EVIDENCE FOR CELL-SURFACE AND INTERNAL PHOSPHOLIPASE-ACTIVITY IN ASCIDIAN EGGS

Upon fertilization, ascidian eggs release a cell surface glycosidase u sed in the block to polyspermy and undergo cortical contractions resul ting from increased intracellular calcium levels. The glycosidase is r eleased by fertilization, calcium ionophores or added phospholipase C (PLC) activity. The PLC inhibitor D609 blocks glycosidase release. Int act Ascidia ceratodes eggs cleave 4-methylumbelliferyl-phospho-choline when it is added to seawater. This yields highly fluorescent 4-methyl umbelliferone. Authentic phospholipase C but not phospholipase D can c leave this substrate. Thus, the authors believe that cleavage of the s ubstrate is specific for PLC activity. Eggs incubated in the fluorogen ic substrate after having been washed and detergent extracted were not fluorescent. Therefore the substrate failed to enter intact cells. Gl ycosidase release and PLC activity were stimulated by ionomycin. Octyl glucoside or Triton X-100 extracts of ascidian eggs had two forms of p hospholipase activity as shown by ion affinity chromatography: PL1 elu ting at 0.25 mol/L NaCl and PL2 eluting at 0.6 mol/L NaCl. The PL1 app eared to be isolated as a single protein. When surface proteins were l abeled with non-penetrating biotin and were subsequently reacted with streptavidin, half of the PLC activity bound. This demonstrates that h alf the ascidian egg PLC activity is located on the surface of either the egg or follicle cell, and half is located within the egg.