Purification of cortical granules from unfertilized sea urchin egg homogenates by zonal centrifugation

Cortical granules were purified from homogenates of unfertilized sea urchin, Strongylocentrotus purpurataus, eggs by rate-zonal centriguation. Purification was evaluated by integrated biochemical and morphological (electron microscopic) analysis. The enzyme β-1,3-glucanase was employed as the biochemical marker for cortical granules. Cortical granule preparations obtained in sucrose gradients constructed in 0.5 M KCl contained acid phosphatase activity and were heavily contaminated by yolk platelets. Highly purified cortical granules isolated in sucrose gradients constructed in 0.5 M KCl with 10^-3 M EDTA at pH 7.0 were almost completely free of contamination by yolk and other cytoplasmic particles, and contained only trace levels of acid phosphatase activity. The purified cortical granules contained 40-45% of the total β-1,3-glucanase activity in the eggs, and 6.7 ± 1.9% of the total egg protein. The isolated cortical granules retained another known chemical constituent, sulfated acid mucopolysaccharides.