TRANSIENT HOLES IN THE ERYTHROCYTE MEMBRANE DURING HYPOTONIC HEMOLYSIS AND STABLE HOLES IN THE MEMBRANE AFTER LYSIS BY SAPONIN AND LYSOLECITHIN

Ferritin and colloidal gold were found to permeate human erythrocytes during rapid or gradual hypotonic hemolysis. Only hemolysed cells contained these particles; adjacent intact cells did not contain the tracers. Ferritin or gold added 3 min after the onset of hypotonic hemolysis did not permeate the ghost cells which had, therefore, become transiently permeable. By adding ferritin at various times after the onset of hemolysis, it was determined that for the majority of the cells the permeable state (or interval between the time of development and closure of membrane holes) existed only from about 15 to 25 sec after the onset of hemolysis. It was possible to fix the transient "holes" in the open position by adding glutaraldehyde only between 10 and 20 sec after the onset of hemolysis. The existence of such fixed holes was shown by the cell entry of ferritin and gold which were added to these prefixed cells. Membrane defects or discontinuities (of the order of 200–500 A wide) were observed only in prefixed cells which were permeated by ferritin subsequently added. Adjacent prefixed cells which did not become permeated by added ferritin did not reveal any membrane discontinuities. Glutaraldehyde does not per se induce or create such membrane defects since cells which had been fixed by glutaraldehyde before the 10-sec time point or after the 180-sec time point were never permeable to added ferritin, and the cell membranes never contained any defects. It was also observed that early in hemolysis (7–12 sec) a small bulge in one zone of the membrane often occurred. Ghost cells produced by holothurin A (a saponin) and fixed by glutaraldehyde became permeated by ferritin subsequently added, but no membrane discontinuities were seen. Ghosts produced by lysolecithin and fixed by glutaraldehyde also became permeated by subsequently added ferritin, and many membrane defects were seen here (about 300 A wide).

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STRUCTURE OF MEMBRANE HOLES IN OSMOTIC AND SAPONIN HEMOLYSIS

The Journal of Cell Biology ◽

10.1083/jcb.56.2.519 ◽

1973 ◽

Vol 56 (2) ◽

pp. 519-527 ◽

Cited By ~ 121

Author(s):

P. Seeman ◽

D. Cheng ◽

G. H. Iles

Keyword(s):

Electron Microscopy ◽

Erythrocyte Membrane ◽

Serial Section ◽

Colloidal Gold ◽

Osmotic Hemolysis ◽

Section Electron ◽

Membrane Defects ◽

Serial Section Electron Microscopy ◽

Section Electron Microscopy

Serial section electron microscopy of hemolysing erythrocytes (fixed at 12 s after the onset of osmotic hemolysis) revealed long slits and holes in the membrane, extending to around 1 µm in length. Many but not all of the slits and holes (about 100–1000 Å wide) were confluent with one another. Ferritin and colloidal gold (added after fixation) only permeated those cells containing membrane defects. No such large holes or slits were seen in saponin-treated erythrocytes, and the membrane was highly invaginated, giving the ghost a scalloped outline. Freeze-etch electron microscopy of saponin-treated membranes revealed 40–50 Å-wide pits in the extracellular surface of the membrane. If these pits represent regions from which cholesterol was extracted, then cholesterol is uniformly distributed over the entire erythrocyte membrane.

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Phospholipid phosphorylation in erythrocyte of spontaneously hypertensive rats

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1982.243.4.h590 ◽

1982 ◽

Vol 243 (4) ◽

pp. H590-H597 ◽

Cited By ~ 2

Author(s):

S. Koutouzov ◽

P. Marche ◽

J. F. Cloix ◽

P. Meyer

Keyword(s):

Erythrocyte Membrane ◽

Spontaneously Hypertensive Rat ◽

Intact Cells ◽

Membrane Function ◽

Spontaneously Hypertensive ◽

Phosphatidylinositol Kinase ◽

Inositol Lipids ◽

Wistar Kyoto Rat ◽

Series Of Experiments ◽

Wistar Kyoto

The rapid turnover of phosphoinositides within membranes suggests that these lipids play an important role in membrane function. Since various abnormalities have been described in the erythrocyte membrane of the spontaneously hypertensive rat (SHR) we have studied the turnover of phosphoinositides in the erythrocyte of SHR and age-matched normotensive Wistar-Kyoto rat (WKY). This was achieved by measuring the incorporation of 32P into inositol lipids after incubation of 1) intact erythrocytes with [32P]orthophosphate and 2) isolated ghost membranes with [gamma-32P]ATP. In both series of experiments more than 99% of the radioactivity incorporated into lipids was into the polyphosphoinositides diphosphoinositide (DPI) and triphosphoinositide (TPI). In both intact erythrocytes and ghost membranes, the levels of 32P incorporated into DPI and TPI were significantly different in SHR than in WKY. Further analysis of factors known to influence the labeling of DPI and TPI indicated that this could be ascribed to decreased activities of phosphatidylinositol kinase and/or DPI kinase, with respect to ATP as substrate. Moreover comparison of data obtained in intact cells with those obtained with ghost membranes suggests that within the SHR erythrocyte, membrane-cytosol interactions may occur that could also be responsible for the alteration of phosphoinositide labeling observed in hypertensive animals. Since phosphoinositides have been reported to be involved in the Ca2+-gating system of membrane, our findings could be associated with the abnormal Ca2+ binding and transport recently described in SHR erythrocyte.

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Erythrocyte membrane fluidity in adult polycystic kidney disease: difference between intact cells and ghost membranes

European Journal of Clinical Investigation ◽

10.1046/j.1365-2362.1996.121259.x ◽

1996 ◽

Vol 26 (2) ◽

pp. 171-173 ◽

Cited By ~ 10

Author(s):

K. VAREESANGTHIP ◽

T. H. THOMAS ◽

P. TONG ◽

R. WILKINSON

Keyword(s):

Kidney Disease ◽

Membrane Fluidity ◽

Polycystic Kidney Disease ◽

Erythrocyte Membrane ◽

Polycystic Kidney ◽

Adult Polycystic Kidney Disease ◽

Intact Cells ◽

Erythrocyte Membrane Fluidity

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Erythrocyte membrane defects in hemolytic anemias found through derivative thermal analysis of electric impedance

Journal of Biochemical and Biophysical Methods ◽

10.1016/j.jbbm.2007.02.004 ◽

2007 ◽

Vol 70 (4) ◽

pp. 641-648 ◽

Cited By ~ 3

Author(s):

I.T. Ivanov ◽

A. Tolekova ◽

P. Chakaarova

Keyword(s):

Thermal Analysis ◽

Erythrocyte Membrane ◽

Electric Impedance ◽

Membrane Defects

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Effect of membrane splitting on transmembrane polypeptides.

The Journal of Cell Biology ◽

10.1083/jcb.102.2.551 ◽

1986 ◽

Vol 102 (2) ◽

pp. 551-559 ◽

Cited By ~ 12

Author(s):

K A Fisher ◽

K C Yanagimoto

Keyword(s):

Erythrocyte Membrane ◽

Periodic Acid ◽

Freeze Fracture ◽

Anion Channel ◽

Band 3 ◽

Intact Cells ◽

Coomassie Blue ◽

Sds Page ◽

Before And After ◽

Polypeptide Backbone

We investigated the effect of membrane splitting on the primary structure of human erythrocyte membrane polypeptides. Monolayers of intact, chemically unmodified cells were freeze-fractured and examined by one-dimensional SDS PAGE. Silver-stained gels revealed all major polypeptides that stain with Coomassie Blue as well as all bands that stain with periodic acid Schiff's reagent. Both nonglycosylated and glycosylated membrane polypeptides could be detected at concentrations of only a few nanograms per band. Membrane splitting had no effect on the position or number of bands. Monolayers of intact erythrocytes that had been enzymatically radioiodinated with lactoperoxidase were examined by electrophoresis, fluorography, and liquid scintillation counting. Radioactivity was quantified before and after monolayer formation and splitting, and at several stages of gel staining, drying, and fluorography. Although overexposed fluorographs revealed several minor radioiodinated bands in addition to band 3 and the glycophorins, no new bands were detected in split membrane samples derived from intact cells. These observations support the conclusion that neither the band 3 anion channel nor the glycophorin sialoglycoproteins are fragmented during freeze-fracturing. Although both band 3 and glycophorin partition to the cytoplasmic side of the membrane, preliminary quantitative observations suggest an enrichment of glycophorin in the split extracellular "half" membrane. We conclude that the process of membrane splitting by planar monolayer freeze-fracture does not cleave the covalent polypeptide backbone of any erythrocyte membrane protein, peripheral or integral.

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Injury-related phaseollin accumulation in Phaseolus vulgaris and its implications with regard to specificity of host–parasite interaction

Canadian Journal of Botany ◽

10.1139/b75-109 ◽

1975 ◽

Vol 53 (9) ◽

pp. 921-928 ◽

Cited By ~ 30

Author(s):

James E. Rahe ◽

Robert M. Arnold

Keyword(s):

Phaseolus Vulgaris ◽

Colletotrichum Lindemuthianum ◽

Living Tissue ◽

Cotyledonary Nodes ◽

Etiolated Seedlings ◽

Parasite Specificity ◽

Per Se ◽

Freezing Injuries ◽

Host Parasite ◽

Time Point

Phaseollin accumulated locally at point-freezing injuries on hypocotyls of intact etiolated seedlings of Phaseolus vulgaris. Maximum amounts occurred within 24 to 30 h after injury. Smaller amounts accumulated at similar sites on hypocotyls excised at the time point-freezing injuries were made, and the accumulation was less localized. Increasing amounts of phaseollin occurred at sites increasingly distant from the cotyledonary nodes in both intact and excised hypocotyls. Much higher levels of phaseollin were elicited by excision per se than by point-freezing. Phaseollin was not detected after freezing of whole hypocotyls, indicating that living tissue adjacent to injuries is required for accumulation. The data are discussed in relation to host–parasite specificity, with particular reference to the interaction between P. vulgaris and Colletotrichum lindemuthianum.

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The Active Transport of Sodium by Ghosts of Human Red Blood Cells

The Journal of General Physiology ◽

10.1085/jgp.45.5.837 ◽

1962 ◽

Vol 45 (5) ◽

pp. 837-859 ◽

Cited By ~ 140

Author(s):

Joseph F. Hoffman

Keyword(s):

Active Transport ◽

Blood Cells ◽

Total Population ◽

Passive Transport ◽

Intact Cells ◽

Exchange Diffusion ◽

Human Red Blood Cells ◽

Hypotonic Hemolysis ◽

Active Transport System ◽

External K

The outflux of Na24 from prelabeled ghosts was measured under various conditions. Prelabeling was accomplished by hypotonic hemolysis of intact human cells in the presence of tracer Na24. The resultant ghosts when subsequently washed were found to retain 10 to 20 per cent of the initial Na24. Separate experiments indicated that this trapped amount resides in only a portion of ghosts comprising the total population. The characteristics of the outflux of this residual Na24 indicated that the ghost system closely resembles intact red cells. The outflux of Na from ghosts could be divided into three components: active and passive transport and exchange diffusion. The active transport system, necessarily driven by metabolism, required the presence of K in the extracellular phase and was blocked by strophanthidin. The concentration dependence of the Na pump flux on the external K and internal Na appeared the same in ghosts as in intact cells. Certain other features of this ghost system are also discussed.

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Characterization of Glycolytic Enzyme Complexes on Murine Erythrocyte Membranes.

Blood ◽

10.1182/blood.v104.11.1571.1571 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1571-1571

Author(s):

Philip S. Low ◽

Estela M. Campanella ◽

William A. Anong ◽

Nancy J. Wandersee ◽

Cheryl A. Hillary ◽

...

Keyword(s):

Confocal Microscopy ◽

Erythrocyte Membrane ◽

Band 3 ◽

Glycolytic Enzyme ◽

Glycolytic Enzymes ◽

Erythrocyte Membranes ◽

Dependent Manner ◽

Intact Cells ◽

Anion Transporter ◽

Enzyme Complexes

Abstract Glycolytic enzymes have been recently shown to exist as multi-enzyme complexes in association with the cytoplasmic domain of band 3 at the inner surface of the human erythrocyte membrane. Because several of the glycolytic enzyme binding sites have been mapped to sequences near the NH2-terminus of band 3 (DDYED and EEYED) that are not conserved in mice (EEVLE and EELEN), the question naturally arose whether the existence of glycolytic enzyme complexes on erythrocyte membranes might be only a product of recent evolution. To test this hypothesis, fresh murine erythrocytes were fixed and stained with antibodies to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH) and carbonic anhydrase II (CA II was used as a control, since it binds to a distant site near the COOH-terminus of band 3). Importantly, analysis of intact murine erythrocytes by confocal microscopy demonstrated that all of the above enzymes are localized to the membrane in oxygenated cells. In contrast, upon deoxygenation of the intact cells, release of the glycolytic enzymes (but not CA II) from the erythrocyte membrane and their uniform redistribution throughout the cytoplasm is observed. Because deoxyhemoglobin has been shown in human erythrocytes to compete with glycolytic enzymes (but not with CA II) for a common binding site at the NH2-terminus of band 3, these data argue that murine band 3, despite its weak homology to human band 3, still constitutes an organization center for glycolytic enzymes on the erythrocyte membrane. To further test this hypothesis, erythrocytes from band 3 knockout mice were similarly examined by confocal microscopy. Not surprisingly, all of the enzymes in all of the cells were evenly distributed throughout the cytoplasm, regardless of the oxygenation state of the cell. Further, immunoblot analyses demonstrated that glycolytic enzyme content of the band 3 knockout erythrocytes was measurably reduced compared to healthy mice, suggesting that the anion transporter may also contribute to enzyme stabilization during the lifetime of the erythrocyte. Finally, to determine whether the integrity of other membrane structures might impact the assembly of glycolytic enzyme complexes on the erythrocyte membrane, α-spectrin deficient mice were also examined for their enzyme distributions. Curiously, > 50% of the cells in any field exhibited glycolytic enzyme staining throughout the cytoplasm, with the remainder showing mainly membrane staining. Conceivably, the stabiity of glycolytic enzyme complexes on the membrane may also depend on the integrity of the membrane skeleton. Taken together, these data argue that glycolytic enzymes assemble in an oxygenation-dependent manner into complexes on murine erythrocyte membranes and that the stability of these complexes depends on the presence of band 3 and to a lesser extent α-spectrin. Supported by NIH grant GM24417.

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