PKG Substrate

Kinase experiment:

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 Km and Vmax 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].

References:

[1]. Wong A, et al. Cyclic GMP-dependent stimulation of serotonin transport does not involve direct transporter phosphorylation by cGMP-dependent protein kinase. J Biol Chem. 2012 Oct 19;287(43):36051-8.