TECHNICAL NOTE: measurement of cAMP-dependent protein kinase activity using a fluorescent-labeled kemptide.
| Autor: | MacAla LJ; Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA., Hayslett JP, Smallwood JI |
|---|---|
| Jazyk: | angličtina |
| Zdroj: | Kidney international [Kidney Int] 1998 Nov; Vol. 54 (5), pp. 1746-50. |
| DOI: | 10.1046/j.1523-1755.1998.00140.x |
| Abstrakt: | 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. |
| Databáze: | MEDLINE |
| Externí odkaz: |
|