Effect of temperature, different detergents and local anesthetics in the isolation of detergent-resistant membranes from human erythrocytes

Abstract

Objectives: To investigate the effects of temperature, various detergents and anesthetics on the isolation of detergent-resistant-membranes (DRMs) from human erythrocytes. Design and Methods: DRMs were isolated by ultracentrifugating detergent-treated whole RBCs in sucrose density gradients. Results: After incubation of cells at 37°C with either Triton X-100 or C12E8, it was possible to isolate DRMs with the same cholesterol content as DRMs prepared at 4°C, but with a strongly reduced content of total and of specific DRM marker proteins (stomatin, flotillin-2), indicating that physiological temperature facilitates solubilisation of membrane proteins while maintaining the resistance of cholesterol. Detergents ASB-14, ASB-16 and CHAPS used at the same concentration as Triton X-100 and C12E8 did not allow the isolation of DRMs from erythrocytes at 4°C. No significant changes in cholesterol and total protein contents were found in DRMs isolated from cells previously treated with lidocaine, prilocaine and bupivacaine and extracted by Triton X-100