Characterization of PKACα enzyme kinetics and inhibition in an HPLC assay with a chromophoric substrate.

ABSTRACT: Here we describe a convenient, inexpensive, and non-hazardous method for the measurement of the kinase activity of the catalytic subunit of cAMP-dependent protein kinase (PKACα). The assay is based on the separation of a substrate peptide labeled with a strong chromophore from the phosphorylated product peptide by high-performance liquid chromatograph (HPLC) and quantification of the product ratiometrically at a wavelength in the visual spectrum (Vis). The utility and reliability of the HPLC-Vis assay were demonstrated by characterizing the kinetic parameters (K_M, V_max) of the new Rh-MAB-Kemptide substrate, a commercially prepared TAMRA-Kemptide substrate, and ATP as well as the potency (IC₅₀, K_i) of the known PKACα inhibitors H89 and PKI(5-24).

SUBMITTER: Luzi NM 

PROVIDER: S-EPMC5889107 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature