Quantification of Protein Kinase A (PKA) Activity by an in vitro Radioactive Assay Using the Mouse Sperm Derived Enzyme

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 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 in vitro testing of drugs on PKA activity and for following changes of PKA activity during the onset of capacitation.

Keywords: Capacitation; Fertilization; Kinase activity assay; Protein Kinase A (PKA); Sperm.