Membrane phospholipid asymmetry in DMPC‐induced human red cell vesicles

Jong, Kitty de; Ott, Peter

doi:10.1016/0014-5793(93)81708-8

Cited by 11 publications

(9 citation statements)

References 41 publications

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“…Phosphatidylserine (PS) Exposure Assayed by Prothrombinase Activity-Exposure of PS was measured using a variation of the method of de Jong and Ott (29). Membrane preparations or erythrocytes were diluted to 3 ϫ 10 8 cells or ghosts/ml with either MBSS or hypotonic MBSS (10% MBSS diluted in water).…”

Section: Methodsmentioning

confidence: 99%

Physical Properties of Erythrocyte Ghosts That Determine Susceptibility to Secretory Phospholipase A2

Harris

Smith

Bell

2001

Journal of Biological Chemistry

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Artificial membranes may be resistant or susceptible to catalytic attack by secretory phospholipase A 2 (sPLA 2 ) depending on the physical properties of the membrane. Living cells are normally resistant but become susceptible during trauma, apoptosis, and/or a significant elevation of intracellular calcium. Intact erythrocytes and ghosts were studied to determine whether the principles learned from artificial systems apply to biological membranes. Membrane properties such as phospholipid and/or protein composition, morphology, and microscopic characteristics (e.g. fluidity) were manipulated by preparing ghosts under different experimental conditions such as in the presence or absence of divalent cations with or without ATP. The properties of each membrane preparation were assessed by biochemical and physical means (fluorescence spectroscopy and electron and two-photon microscopy using the membrane probes bis-pyrene and laurdan) and compared with sPLA 2 activity. The properties that appeared most relevant were the degree of phosphatidylserine exposure on the outer face of the membrane and changes to the membrane physical state detected by bis-pyrene and laurdan. Specifically, vulnerability to hydrolysis by sPLA 2 was associated with an increase in bilayer order apparently reflective of expansion of membrane regions of diminished fluidity. These results argue that the general principles identified from studies with artificial membranes apply to biological systems.

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Section: Methodsmentioning

confidence: 99%

Physical Properties of Erythrocyte Ghosts That Determine Susceptibility to Secretory Phospholipase A2

Harris

Smith

Bell

2001

Journal of Biological Chemistry

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“…Prothrombinase Assay. The prothrombinase assay, originally designed by Bevers et al (1982), was used to obtain semiquantitative information on the orientation of PS in cell membranes and was performed essentially as described earlier (De Jong & Ott, 1993). Briefly, intact oxidant-treated and control cells were resuspended at 4% hematocrit in PBS.…”

Section: Methodsmentioning

confidence: 99%

Oxidative Damage Does Not Alter Membrane Phospholipid Asymmetry in Human Erythrocytes

1997

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Oxidant-induced damage has been proposed to be the underlying mechanism for loss of membrane phospholipid asymmetry in the erythrocyte membrane. In sickle cell disease, thalassemia, and diabetes as well as in senescent erythrocytes, an apparent correlation between oxidative damage and loss of phosphatidylserine asymmetry has been reported. In the present study, erythrocytes were subjected to various levels of oxidative stress and/or sulfhydryl modifying agents. The transmembrane location of phosphatidylserine (PS) was assessed by FITC-conjugated annexin V labeling and the PS-dependent prothrombinase assay. Transbilayer movement of spin-labeled PS was used to determine aminophospholipid translocase activity. Our data show that cells did not expose PS as the result of oxidative stress induced by phenylhydrazine, hydrogen peroxide, tert-butyl hydroperoxide, cumene hydroperoxide, or sulfhydryl modification by N-ethylmaleimide (NEM) and diamide, even under conditions that led to severe cellular damage and impairment of aminophospholipid translocase activity. In contrast, the increase of intracellular calcium induced by treatment with calcium and ionophore A23187 leads to a rapid scrambling of the lipid bilayer and the exposure of PS, which can be exacerbated by the inhibition of aminophospholipid translocase activity. Oxidation of the cells with hydrogen peroxide or phenylhydrazine did not affect A23187-induced uptake of calcium, but partly inhibited calcium-induced membrane scrambling. In conclusion, oxidative damage of erythrocytes does not induce exposure of phosphatidylserine on the membrane surface, but can interfere with both aminophospholipid translocase activity and calcium-induced randomization of membrane phospholipids.

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“…Phosphatidylserine Exposure Assayed by Prothrombinase ActiVity. Exposure of phosphatidylserine was measured using a variation of the method of de Jong and Ott (22). Erythrocytes were diluted to 3 × 10 8 cells/ml in MBSS.…”

mentioning

confidence: 99%

Relationship between Erythrocyte Membrane Phase Properties and Susceptibility to Secretory Phospholipase A₂

et al. 2002

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Normally, cell membranes resist hydrolysis by secretory phospholipase A 2 . However, upon elevation of intracellular calcium, the cells become susceptible. Previous investigations demonstrated a possible relationship between changes in lipid order caused by increased calcium and susceptibility to phospholipase A 2 . To further explore this relationship, we used temperature as an experimental means of manipulating membrane physical properties. We then compared the response of human erythrocytes to calcium ionophore at various temperatures in the range of 20-50 °C using fluorescence spectroscopy and two-photon fluorescence microscopy. The steady state fluorescence emission of the environmentsensitive probe, laurdan, revealed that erythrocyte membrane order decreases systematically with temperature throughout this range, especially between 28 and 45 °C. Furthermore, the ability of calcium ionophore to induce increased membrane order and susceptibility to phospholipase A 2 depended similarly on temperature. Both responses to calcium influx were enhanced as membrane fluidity increased. Analysis of the spatial distribution of laurdan fluorescence at several temperatures indicated that the ordering effect of intracellular calcium on fluid membranes generates an increase in the number of fluid-solid boundaries. Hydrolysis of the membrane appeared to progress outward from these boundaries. We conclude that phospholipase A 2 prefers to hydrolyze lipids in fluid regions of human erythrocyte membranes, but primarily when those regions coexist with domains of ordered lipids.

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Membrane phospholipid asymmetry in DMPC‐induced human red cell vesicles

Cited by 11 publications

References 41 publications

Physical Properties of Erythrocyte Ghosts That Determine Susceptibility to Secretory Phospholipase A2

Physical Properties of Erythrocyte Ghosts That Determine Susceptibility to Secretory Phospholipase A2

Oxidative Damage Does Not Alter Membrane Phospholipid Asymmetry in Human Erythrocytes

Relationship between Erythrocyte Membrane Phase Properties and Susceptibility to Secretory Phospholipase A₂

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Membrane phospholipid asymmetry in DMPC‐induced human red cell vesicles

Cited by 11 publications

References 41 publications

Physical Properties of Erythrocyte Ghosts That Determine Susceptibility to Secretory Phospholipase A2

Physical Properties of Erythrocyte Ghosts That Determine Susceptibility to Secretory Phospholipase A2

Oxidative Damage Does Not Alter Membrane Phospholipid Asymmetry in Human Erythrocytes

Relationship between Erythrocyte Membrane Phase Properties and Susceptibility to Secretory Phospholipase A2

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Relationship between Erythrocyte Membrane Phase Properties and Susceptibility to Secretory Phospholipase A₂