Lipid Analysis of low-density caveolae-like membranas from Bufo arenarum oocytes

• BUSCHIAZZO J., ALONSO T.S. Y BONINI I.C.

ROSARIO

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2006

SAIB

LIPID ANALYSIS OF LOW-DENSITY CAVEOLAE-LIKE MEMBRANES FROM Bufo arenarum OOCYTES Buschiazzo, Jorgelina; Alonso, Telma S. and Bonini, Ida C. Instituto de Investigaciones Bioquímicas de Bahía Blanca, C.C. 857, B. Blanca, Argentina. jbusch@criba.edu.ar Lipid microdomains (rafts) are dynamic assemblies of cholesterol, sphingolipids and specific proteins involved in cell signaling regulation. We conducted a quantitative analysis of lipids and proteins of detergent-free low-density membranes isolated from B. arenarum oocytes in order to elucidate how these domains perform their regulatory functions in the amphibian system. Membrane fractions were separated in the absence of detergent by sucrose gradient centrifugation and characterized by Coomassie staining and Western-blot. Lipids were analyzed by chromatography techniques. The fraction corresponding to "light" membranes (10-22.5% sucrose interface) derived from plasma membrane as suggested by the activity of 5’nucleotidase. In addition, this fraction is enriched in sphingomyelin (0.12 nmol/mg prot.) and cholesterol (0.48 nmol/mg prot.) with respect to the other membrane fractions. Extra heavy membranes are enriched in cardiolipin, a specific component of the inner mitochondrial membrane and evidence a low level of cholesterol. Staining of proteins showed differential bands mainly between light and the other two membrane fractions. The finding of a single 21-kDa caveolin in light membranes indicates the presence of caveolae-like structures in B. arenarum oocytes. In support of this finding, lipid composition of light membrane fraction is consistent with that proposed for lipid rafts.