Thesis

ATPase and G-6-Pase Activity in Sea Urchin Egg Membranes

Fertilized and unfertilized Strongylocentrotus purpuratus and Lytechinus pictus egg membrane preparations including plasmamembranes and egg envelopes, were obtained via gentle homogenization and centrifugation in Tris- MgCl2 solutions. Membrane preparations were then assayed for Mg++ and Na+-K+ and glucose-6-phosphatase (G-6-Pase) activity with pH optima and enzyme-substrate affinity being investigated. The pH optima for G-6-Pase, Mg++ ATPase and Na+-K+ ATPase were found to be 5. 21, 6.3, and 6.91 respectively. The membrane-associated G-6-Pase activity was found to be higher in the unfertilized membrane preparations, while the Na+- K+ ATPase activity was higher in the fertilized preparations. A discontinuous sucrose density gradient (20-67% w/w) separated the membrane preparations into four major protein peaks which were assayed for G-6-Pase activity. A change in distribution of the G-6-Pase activity was noted after fertilization. The effects of pretreatment with mycostatin, NH4OH, phospholipase A, and acetone-4% water upon the activity of membrane-associated G-6-Pase were examined. Mycostatin pretreatment of unfertilized eggs was found to inhibit G-6-Pase and both of the ATPases. The G-6-Pase activities of fertilized and unfertil ized membrane preparations were unaffected by pretreatment with phospholipase A and were inhibited by NH4OH pretreatment. Acetone-4% water pretreatment proved to have no influence upon membrane-associated G-6-Pase activity in fertilized preparations, but inhibited the activity in unfertilized preparations.

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