Isolation and partial characterization of the plasma membrane of the sea urchin egg.

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Abstract
The cell surface complex of the sea urchin [Strongylocentrotus purpuratus] egg consists of 2 subcellular organelles: the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles. A method of isolating the plasma membrane from this complex and its biochemical characterization are presented. Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na+/K+ ATPase and 2 cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles. The purified plasma membranes are highly enriched in the Na+/K+ ATPase but contain very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a fluffy proteinaceous layer on the external surface, which probably represents peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with KI removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes and KI-extracted membranes indicates that the plasma membrane contains 5-6 major protein species, and a large number of minor species, that are not extractable with KI. The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least 6-7 polypeptide components. The phospholipid composition of the KI-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was a major component of the plasma membrane. Before KI extraction the purified plasma membranes retain the same species-specific sperm binding property as in the intact egg, indicating that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation.