Stival Cintia, Graf Carolina Baro, Visconti Pablo E, Krapf Dario
Laboratory of Cell Signal Transduction Networks, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET-UNR, Rosario 2000, Argentina.
Department of Veterinary and Animal Sciences, University of Massachusetts, 01003 Amherst, Massachusetts, USA.
Bio Protoc. 2020 Jun 20;10(12):e3658. doi: 10.21769/BioProtoc.3658.
In order to acquire fertilizing potential, mammalian sperm must undergo a process known as , 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, 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 P into a peptidic substrate. This approach easily allows the analysis of different agonists or antagonists of PKA. Since mouse sperm is a rich source of soluble PKA, this assay allows a simple fractionation that renders PKA usable both for testing of drugs on PKA activity and for following changes of PKA activity during the onset of capacitation.
为了获得受精能力，哺乳动物精子必须经历一个称为 的过程，该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，通过蛋白质印迹分析其底物的磷酸化状态来评估PKA活性。该技术面临两个主要缺点：它不是激酶活性的直接测量方法，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，因此可以使用天然酶进行诸如“放射性测定”等 测定。与蛋白质印迹不同，放射性测定是一种通过量化肽底物中掺入的磷来评估PKA活性的直接技术。这种方法很容易分析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，该测定允许进行简单的分级分离，使PKA既可以用于测试药物对PKA活性的影响，也可以用于跟踪获能开始期间PKA活性的变化。
