TECHNICAL NOTE: measurement of cAMP-dependent protein kinase activity using a fluorescent-labeled kemptide.
BACKGROUND
Traditional protein kinase assays include the use of [32P] labeled ATP as phosphate donor and a substrate protein or peptide as phosphoreceptor. Since this approach has a number of drawbacks in addition to generating ionizing radiation, several non-isotopic methods have been developed. Although shown to reflect the activity of purified enzymes, none have been demonstrated to detect physiological changes in endogenous enzyme activity in cell homogenates.


METHODS
Studies were performed to examine the kinetics, reproducibility, and optimal assay conditions of a novel non-radioisotopic kinase assay that detects PKA activity by phosphorylation of the peptide substrate Kemptide covalently bound to a fluorescent molecule (f-Kemptide). Basal and agonist-induced PKA activity in epithelial cell homogenates was measured.


RESULTS
The kinetics of f-Kemptide were similar to the standard radioisotopic method with intraassay and interassay variations of 5.6 +/- 0.8% and 14.3 +/- 2.6%, respectively. Neither fluorescence quenching nor enhancing effects were found with consistent amounts of homogenate protein. Specific PKA activity was determined as the IP20-inhibitable fraction to account for nonspecific phosphorylation, perhaps due to S6 kinase or a similar enzyme. The basal activity of 38% of total PKA in A6 cells increased by 84% after exposure to vasopressin and by 58% after short exposure to forskolin. In T84 cells exposed to VIP there was a 360% increase over basal activity.


CONCLUSIONS
These results show that f-Kemptide exhibits acceptable kinetics, and that the assay system can quantitatively and reproducibly measure basal and stimulated PKA activity in cell homogenates.
MacAla
L J
LJ
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA.
Hayslett
J P
JP
Smallwood
J I
JI
D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D009842:Oligopeptides / Q000378:metabolism*; D015203:Reproducibility of Results
D009842:Oligopeptides; C037642:kemptide; D017868:Cyclic AMP-Dependent Protein Kinases