Calcium-mediated release of glucanase activity from cortical granules of sea urchin eggs

Abstract

Confluent monolayers of sea urchin eggs were bonded to culture dishes coated with protamine sulfate. The cytoplasm was then sheared away by a jet of isosmotic buffer. About 326,000 circular fragments of individual egg cortices (430 μg protein) remained bound to each dish. The fragments are composed of cortical granules (CG), plasma membrane, and vitelline layer. A single dish contains 7.7 × 10 8 CG and is referred to as a CG lawn (CGL). Ca 2+-EGTA buffers of estimated free-Ca 2+ concentrations (0.06–25.7 μ M) were applied to CGL and samples removed and assayed for the CG marker enzyme exo-β(1 → 3)-glucanohydrolase (glucanase). Estimated free-Ca 2+ concentrations above 2.75 μ M caused the total release of the glucanase to the supernatant within 4 min. The half-maximal rate of appearance of glucanase occurred in 2.5 μ M Ca 2+. At all Ca 2+ concentrations tested, the appearance of enzyme activity exhibited sigmoidal kinetics. The visual disappearance of CG correlated with the appearance of glucanase in the Ca 2+ buffer. In response to Ca 2+ the CG probably lyse, fuse with adjacent CG, or fuse with the underlying plasma membrane. The calmodulin antagonist trifluoperazine inhibited Ca 2+-mediated glucanase release from CGL (I 50 8 μ M). The sensitivity of the CGL to Ca 2+ in the 1–10 μ M range is rapidly lost during incubation of CGL in the isolation buffer. ATP and low temperature retard the rate of loss of Ca 2+ sensitivity. These secretory granules are a model for studying the mechanism of Ca 2+-induced secretion. In addition, they contain structural proteins and enzymes which function in the fertilization process. CGL preparations should be useful in studies dealing with the processing of CG components after their release in response to micromolar concentrations of Ca 2+.