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Author:
Date:
2020-06-20
[Abstract] In order to acquire fertilizing potential, mammalian sperm must undergo a process known as capacitation, which relies on the early activation of Protein Kinase A (PKA). Frequently, PKA activity is assessed in whole-cell experiments by analyzing the phosphorylation status of its substrates in a western-blot. This technique faces two main disadvantages: it is not a direct measure of the kinase activity and it is a time-consuming approach. However, since PKA can be readily obtained from sperm extracts, in vitro assays such as the “radioactive assay” can be performed using the native enzyme. Unlike western-blot, the radioactive assay is a straightforward technique to evaluate PKA activity by quantification of incorporated 32P into a peptidic ...
[摘要] [Abstract]为了获得受精潜能，哺乳动物的精子必须经历一个被称为电容的过程，这个过程依赖于蛋白激酶A（PKA）的早期激活。通常，在全细胞实验中，通过分析其底物在western-blot中的磷酸化状态来评估PKA的活性。这种技术面临着两个主要的缺点：它不是对激酶活性的直接测量，而且是一种耗时的方法。然而，由于PKA可以很容易地从精子提取物中获得，体外检测如"放射性检测"可以使用原生酶进行。与western-blot不同的是，放射性测定法是一种直接的技术，通过将整合的32P定量到肽类底物中来评价PKA的活性。这种方法很容易允许分析PKA的不同激动剂或拮抗剂。由于小鼠精子是可溶性PKA的丰富来源，这种测定法可以进行简单的分馏，使PKA既可用于体外测试药物对PKA活性的影响，也可用于跟踪PKA活性在增容开始时的变化。
[Background] ...
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