The rapid purification and partial characterization of human sperm proacrosin using an automated fast protein liquid chromatography (FPLC) system.
The rapid purification and partial characterization of human sperm proacrosin using an automated fast protein liquid chromatography (FPLC) system.
复制标题
使用自动化快速蛋白液相色谱 (FPLC) 系统对人精子顶体蛋白原进行快速纯化和部分表征。
DOI：
10.1016/0304-4165(86)90298-9
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Polakoski,KL
中科院分区：
文献类型：
--
作者：
Siegel,MS;Bechtold,DS;Kopta,CI;Polakoski,KL
开发了一种快速有效的程序来获得高度纯化的人顶体素原。通过含有50mM苯甲脒的1M蔗糖离心洗涤射出的精子，并在苯甲脒存在下酸提取。将溶解的材料透析然后冻干。将样品重悬于 8 M 盐酸胍的乙酸 (0.5 M) pH 2.5 溶液中，然后使用自动快速蛋白质液相色谱系统进行凝胶渗透色谱分析，该系统使用串联设置的两根 Pharmacia Superose 12 柱，并在相同的缓冲液中进行平衡。顶体素原作为单独的峰洗脱，与另一种被称为精蛋白原的蛋白酶原很好地分离。当在银染的 SDS-聚丙烯酰胺凝胶以及明胶-SDS-聚丙烯酰胺凝胶上观察时，确定顶体素原制剂是高度纯化的。顶体素原在这两个系统上均以双联体形式出现（Mr= 55 000 和 53 000）。在 pH 8 时顶体素原自动转化为顶体素，产生典型的 S 形自动激活曲线。对 SDS-聚丙烯酰胺凝胶进行蛋白质染色后，结果表明，在纯化的顶体素原制剂活化后，55 000 和 53 000 分子量的蛋白质最初降解为 49 000 形式，然后降解为几种较低分子量形式 (Mr= 40 000 – 34 000)。在明胶-SDS-聚丙烯酰胺酶谱分析后观察到关于蛋白水解消化的类似结果，除了还观察到蛋白酶强度在58000和53000之间随时间增加。研究发现，钴和钙是顶体素原转化为顶体素的有效抑制剂，而钠对这一过程的抑制作用要小得多。研究发现钙能显着增强人顶体素的蛋白水解活性，而对苯甲酰精氨酸乙酯的顶体素水解没有明显影响。因此，所描述的纯化程序产生了高度纯化的顶体素原制剂，其产率足以允许其部分表征。
A rapid efficient procedure was developed for obtaining highly purified human proacrosin. Ejaculated spermatozoa were washed via centrifugation through 1 M sucrose containing 50 mM benzamidine and acid-extracted in the presence of benzamidine. The solubilized material was dialyzed then lyophilized. The sample was resuspended in 8 M guanidine hydrochloride in acetic acid (0.5 M) pH 2.5 and then subjected to gel permeation chromatography with an automated fast protein liquid chromatography system utilizing two Pharmacia Superose 12 columns set in tandem that were equilibrated in the same buffer. The proacrosin eluted as an individual peak that was well separated from another proteinase zymogen referred to as sperminogen. The proacrosin preparation was determined to be highly purified when observed on silverstained SDS-polyacrylamide gels as well as on gelatin-SDS-polyacrylamide gels. The proacrosin appeared as a doublet (Mr= 55 000 and 53 000) on both of these systems. The autoconversion of proacrosin to acrosin at pH 8 resulted in a typical sigmoidal autoactivation curve. Following protein staining of SDS-polyacrylamide gels, it was shown that upon activation of purified proacrosin preparations the 55 000 and 53 000 molecular weight proteins were initially degraded to a 49 000 form and then to several lower molecular weight forms (Mr= 40 000 – 34 000). Similar findings with regard to proteolytic digestion were observed following gelatin-SDS-polyacrylamide zymography except that an increase with time in proteinase intensity between 58 000 and 53 000 was also observed. Cobalt and calcium were found to be potent inhibitors of the conversion of proacrosin into acrosin, while sodium resulted in much less inhibition of this process. Calcium was found to markedly enhance the proteolytic activity of human acrosin, while it had no observable influence on the acrosin hydrolysis of benzoylarginine ethyl ester. Thus, the described purification procedure resulted in a highly purified proacrosin preparation in sufficient yields to allow for its partial characterization.