the red cell membrane occurs in the absence of LPO or by the deletion of any of the other reagents. A 6 million-fold excess of chloride ions inhibits iodination by no more than 50%. Incorporation of up to 1 x 10(6) iodide atoms into a single erythrocyte membrane results in no significant cell lysis. The incorporated label is exclusively in tyrosine residues as monoiodotyrosine. 10-15% of the trichloroacetic acid-precipitable radioactivity can be extracted with lipid solvents but is present as either labeled protein or (125)I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins reveals only two labeled protein bands out of the 15 present, and the presence of 50-1 x 10(6) iodide atoms per ghost does not alter this pattern. Component a has a molecular weight of 110,000, is carbohydrate poor, and represents 40% of the total label. Component b has an apparent molecular weight of 74,000, contains all of the demonstrable sialic acid, and accounts for 60% of the total label. Trypsinization of iodinated, intact red cells results in the disappearance of only component b, the appearance of labeled glycopeptides in the medium, and the absence of smaller, labeled peptides remaining in the membrane. Pronase treatment hydrolyzes component b in a similar fashion, but also cleaves component a to a 72,000 mol wt peptide which is retained in the membrane. A combination of protease treatment and double labeling with (125)I and (131)I does not reveal the appearance of previously unexposed proteins.
Article
Pronase degrades proteins on the outer surface of the human erythrocyte membrane which run in polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate at a molecular weight of approximately 125,000. Carbohydrate and sialic acid are removed, but fragments of molecular weight 50,000 to 100,000 remain attached to the membrane. The most prominent fragment, one of molecular weight about 73,000, can be labeled with a membrane-impermeable reagent (sulfanilic acid diazonium salt), so it is still accessible from the outside of the cell. Pronase rapidly inactivates membrane-bound acetylcholinesterase, but it has relatively little effect on the facilitated diffusion of glucose; both are inhibited by the diazonium salt. Extensive digestion leads to potassium loss and osmotic lysis. Ghosts prepared in 15 mosm-Tris (pH 7.6) are extensively degraded by pronase: essentially all the protein shifts to low molecular weight. Pronase is even more potent in 3% sodium dodecyl sulfate. Ghosts prepared from intact cells which have been treated with the enzyme hydrolyze when dissolved in the detergent unless steps are taken to inhibit proteolysis.
Article
A very reactive, highly radioactive (about 10 Ci/m-mole) reagent designed to acylate amino groups has been synthesized. This compound, the sulphone of 35S-labelled formylmethionyl methyl phosphate, cannot pass through the red blood cell membrane. When added to intact red blood cells, it reacts with, and labels, two proteins on the outside surface of the cell membrane: these have molecular weights of about 105,000 and 90,000 daltons. All membrane proteins appear to be labelled if erythrocyte ghosts are exposed to the reagent. The two proteins exposed on the outside surface of the erythrocyte are major membrane components: they probably cover a significant portion of the surface of the cell. The smaller of these two proteins carries the bulk of the cell surface sialic acid and a large portion of cell surface carbohydrate. The other, a larger protein, carries very little, if any, carbohydrate or sialic acid.
Article
A protein from the water-soluble fraction of bovine erythrocyte ghosts has been purified and characterized. This protein contains two high molecular weight polypeptide chains (MW ∼ 220,000 and 240,000); its physical properties suggest that it is a rod-like molecule. It makes up at least one-fifth of the total ghost protein.