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Effect of Cholesterol Depletion and Temperature on the Isolation of Detergent-Resistant Membranes from Human Erythrocytes

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Abstract

Transient lateral microdomains or lipid rafts play important roles in many physiological membrane-mediated cell processes. Detergent-resistant membranes (DRMs) are good models for the study of lipid rafts. Here we report that DRMs can be obtained by treating human erythrocytes with the nonionic detergents Triton X-100 or octaethylene glycol monododecyl ether (C12E8) at 37°C, and by treatment at 4°C of cholesterol-depleted erythrocytes. Electron paramagnetic resonance with spin labels inserted at different membrane depths (5- and 16-doxyl stearic acids, 5-SASL and 16-SASL) were used to measure the order parameter (S) of the cell membranes and DRMs. We previously reported significantly higher S values in DRMs with respect to intact erythrocyte membranes. Here we show that higher S values were still measurable in DRMs prepared from intact erythrocytes at 37°C, or from cholesterol-depleted cells at 4°C, for both detergents. For 5-SASL only, increased S values were measured in 4°C DRMs obtained from cholesterol-depleted versus intact erythrocytes. Flotillin-2, a protein marker of lipid rafts, was found in DRMs from intact cells in trace amounts but it was sensitively increased in C12E8 DRMs prepared at 4°C from cholesterol-depleted erythrocytes, while the membrane-skeletal proteins spectrin and actin were excluded from both Triton X-100 and C12E8 DRMs. However, contrary to the 4°C treatment results, flotillin-2 and stomatin were not resistant to Triton X-100 and C12E8 treatment at physiological temperature. The role of cholesterol in DRMs formation is discussed and the results presented provide further support for the use of C12E8 to the study of DRMs.

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Notes

  1. Free cholesterol in human red blood cells is 3.15 μmol/ml cells, while total lipid phosphorus is 3.90 μmol/ml cells (Dodge and Phillips 1967). Cholesterol is therefore present in the cell membrane at approximately 45 mol% with respect to total lipids. In DRMs from normal cells, we have 30% of the cell cholesterol, corresponding to approximately 0.945 μmol (per milliliter of cells). If we assume that the sphingolipid-enriched, detergent-resistant phase early described in the literature (before the notion of lipid rafts was introduced) contained what we now call DRMs (and can be released from the membrane skeleton only by treatment with carbonate), then the sphingomyelin in the detergent-resistant portion of the erythrocyte, obtained with Triton X-100 at concentrations comparable to those we used, corresponds approximately to 70–80% of the cell sphingomyelin (Yu et al. 1973; Sheetz 1979). Because the latter corresponds to 25 mol% of total cell lipid phosphorus (Dodge and Phillips 1967), it amounts to 1.0 μmol (per milliliter of cells), and its content in the DRMs should be approximately 0.7–0.8 μmol. Therefore, the cholesterol percentage with respect to the total of DRM lipids (i.e., sphingomyelin plus cholesterol, with the reasonable approximation that the other phospholipids are present in DRMs in much lower amount) is 54–57%.

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Acknowledgments

This work was supported by PRIN funds of the “Ministero dell’Università e della Ricerca,” Italy, and FAPESP (Proc. 09/00904-1), Brazil. CCD acknowledges the fellowships from CAPES (Proc. 3597/06-7) and CNPq (Proc. 141618/2005-1), Brazil.

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  1. Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, Cid. Univ. Zeferino Vaz s/n, C.P. 6109, CEP 13083-970, Campinas, SP, Brazil

    Cleyton C. Domingues, Bruna Renata Casadei & Eneida de Paula

  2. Dipartimento di Biochimica “A. Castellani”, Università di Pavia, via Agostino Bassi, 21, 27100, Pavia, Italy

    Annarita Ciana, Cesare Balduini & Giampaolo Minetti

  3. Dipartimento di Chimica Generale, Università di Pavia, via Taramelli, 12, 27100, Pavia, Italy

    Armando Buttafava

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  1. Cleyton C. Domingues
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  2. Annarita Ciana
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  3. Armando Buttafava
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  4. Bruna Renata Casadei
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Correspondence to Giampaolo Minetti.

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C. C. Domingues and A. Ciana have contributed equally to this article.

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Domingues, C.C., Ciana, A., Buttafava, A. et al. Effect of Cholesterol Depletion and Temperature on the Isolation of Detergent-Resistant Membranes from Human Erythrocytes. J Membrane Biol 234, 195–205 (2010). https://doi.org/10.1007/s00232-010-9246-5

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