Glycophorin in Lipid Bilayers

Grant, Chris W.M.; McConnell, Harden M.

doi:10.1073/pnas.71.12.4653

Cited by 147 publications

(35 citation statements)

References 32 publications

(31 reference statements)

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“…Reconstitution experiments showed also that membrane particles could only be observed in systems which successfully recombined an integral membrane protein into a lamellar lipid phase (26)(27)(28)(29)(30) In some membranes, the particles have been shown to be involved in transmembrane phenomena (23,25,41,42). Should it also be the case in myelin, the membrane particles and/or the particles of the radial component would provide functional shortcuts across the myelin sheath.…”

Section: Resultsmentioning

confidence: 99%

Membrane particles on fracture faces of frozen myelin.

Silva¹,

Miller²

1975

Proc. Natl. Acad. Sci. U.S.A.

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Freeze-fracture electron microscopy reveals constant and widespread presence of membrane particles on the fracture faces of frozen myelin. In unfixed myelin frozen shortly after dissection the distribution of the particles is uniform. In glutaraldehyde-fixed and/or glycerol-impregnated myelin the particles frequently occupy a network interspersed with circumscribed particle-free ateas of variable dimension. In these membranes the network of particles is propagated radially across many lamellae. Particle-rich areas are closely apposed and contrast with frequent delamination of adjacent particle-free regions. We propose that, as in other plasma membranes, the particles of myelin represent protein-containing structures intercalated across the hydrophobic matrix of a membrane with bilayer organization. Our results indicate that these structures contain sites which mutually interact at the surface of apposed membranes and may be important in maintaining the organizational integrity of myelin. Freeze-fracture splits the lipid bilayer regions of the membrane and allows electron microscopic examination of hydrophobic membrane domains and the detection of sites (membrane-intercalated particles) where the bilayer is interrupted (1). The single exception to the presence of particles in plasma membranes was reported by Branton in a study of the fracture faces of frozen myelin (2). These were reported as being totally free of particles, and their featureless appearance made them virtually undistinguishable from the fracture faces observed in lamellar phase, unsaturated phospholipids (3). Interpretation of the x-ray diffraction patterns of myelin supported also the assumption of an uninterrupted bilayer (4) and, although admitting the possibility of small amounts of protein extending across it, explained asymmetries of the electron density profile as the result of an asymmetric lipid distribution (4). These electron microscopic and x-ray diffraction interpretations were consistent with the accepted function of myelin, because they provided a sheath of high electrical resistance which insulated the axon (5, 6). Anatomically, however, electron microscopic examination of myelin from central and/or peripheral nervous systems revealed structural complexities not expected from simple multilayer systems: (a) paranodal myelin/axon and myelin/ myelin specializations (7) MATERIALS AND METHODSRat (Lewis; 12-15 weeks) sciatic and optic nerves were dissected from sodium-pentobarbital-anesthetized animals and fixed immediately in 2% glutaraldehyde (Fisher Scientific Co., Pittsburgh, Pa.) in phosphate-buffered saline at pH 7.3 for 1 hr at 37'. Frog (Rana pipiens) sciatic nerves were dissected from pithed animals and fixed as above. Garfish (Lepisosteus osseus) olfactory and IV nerves were dissected from decapitated fish and fixed in 2% glutaraldehyde in Ringer's solution (12). After fixation the material was gradually impregnated by dropwise addition of 25% glycerol in phosphate-buffered saline or Ringer's solution over a period o...

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Section: Resultsmentioning

confidence: 99%

Membrane particles on fracture faces of frozen myelin.

Silva¹,

Miller²

1975

Proc. Natl. Acad. Sci. U.S.A.

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“…Electroinsertion effi-ciency increased with the temperature. In lipid layers, the glycophorin molecules have a tendency to occupy fluid regions of the bilayer (Grant and McConnell, 1974). By lowering the temperature, the membrane fluidity decreases and counteracts protein incorporation efficiency.…”

Section: Discussionmentioning

confidence: 99%

Electropermeabilization mediates a stable insertion of glycophorin A with Chinese hamster ovary cell membranes

Ouagari

Benoist

Sixou

et al. 1994

European Journal of Biochemistry

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Electropulsation allowed us to incorporate glycophorin A, an integral membrane protein, into mammalian nucleated cell membranes (Chinese hamster ovary cells). The induction of stable protein association is effective only when the field intensity is higher than its threshold value, creating membrane permeabilization to small molecules. Under controlled conditions, cell viability was only slightly altered by this treatment. Pulse number and duration controlled both the number of modified cells and incorporated molecules. The phenomena was temperature dependent. An average of 5 X104 moleculeskell was bound. About 80% of cells in the pulsed population were observed to incorporate glycophorin. The protein incorporation was shown to be stable 48 h after electroassociation. Electrically bound proteins were shared between the cells after each division. As enhanced binding is detected if glycophorin is added after the pulses, it is the long-lived alteration of the membrane mediated by the pulses which supports the association.It is commonly accepted that the driving force for the spontaneous incorporation of proteins is hydrophobic effect. However, a number of other effects may support or counteract this hydrophobic effect. Generally, the free energy of protein incorporation includes four contributions which arise from a change of water structure, protein state, lipid state, bonds between protein and water or lipid molecules. Nevertheless, the central questions of how hydrophobic sequences would be transferred from an aqueous to an hydrophobic environment and how the polar regions of protein that span the membrane would be transferred across the hydrocarbon core of the bilayer, have not been emphasized. So, this makes it exceedingly unlikely that insertion of integral protein occurs directly across the lipid bilayer (Singer, 1990;Singer and Yaffe, 1990; Singer et al., 1987a, b). The membrane contribution, which has been neglected until now, necessarily appears important allowing the penetration of macromolecules with strongly polar segments. Thermodynamics of protein insertion in lipid systems imply the occurrence of the lipid matrix perturbation. Thus, protein incorporation thermodynamics ( J i m and Zakim, 1987;Jahnig, 1983) show a favourable contribution of electrostatic forces and hydrophobic forces which occur on the membrane-spanning sequence, whereas hydration forces (Pargesian and Rau, 1984) and hydrophobic forces which occur on the protein polar segments prevent incorporation. Spontaneous insertion takes place if the first component is stronger than the second one. Such a case would occur only if the membrane is in an 'excited' state.Electropulsation can create such an 'excited' state of the membrane. Recent studies reported the so-called 'electroinCorrespondence to J. Teissie,

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“…Both are transmembrane polypeptides, but direct identification of the particles with either has been difficult. Reconstitution experiments indicate that purified glycophorin (30) or aggregates of the hydrophobic peptide of glycophorin (31) can give the otherwise smooth fracture faces of liposomes a rough, bumpy, or rugose character. On the other hand, our experiments show that Band 3 alone can reconstitute the appearance of discrete particles indistinguishable from most of those in the erythrocyte membrane.…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of intramembrane particles in recombinants of erythrocyte protein band 3 and lipid: effects of spectrin-actin association.

Yu¹,

Branton²

1976

Proc. Natl. Acad. Sci. U.S.A.

157

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The integral membrane protein Band 3 of the human erythrocyte, either purified or in a crude Triton X-100 extract of ghosts, was combined with egg lecithin in a cholate solution. During dialysis to remove cholate, lipid bilayer vesicles formed in which Band 3 existed as a dimer and in which intramembrane particles indistinguishable from those in the native membrane were exposed by freeze-fracturing. The recombinant vesicles were stable in both high and low salt concentrations, sedimented at a density that increased in proportion to their protein content, and bound spectrin-actin extracted from erythrocyte ghosts. When spectrin-actin was associated with the vesicles, the behavior of the recombinant intramembrane particles simulated that of the erythrocyte ghost intramembrane particles: they were dispersed at pH 7.6 and aggregated at pH 5-5.5. Thus, some of the characteristics of the native membrane have been reconstituted in the recombinant.Band 3* is one of the major transmembrane polypeptides of the human erythrocyte (2, 3). It may be involved in facilitated anion movements (4, 5) and glucose transport (6-8), and its interactions in and at the surface of the erythrocyte plasma membrane may be the basis of important structural and rheological properties. Its binding characteristics may affect the distribution of other membrane proteins (9, 10); its transmembrane disposition suggests that it may contribute to the appearance of intramembrane particles (2, 11, 12); and its possible interaction with spectrin and erythrocyte actin may influence cell shape, elastic properties, and surface topology (13-15). Now that methods for purifying the Band 3 polypeptides in an apparently undenatured form are available (3, 16), direct verification and analysis of these many functional attributes should be possible.Previous work in this laboratory has implied an interaction between the integral transmembrane protein(s) responsible for the intramembrane particles of the human erythrocyte and the peripheral proteins spectrin and actin. Reasoning that Band 3 polypeptides may form the intramembrane particles, we have inserted purified or partially purified Band 3 into the lipid bilayers of egg yolk lecithin liposomes and evaluated integralperipheral protein interaction by observing particle distribution patterns in the protein-lipid recombinants. Our results demonstrate that Band 3 may form many of the intramembrane particles of the native membrane, show that the Band 3-lipid recombinants can bind spectrin and erythrocyte actin, and suggest that some of the controls that regulate intramembrane particle distribution in the erythrocyte membrane can be reconstituted by binding spectrin-actin to the recombinant. Integral membrane proteins were solubilized and partially purified by incubating a sample of packed ghosts in an equal volume of 1.5% Triton X-100 in 5 mM sodium phosphate buffer, pH 8.0, as previously described (19). The concentration of Triton X-100 in the resultant protein solution was then lowered to its critical micel...

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Glycophorin in Lipid Bilayers

Cited by 147 publications

References 32 publications

Membrane particles on fracture faces of frozen myelin.

Membrane particles on fracture faces of frozen myelin.

Electropermeabilization mediates a stable insertion of glycophorin A with Chinese hamster ovary cell membranes

Reconstitution of intramembrane particles in recombinants of erythrocyte protein band 3 and lipid: effects of spectrin-actin association.

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