Factors controlling the resealing of the membrane of human erythrocyte ghosts after hypotonic hemolysis

Bodemann, H. H.; Passow, Hermann

doi:10.1007/bf01868092

Cited by 278 publications

(105 citation statements)

References 11 publications

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“…This fact justifies the use of ghosts to help access a basis for the red blood cell's biconcave shape. The procedure used to make ghosts was principally that described by Bodemann and Passow (13) and Lepke and Passow (14). Details of the procedures used are given in Materials and Methods.…”

Section: Resultsmentioning

confidence: 99%

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Biconcave shape of human red-blood-cell ghosts relies on density differences between the rim and dimple of the ghost's plasma membrane

Hoffman

2016

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

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The shape of the human red blood cell is known to be a biconcave disk. It is evident from a variety of theoretical work that known physical properties of the membrane, such as its bending energy and elasticity, can explain the red-blood-cell biconcave shape as well as other shapes that red blood cells assume. But these analyses do not provide information on the underlying molecular causes. This paper describes experiments that attempt to identify some of the underlying determinates of cell shape. To this end, red-blood-cell ghosts were made by hypotonic hemolysis and then reconstituted such that they were smooth spheres in hypo-osmotic solutions and smooth biconcave discs in iso-osmotic solutions. The spherical ghosts were centrifuged onto a coated coverslip upon which they adhered. When the attached spheres were changed to biconcave discs by flushing with an iso-osmotic solution, the ghosts were observed to be mainly oriented in a flat alignment on the coverslip. This was interpreted to mean that, during centrifugation, the spherical ghosts were oriented by a dense band in its equatorial plane, parallel to the centrifugal field. This appears to be evidence that the difference in the densities between the rim and the dimple regions of red blood cells and their ghosts may be responsible for their biconcave shape.red-blood-cell ghosts | membrane/cytoskeletal complex | biconcave discs | spheres T his paper is concerned with identifying a possible determinant responsible for the biconcave shape of human red blood cells (RBCs). Although RBCs ("globules") were first discovered in the latter part of the 17th century (1), it was not until 1827 that they were definitively shown by Hodgkin and Lister (2) to be biconcave discs. Since that time much has been learned about RBC composition (3), in particular identification of its membrane/cytoskeletal (M/CS) elements and structure (4). Our particular interest focuses on whether the symmetry of the M/CS is the same or different between the cell's dimple and rim regions. If different, such an observation might provide insight into the structural basis for the cell's biconcave shape. The approach used was to centrifuge hypotonically sphered red-blood-cell ghosts onto a coverslip upon which they adhered. The question was, what was the orientation of the stuck ghosts when they were made into biconcave discs upon exposure to an isotonic solution?It is important to mention that others have used a quite different approach to understand the basis for the red blood cell's biconcave shape. These types of studies have used solutions of theoretical models that are mainly based on the red blood cell's known physical parameters, such as the membrane's bending energy and elastic properties (e.g., 5-11) in addition to lateral inhomogeneities in the M/CS (12). These analyses have remarkably generated not only the cell's biconcave shape but also many other types of shapes that the cells are known to assume. Thus, they provide insight into and understanding of the types of forces that characteri...

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Section: Resultsmentioning

confidence: 99%

“…After testing various procedures that we previously used for the preparation of ghosts, we settled on those described by Bodemann and Passow (13) and Lepke and Passow (14). These methods, with modifications, provided ghosts the shapes of which were best preserved and could be manipulated as desired as described in the text.…”

Section: Methodsmentioning

confidence: 99%

Biconcave shape of human red-blood-cell ghosts relies on density differences between the rim and dimple of the ghost's plasma membrane

Hoffman

2016

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

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“…Thus, Ca 2ϩ at physiological concentrations (1.0-2.0 mM) is known to inhibit recovery of K ϩ impermeability in RBC ghosts produced by hyposmotic lysis (15). Paradoxically, Ca 2ϩ is also demonstrated to increase the final degree (14,25) and rate (26,27) of resealing of osmotically lysed RBCs, when present at subphysiological concentrations (0.005-0.05 mM). We did not assess resealing in laser-wounded RBCs as a function of Ca 2ϩ concentration.…”

Section: Discussionmentioning

confidence: 99%

The endomembrane requirement for cell surface repair

McNeil

Miyake

Vogel

2003

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

136

107

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The capacity to reseal a plasma membrane disruption rapidly is required for cell survival in many physiological environments. Intracellular membrane (endomembrane) is thought to play a central role in the rapid resealing response. We here directly compare the resealing response of a cell that lacks endomembrane, the red blood cell, with that of several nucleated cells possessing an abundant endomembrane compartment. RBC membrane disruptions inflicted by a mode-locked Ti:sapphire laser, even those initially smaller than hemoglobin, failed to reseal rapidly. By contrast, much larger laser-induced disruptions made in sea urchin eggs, fibroblasts, and neurons exhibited rapid, Ca 2؉ -dependent resealing. We conclude that rapid resealing is not mediated by simple physiochemical mechanisms; endomembrane is required.

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“…A certain fraction of the ghost population is capable of resealing, whereas the rest of the ghosts remain leaky. Bodemann and Passow [37] and Schwoch and Passow [38] have distinguished between three types of ghosts, one of these (called Type II) representing a ghost population which reseals after reversion of the haemolysis procedure by addition of alkali ions. It needs no argument that for studying phospholipase action at the inside of resealed ghosts, the fraction of Type II ghosts should be as high as possible.…”

Section: Phospholipase a 2 Action Within Resealed 9hostsmentioning

confidence: 99%

Organization of phospholipids in human red cell membranes as detected by the action of various purified phospholipases

Zwaal

Roelofsen

Comfurius

et al. 1975

Biochimica et Biophysica Acta (BBA) - Biomembranes

350

119

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SUMMARY1. The action of eight purified phospholipases on intact human erythrocytes has been investigated. Four enzymes, e.g. phospholipases A2 from pancreas and Crotalus adamanteus, phospholipase C from Bacillus cereus, and phospholipase D from cabbage produce neither haemolysis nor hydrolysis of phospholipids in intact cells. On the other hand, both phospholipases A2 from bee venom and Naja naja cause a non-haemolytic breakdown of more than 50 ~ of the lecithin, while sphingomyelinase C from Staphylococcus aureus is able to produce a non-lytic degradation of more than 80 ~o of the sphingomyelin.2. Phospholipase C from Clostridium welchii appeared to be the only lipolytic enzyme tested, which produces haemolysis of human erythrocytes. Evidence is presented that the unique properties of the enzyme itself, rather than possible contaminations in the purified preparation, are responsible for the observed haemolytic effect.3. With non-sealed ghosts, all phospholipases produce essentially complete breakdown of those phospholipids which can be considered as proper substrates for the enzymes involved.4. Due to its absolute requirement for Ca 2 ÷, pancreatic phospholipase A2 can be trapped inside resealed ghosts in the presence of EDTA, without producing phospholipid breakdown during the resealing procedure. Subsequent addition of Ca 2 ÷ stimulates phospholipase A z activity at the inside of the resealed cell, eventually leading to lysis. Before lysis occurs, however, 25 ~o of the lecithin, half of the phosphatidylethanolamine and some 65 ~ of the phosphatidylserine can be hydrolysed. This observation is explained in relation to an asymmetric phospholipid distribution in red cell membranes.Phospholipases used: phospholipase A2 (phosphatide acyl-hydrolyse, EC 3.1.1.4) from pig pancreas, bee venom, Naja naja, and Crotalus adamanteus, respectively. Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Bacillus cereus and Clostridium welchii. Sphingomyelinase C (sphingomyelin cholinephosphohydrolase) from Staphylococcus aureus. Phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) from Savoy cabbage. 84 5. It is concluded that the ability of the various phospholipases to attack the red cell membrane is dependent upon: (i) substrate specificity of the phospholipases, (ii) sidedness of the phospholipids when only one side of the membrane is exposed to phospholipase action, and (iii) compression state of the membranous lipid layer. The latter point is dealt with in more detail in the accompanying paper (Demel, R.

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Factors controlling the resealing of the membrane of human erythrocyte ghosts after hypotonic hemolysis

Cited by 278 publications

References 11 publications

Biconcave shape of human red-blood-cell ghosts relies on density differences between the rim and dimple of the ghost's plasma membrane

Biconcave shape of human red-blood-cell ghosts relies on density differences between the rim and dimple of the ghost's plasma membrane

The endomembrane requirement for cell surface repair

Organization of phospholipids in human red cell membranes as detected by the action of various purified phospholipases

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