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

Author(s)

Domingues, Cleyton C. ^[1] ; Ciana, Annarita ^[2] ; Buttafava, Armando ^[3] ; Casadei, Bruna Renata ^[1] ; Balduini, Cesare ^[2] ; de Paula, Eneida ^[1] ; Minetti, Giampaolo ^[2]

Affiliation

^[1] Univ Estadual Campinas, Dept Bioquim, Inst Biol, BR-13083970 Campinas, SP - Brazil

^[2] Univ Pavia, Dipartimento Biochim A Castellani, I-27100 Pavia - Italy

^[3] Univ Pavia, Dipartimento Chim Gen, I-27100 Pavia - Italy

• Document type: Journal article
• Source: Journal of Membrane Biology; v. 234, n. 3, p. 195-205, APR 2010.
• Web of Science Citations: 18

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 (C(12)E(8)) at 37A degrees C, and by treatment at 4A degrees 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 37A degrees C, or from cholesterol-depleted cells at 4A degrees C, for both detergents. For 5-SASL only, increased S values were measured in 4A degrees 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 C(12)E(8) DRMs prepared at 4A degrees C from cholesterol-depleted erythrocytes, while the membrane-skeletal proteins spectrin and actin were excluded from both Triton X-100 and C(12)E(8) DRMs. However, contrary to the 4A degrees C treatment results, flotillin-2 and stomatin were not resistant to Triton X-100 and C(12)E(8) treatment at physiological temperature. The role of cholesterol in DRMs formation is discussed and the results presented provide further support for the use of C(12)E(8) to the study of DRMs. (AU)

• FAPESP's process: 09/00904-1 - Effect of different surfactants and protocol changes (temperature and ionic strength changes, cholesterol depletion and local anesthetics) in detergent resistant membranes of erythrocytes
• Grantee: Eneida de Paula
• Support Opportunities: Regular Research Grants