Unknown

Dataset Information

0

Characterization of PKAC? 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 (PKAC?). 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 (KM, Vmax) of the new Rh-MAB-Kemptide substrate, a commercially prepared TAMRA-Kemptide substrate, and ATP as well as the potency (IC50, Ki) of the known PKAC? 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 ?-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.

SUBMITTER: Luzi NM 

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

REPOSITORIES: biostudies-literature

altmetric image

Publications

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

Luzi Nicole M NM   Lyons Charles E CE   Peterson Darrell L DL   Ellis Keith C KC  

Analytical biochemistry 20170605


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  ...[more]

Similar Datasets

| S-EPMC5097697 | biostudies-literature
| S-EPMC6641702 | biostudies-literature
| S-EPMC4871228 | biostudies-literature
| S-EPMC2964420 | biostudies-literature
| S-EPMC8071115 | biostudies-literature
| S-EPMC7022172 | biostudies-literature
| S-EPMC7609449 | biostudies-literature
| S-EPMC7041903 | biostudies-literature
| S-EPMC7604385 | biostudies-literature
| S-EPMC4522773 | biostudies-literature