Veron M, Shapiro B M
J Biol Chem. 1977 Feb 25;252(4):1286-92.
The binding of concanavalin A (Con A) to Strongylocentrotus purpuratus eggs has been investigated using 125I-concanavalin A (125I-Con A). The lectin binds specifically to the egg surface and does not produce agglutination of the eggs. High 125I-Con A concentrations are necessary to saturate all of the surface binding sites. Scatchard plots of the binding data are biphasic and may be interpreted as showing two main classes of sites. Unfertilized eggs have 4 X 10(8) high affinity sites/egg (Ka=8 X 10(-7) M) and 4.5 X 10(8) istes of lower affinity (Kb=4 X 10(-6) M). The sites may be assigned to different layers of the egg surface by studying the effects of removal of egg coats on the binding reaction. Removal of the jelly coat by washing eggs at pH 4.5 has no effect on binding. However, disruption of the vitelline layer with dithiothreitol leads to a decrease in the number of high affinity sites. After fertilization, the high affinity sites are found upon the fertilization membrane produced from the vitelline layer. Low affinity sites predominate in the plasma membrane, and are not affected by treatments which alter the vitellinelayer. The number of low affinity sites double upon fertilization, with the insertion of new membrane into the egg surface, as a result of cortical granule exocytosis. The doubling of sites is not due to hyaline material released from the cortical vesicles at fertilization, and thus these sites appear to reside upon the new membrane that is inserted from the cortical vesicles. If eggs are activated with ammonia, bypassing the cortical reaction, no change in the binding of ConA occurs. Con A inhibits fertilization at concentrations higher than 0.1 mg/ml, where less than 50% of the high affinity (viteline layer) binding sites are occupied, and there is little binding to the low affinity (plasma membrane) sites. Thus, the interaction of sperm with vitelline layer components may be an obligatory step in the fertilization process. A fraction of the 125I-Con A binding sites is cleaved from the egg surface upon fertilization or after activation by the calcium ionophore A23187. This release of Con A binding sites occurs during the limited proteolysis of surface components that accompanies the cortical reaction, and does not occur with ammonia activation of the egg, where the cortical reaction does not occur. Thus, the changes in Con A binding at fertilization are caused by the massive cortical granule exocytosis that occurs within minutes of sperm penetration, and not by activation of the egg per se.
利用¹²⁵I - 伴刀豆球蛋白A（¹²⁵I - Con A）研究了伴刀豆球蛋白A（Con A）与紫海胆卵的结合情况。该凝集素特异性结合卵表面，不会使卵发生凝集。需要高浓度的¹²⁵I - Con A才能饱和所有表面结合位点。结合数据的Scatchard图呈双相性，可解释为显示出两类主要的位点。未受精卵每个卵有4×10⁸个高亲和力位点（Ka = 8×10⁻⁷M）和4.5×10⁸个低亲和力位点（Kb = 4×10⁻⁶M）。通过研究去除卵膜对结合反应的影响，可将这些位点归属于卵表面的不同层。在pH 4.5下洗涤卵去除胶膜对结合没有影响。然而，用二硫苏糖醇破坏卵黄膜会导致高亲和力位点数量减少。受精后，高亲和力位点存在于由卵黄膜产生的受精膜上。低亲和力位点在质膜中占主导地位，不受改变卵黄膜的处理影响。由于皮质颗粒胞吐作用，受精时随着新膜插入卵表面，低亲和力位点的数量翻倍。位点翻倍不是由于受精时从皮质小泡释放的透明物质，因此这些位点似乎位于从皮质小泡插入的新膜上。如果用氨激活卵，绕过皮质反应，ConA的结合没有变化。Con A在浓度高于0.1mg/ml时抑制受精，此时不到50%的高亲和力（卵黄膜层）结合位点被占据，且与低亲和力（质膜）位点几乎没有结合。因此，精子与卵黄膜层成分的相互作用可能是受精过程中的一个必要步骤。受精或经钙离子载体A23187激活后，一部分¹²⁵I - Con A结合位点从卵表面被切割下来。Con A结合位点的这种释放发生在伴随皮质反应的表面成分有限的蛋白水解过程中，而在卵的氨激活过程中不发生，因为氨激活时不发生皮质反应。因此，受精时Con A结合的变化是由精子穿透后几分钟内发生的大量皮质颗粒胞吐作用引起的，而不是由卵本身的激活引起的。
