Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrotus purpuratus by a simple, rapid procedure.Electron microscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that the cortical granules were associated with these cell surface layers was obtained by lz~I-labeling techniques. The cortical granule preparations were found to be rich in a proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a lzsI-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate that the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent.Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycoprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by ~zsI labeling of the intact egg. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca z+ ion is present. When lysis under isotonic conditions is induced by addition of Ca 2+ ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the a2~I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All of these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization. KEY WORDS egg surface isolationThe eggs of many species of invertebrates and egg surface iodination 9 cortical granule enzymes vertebrates contain specialized organelles, the corvitelline layer 9 plasma membrane tical granules (perhaps more appropriately termed cortical granules T8E JOURNAL OF CELL BIOLOGY ' VOLUME 75, 1977" pages 899-914

The cell surface complex (Detering et al ., 1977, J. Cell Biol . 75, 899-914) of the sea urchin egg consists of two subcellular organelles : the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles . We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization . Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na'/K+ ATPase, as well as two 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 only 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 represent 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 KIextracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as 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 six to seven polypeptide components . The phospholipid composition of the KI-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol . Cholesterol was found to be a major component of the plasma membrane . Before KI extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation .The sea urchin egg has been the object of much study in the areas of molecular and developmental biology during the last several decades. Recently, this cell type has gained increasing importance in studies in cell-cell adhesion, exocytosis, and other membrane-associated events. It is clear that the sea urchin egg provides an excellent model for these studies of more general biological interest, as well as for studies related to developmental and reproductive biology per se.Despite the interest in this cell type, the cell surface of the echinoderm egg has received little attention and is poorly understood at the biochemical level. The available information indicates that the egg is surrounded by a morphologically 248