Evidence for the involvement of metalloendoproteases in the acrosome reaction in sea urchin sperm.
Evidence for the involvement of metalloendoproteases in the acrosome reaction in sea urchin sperm.
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DOI：
10.1016/s0021-9258(18)45597-4
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发表时间：
1987-04
期刊：
影响因子：
--
通讯作者：
H. Farach;D. Mundy;W. Strittmatter;W. Lennarz
中科院分区：
文献类型：
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作者：
H. Farach;D. Mundy;W. Strittmatter;W. Lennarz
海胆受精的一个重要的初始步骤是精子中的细胞内膜融合事件，称为顶体反应。这种依赖于Ca 2+的胞吐过程涉及顶体囊泡膜和质膜的融合。最近，需要二价金属的金属内切蛋白酶已被牵连在其他生物系统中的几个Ca 2+依赖的膜融合事件。鉴于所提出的Zn 2+参与海胆精子顶体反应（Clapper，D.L.，戴维斯，J.A.，拉莫特，P. J.，巴顿角，澳-地和Epel，D.（1985）J. Cell Biol.100，1817-1824）和Zn 2+是金属内切蛋白酶的金属辅因子的事实，我们研究了这种蛋白酶在顶体反应中的潜在作用。一种可溶性金属内切蛋白酶被证明和特点，在精子匀浆中使用荧光蛋白酶底物琥珀酰丙氨酸苯丙氨酸-4-氨甲基香豆素。金属螯合剂EDTA和1，10-菲咯啉对该蛋白酶有抑制作用，Zn ~（2+）可使失活的脱辅基酶恢复活性。金属内切蛋白酶底物和抑制剂阻断了由卵冻膜或离子载体诱导的顶体反应，但对Ca 2+内流没有影响。这些观察结果表明，抑制发生在一个独立的步骤中的Ca 2+进入。总的来说，这项研究的结果提供了强有力的间接证据，顶体反应需要金属内切蛋白酶的作用。
An essential initial step in fertilization in the sea urchin Strongylocentrotus purpuratus is an intracellular membrane fusion event in the sperm known as the acrosome reaction. This Ca2+-dependent, exocytotic process involves fusion of the membrane of the acrosomal vesicle and the plasma membrane. Recently, metalloendoproteases requiring divalent metals have been implicated in several Ca2+-dependent membrane fusion events in other biological systems. In view of the suggested involvement of Zn2+ in the sea urchin sperm acrosome reaction (Clapper, D.L., Davis, J.A., Lamothe, P.J., Patton, C., and Epel, D. (1985) J. Cell Biol. 100, 1817-1824) and the fact that Zn2+ is a metal cofactor for metalloendoproteases, we investigated the potential role of this protease in the acrosome reaction. A soluble metalloendoprotease was demonstrated and characterized in sperm homogenates using the fluorogenic protease substrate succinyl-alanine-alanine-phenylalanine-4-aminomethylcoumarin. The protease was inhibited by the metal chelators EDTA and 1,10-phenanthroline, and activity of the inactive apoenzyme could be reconstituted with Zn2+. The metalloendoprotease substrate and inhibitors blocked the acrosome reaction induced either by egg jelly coat or by ionophore, but had no effect on the influx of Ca2+. These observations suggest that inhibition occurs at a step independent of Ca2+ entry. Overall, the results of this study provide strong indirect evidence that the acrosome reaction requires the action of metalloendoprotease.