PKG Substrate

Kinase activity is measured by determining the amount of ³³P radioactivity incorporated from [³³P]ATP or [³³P]N⁶-benzyl-ATP into a PKG specific peptide substrate (RKRSRAE). The standard 75 μL assay mixture contains 0.15 μCi of [³³P]ATP, 10 μM ATP, 15 μM PKG peptide substrate, 2 μM PKI (a synthetic peptide inhibitor of cAMP-dependent protein kinase), 1 μg of purified kinase, and 100 μM 8-Br-cGMP in 50 mM HEPES buffer, pH 7.4, containing 10 mM MgCl₂, 0.1% Tween 20, and 1 mM DTT. After incubation at 30°C for 2 min, the reaction is immediately put on ice, and 20 μL of the assay mixture is spotted onto P81 phosphocellulose paper and then quenched in 0.42% H₃PO₄. The paper is further washed three times in 0.42% H₃PO₄ for 10 min with gentle agitation and rinsed once with acetone. After air drying, radioactivity on the paper is measured with a Beckman LS6500 liquid scintillation counter. For measuring the effect of N⁶-benzyl-ATP on the activity of PKG I utilizing ATP as a co-substrate, unlabeled N⁶-benzyl-ATP is added to each reaction at the indicated concentrations. Saturation kinetic analyses for K_m and V_max with ATP or N⁶-benzyl-ATP are performed over a concentration range (0.0015-100 μM) by adding unlabeled ATP or N⁶-benzyl-ATP to a given amount of [³³P]ATP or [³³P]N⁶-benzyl-ATP, respectively^[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.