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

VISCONTI, PABLO E.; STIVAL, CINTIA; KRAPF, DARIO; BARO GRAF, CAROLINA

Bio-protocol

Bio-protocol LLC

Lugar: Sunnyvale, CA,; Año: 2020 vol. 10 p. 3658 - 3671

2331-8325

] In order to acquire fertilizing potential, mammalian sperm must undergo a process known ascapacitation, which relies on the early activation of Protein Kinase A (PKA). Frequently, PKA activity isassessed in whole-cell experiments by analyzing the phosphorylation status of its substrates in awestern-blot. This technique faces two main disadvantages: it is not a direct measure of the kinaseactivity and it is a time-consuming approach. However, since PKA can be readily obtained from spermextracts, 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 byquantification of incorporated 32P into a peptidic substrate. This approach easily allows the analysis ofdifferent agonists or antagonists of PKA. Since mouse sperm is a rich source of soluble PKA, this assayallows a simple fractionation that renders PKA usable both for in vitro testing of drugs on PKA activityand for following changes of PKA activity during the onset of capacitation.