Influence of cytoskeleton and temperature on the interaction between detergents and the erythrocyte membrane

V.M. TRUCCO,; P. M. RODI; A. M. GENNARO

Buzios, Brasil

Congreso; VII Iberoamerican Congress of Biophysics, Buzios, Brasil, 30/9 al 3/10/09; 2009

Sociedade Brasileira de Biofisica

<!-- /* Font Definitions */ @font-face {font-family:"Comic Sans MS"; panose-1:3 15 7 2 3 3 2 2 2 4; mso-font-charset:0; mso-generic-font-family:script; mso-font-pitch:variable; mso-font-signature:647 0 0 0 159 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Comic Sans MS"; mso-fareast-font-family:"Times New Roman"; mso-bidi-font-family:"Times New Roman";} @page Section1 {size:595.3pt 841.9pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:35.4pt; mso-footer-margin:35.4pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Objective: To evaluate the influence of cytoskeleton and temperature on the solubility of the erythrocyte membrane to the detergent Triton X-100 (TX-100). Methods: Human and bovine erythrocytes were incubated with different amounts of TX-100 at different conditions (4 and 37ºC; with or without Na2CO3 to disrupt the cytoskeleton integrity). Phospholipids in the insoluble fraction were quantified, and protein composition was analyzed by SDS-PAGE.  Acyl chain order was determined by EPR spectroscopy. Results: Effect of temperature: plot of insoluble phospholipid vs TX-100 concentration, fitted to an exponential decay, yielded a parameter D0 accounting for the membrane detergent resistance. In previous results (4ºC), D0 for bovine erythrocytes was more than twice the value for human ones; cholesterol depletion (with methyl-b-cyclodextrin) decreased bovine D0 to 75% and human to 48% (Biophys. Chem. 135:14-18, 2008). For the 37ºC extraction (present results), bovine D0 was decreased to 27%, and human D0 to 35% of their values at 4ºC. Insoluble phospholipids for 1% TX-100 were negligible for human erythrocytes, but a significant amount was obtained for bovine ones. Effect of cytoskeleton disruption: analyzed only in human erythrocytes, 1% TX-100, 4ºC. We found a 50% reduction in phospholipid of the insoluble pellet, and an important reduction in protein content. EPR showed increased disorder of lipid chains at carbon 12 level. Conclusions: Extraction at physiological temperature causes an increase in membrane solubilization, more important than that caused by cholesterol depletion at 4ºC. Human erythrocyte membranes are almost fully solubilized at 1% TX-100, 37ºC. However, an insoluble pellet can still be obtained for bovine erytrhocytes under 37ºC extraction. This fact is correlated to the high sphingomyelin content in these cells. Cytoskeleton disruption also led to decreased resistance to TX-100, and to a more disordered lipid assembly in the insoluble pellet, suggesting involvement of cytoskeleton proteins in raft structuration.