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). The advantages of this assay are that it is convenient and inexpensive, uses readily synthesized or commercially available substrates that are shelf-stable, uses a common piece of laboratory equipment, and does not require any hazardous materials such as radioactive γ-³²P-ATP. The assay format is also highly flexible and could be adapted for the testing of many different kinases by changing the peptide substrate sequence.

Keywords: Chromophore; HPLC; Inhibition assay; Kinase assay; Kinetics assay; cAMP-dependent protein kinase.

Fig. 1

Structures of Rh-MAB-Kemptide (1) and TAMRA-Kemptide (2), PKACα substrates with strong chromophores.

Fig 2

UV/Vis spectra of Rh-MAB-Kemptide (1) and TAMRA-Kemptide (2). Both samples are 100 μM in 25 mM phosphate buffer (pH 7.4).

Fig. 3

HPLC traces of Rh-MAB-Kemptide (1) and TAMRA-Kemptide (2) at 560 nm. Both samples are 10 μM in 25 mM phosphate buffer (pH 7.4). HPLC analysis was carried out on the 2.1×100mm C18 column (50 μL injection volume, 0.5 nmoles) as described in the Materials and Methods section. Retention times were 8.45 min for Rh-MAB-Kemptide (1) and 7.81 min for TAMRA-Kemptide (2).

Fig. 4

HPLC trace of a kinase reaction with Rh-MAB-Kemptide (1) at multiple wavelengths. Kinase reaction conditions were: PKACα (0.5 nM), Rh-MAB-Kemptide (1) (10 μM), ATP (25 μM), MgCl₂ (10 mM), in 50 mM MOPS (pH 7.4) in a 200 μL final volume for 1 hour. HPLC analysis was carried out on the 2.1×100mm C18 column (80 μL injection volume, 0.5 nmoles) as described in the Materials and Methods section. Retention times were 8.43 min for Rh-MAB-Kemptide (1) and 8.84 min for phospho-Rh-MAB-Kemptide. Phosphorylation as measured at 560 nm was 33.6%.

Fig. 5

Kinetic analysis of the phosphorylation of Rh-MAB-Kemptide (A) and TAMRA-Kemptide (B) by recombinant human PKACα at a fixed concentration of ATP for determination of K_M,Peptide and other kinetic parameters.

Fig. 6

Kinetic analysis of the phosphorylation of Rh-MAB-Kemptide by recombinant human PKACα at varying ATP concentrations for determination of K_M,ATP and other kinetic parameters.

Fig. 7

Inhibition of recombinant human PKACα by PKI(5-24) (A) and H89 (B).