Department of Animal Science, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan.
Poult Sci. 2009 Dec;88(12):2636-43. doi: 10.3382/ps.2009-00292.
In the eggs of the quail Coturnix japonica, the limiting membrane demarcates the shell membrane at the interface with the albumen and decreases in width during the hatching process. This study was done to identify agents that affect the width of this limiting membrane. Zymography tests on extracts from extraembryonic tissues, yolk sacs, or chorioallantoic membranes, or all three, showed proteolytic activities during d 4 to 10 of incubation. Localization experiments on these activities, performed on d 5 eggs, indicated that they were located in an avascular chorion. Electron microscopic analysis showed there were secretory cells specifically located in the avascular chorion. After partial purification of d 5 avascular chorion extracts using QA52 and Sephadex G-200 column chromatography, the proteolytic activity of 20 kDa was isolated. The protease showed a high level of activity toward succinyl-Gly-Pro-Leu-Gly-Pro-4-methylcoumaryl-7-amide. It had an optimal pH of 9 and digested the limiting membrane. These enzymatic activities were inhibited moderately by EDTA and strongly by leupeptin and aprotinin. It was concluded that it is the 20-kDa protease, showing collagenase-like activity produced by the avascular chorion, that affects the limiting membrane.
在鹌鹑 Coturnix japonica 的卵中，限制膜在与蛋白的界面处划定壳膜，并在孵化过程中宽度减小。本研究旨在确定影响该限制膜宽度的因素。对胚胎外组织、蛋黄囊或绒毛尿囊膜或三者的提取物进行的酶谱试验显示，在孵育第 4 至 10 天期间存在蛋白水解活性。在第 5 天的卵上进行的这些活性的定位实验表明，它们位于无血管的绒毛膜中。电子显微镜分析显示，有专门位于无血管绒毛膜中的分泌细胞。在用 QA52 和 Sephadex G-200 柱层析对第 5 天无血管绒毛膜提取物进行部分纯化后，分离出 20 kDa 的蛋白水解活性。该蛋白酶对琥珀酰-Gly-Pro-Leu-Gly-Pro-4-甲基香豆酰-7-酰胺表现出高活性。它的最适 pH 为 9，可消化限制膜。这些酶活性被 EDTA 适度抑制，被亮抑酶肽和抑肽酶强烈抑制。结论是，无血管绒毛膜产生的 20 kDa 蛋白酶具有胶原酶样活性，它影响限制膜。
