Advances in the characterization of plasma membrana-enriched fractions and lipid microdomains from Bufo arenarum oocytes

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

LOS COCOS, CORDOBA

Workshop; Latest concepts in developmental biology, International Workshop; 2006

SOCIETY OF DEVELOPMENTAL BIOLOGY AND INTERNATIONAL BRAIN RESEARCH ORGANIZATION

ADVANCES IN THE CHARACTERIZATION OF PLASMA MEMBRANE-ENRICHED FRACTIONS AND LIPID MICRODOMAINS FROM Bufo arenarum OOCYTES Buschiazzo, Jorgelina; Bonini, Ida and Alonso, Telma INIBIBB (UNS-CONICET), CC 857, 8000 Bahía Blanca, Argentina. E-mail: jbusch@criba.edu.ar Lipid microdomains, which are known as “rafts” are low-density plasma membrane domains that contain high levels of sphingolipids and cholesterol. Signaling molecules and transmembrane proteins have also been shown to be enriched in lipid rafts. The selective localization of signaling molecules in these specialized regions of the plasma membrane appear to facilitate signal transduction and/or play a role in physiological responses in hormone-sensitive cells such as the amphibian oocyte system. The lipid microdomain involvement in amphibian oocyte maturation and fertilization transduction pathways has not been elucidated to date. The aim of the present study is to analyze the lipid and protein components of plasma membrane enriched-fractions and lipid microdomains from full-grown Bufo arenarum oocytes. Plasma membrane-enriched fractions were obtained either by centrifugation (Blondeau and Baulieu, 1985; Luria et al, 2002) or microdissection. Lipid microdomains were isolated either in the presence or in the absence of detergent by sucrose gradient centrifugation. Proteins contained in sucrose gradient fractions were resolved by SDS-PAGE and characterized by Coomassie and Ponceau S staining as well as by Western-blotting. Lipids were extracted according to Folch et al. (1957), they were separated by thin-layer chromatography and derivatized by methanolysis for compositional studies. Fatty acids were identified in a gas-liquid chromatograph. Phospholipid phosphorus and proteins were measured as described by Rouser et al. (1970) and Lowry et al. (1951), respectively. Cholesterol was quantified using an enzymatic assay. The activity of 5’nucleotidase as an enzyme marker of plasma membrane was tested. Results evidenced that membrane preparations obtained by different methods are enriched in plasma membrane as suggested by the activity of 5’nucleotidase and the level of typical membrane components such as sphingomyelin (SM), phosphatidylserine (PS), phosphatidylinositol and phosphatidic acid (PA). PS and PA showed the highest unsaturation index in whole oocytes and plasma membrane fractions, respectively. SM was the most saturated phospholipid in both cases. Density gradients of detergent-free and detergent-treated membranes showed similar protein profiles. Staining of proteins contained in sucrose gradient fractions from whole oocytes showed the presence of two bands with molecular weights coincident with those proteins stored in yolk platelets. A distinctive band with apparent molecular weight of 66.2 kDa was observed only in low-density membrane fractions and it was evidenced mainly in pellets obtained from total plasma membrane, suggesting its association with lipid rafts.