Crabb J H, Jackson R C
J Membr Biol. 1986;91(1):85-96. doi: 10.1007/BF01870218.
The Ca2+-stimulated release of vesicle contents from cortical fragments prepared from sea urchin eggs is an in vitro model for exocytosis. Cortical fragments have been isolated either in suspension (cell surface complex, CSC preparation), or attached to polycation-coated surfaces (cortical lawn, CL preparation). CL, but not CSC, have been reported to undergo a rapid "aging" process whereby they fail to respond to micromolar free Ca2+. Since, in principle, the only difference between the two preparations is the use of polycations in the CL preparation, polycations were suspected of being inhibitory. This hypothesis was tested by evaluating the effects of polycation-containing buffers on the Ca2+ threshold, rate, and extent of exocytosis in CL prepared from the eggs of Strongylocentrotus purpuratus. A sensitive microphotometric assay, based on light scattering by the individual cortical vesicles in the CL, was used to quantitate the exocytotic response. Buffers containing polylysine were found to be potent inhibitors of cortical exocytosis. The Ca2+ threshold of CL that had been treated for 15 min at room temperature with 50 micrograms/ml of polylysine was more than three orders of magnitude greater than that of freshly prepared CL. The other polycations tested (protamine, spermine and neomycin) were also found to be inhibitory, but to a lesser degree than polylysine. Two lines of evidence suggested that the polycations used in the preparation of CL are responsible for the rapid "aging" phenomenon: CSC fragments that had been affixed to polylysine-coated coverslips were shown to acquire "aging" characteristics similar to the CL preparations; control CSC that had been maintained in suspension did not. Radiolabeled poly-L-lysine was shown to dissociate from coated coverslips and redistribute onto CL.
海胆卵制备的皮质碎片中，Ca2+刺激的囊泡内容物释放是一种胞吐作用的体外模型。皮质碎片可以悬浮分离（细胞表面复合物，CSC制备），也可以附着在聚阳离子包被的表面（皮质片层，CL制备）。据报道，CL而非CSC会经历快速的“老化”过程，在此过程中它们对微摩尔浓度的游离Ca2+不再有反应。原则上，两种制备方法的唯一区别在于CL制备中使用了聚阳离子，因此怀疑聚阳离子具有抑制作用。通过评估含聚阳离子缓冲液对紫海胆卵制备的CL中Ca2+阈值、胞吐速率和程度的影响，对这一假设进行了验证。基于CL中单个皮质囊泡的光散射的灵敏显微光度测定法，用于定量胞吐反应。发现含聚赖氨酸的缓冲液是皮质胞吐作用的有效抑制剂。在室温下用50微克/毫升聚赖氨酸处理15分钟的CL的Ca2+阈值比新鲜制备的CL高出三个数量级以上。测试的其他聚阳离子（鱼精蛋白、精胺和新霉素）也有抑制作用，但程度比聚赖氨酸小。有两条证据表明，CL制备中使用的聚阳离子是导致快速“老化”现象的原因：附着在聚赖氨酸包被盖玻片上的CSC碎片表现出与CL制剂相似的“老化”特征；保持悬浮状态的对照CSC则没有。放射性标记的聚-L-赖氨酸从包被的盖玻片上解离并重新分布到CL上。
