A CE assay for the detection of agonist-stimulated adenylyl cyclase activity

Jennifer M. Cunliffe, Matthew R. Whorton, Roger K. Sunahara, Robert T. Kennedy

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

A CE assay was a developed for the detection of adenylyl cyclase (AC) activity stimulated at the AC and G protein-coupled receptor (GPCR) level. In the assay, cell membranes over-expressing GPCR and/or AC were incubated with modulators and substrate ATP to produce cAMP in a dose-dependent manner. In both the CE-UV and a radiochemical assay, the addition of forskolin (FSK) resulted in a two- to three-fold maximum increase in AC activity with EC50s of 4.2 ± 0.7 and 2.4 ± 0.7 μM, respectively, demonstrating that similar results were obtained by both assays. GPCR activation was also detected using cell membranes overexpressing AC and the β2-adrenergic receptor (β2AR) fused to the stimulatory G protein. Terbutaline (β2AR agonist) increased the basal rate of cAMP formation 1.7 ± 0.1-fold resulting in an EC50 of 62 ± 10 nM. The assay's ability to detect antagonists is demonstrated by the expected right-shifted EC50 of terbutaline by the β2AR antagonists propranolol The CE-UV assays offers advantages over the traditional radioactivity assay in terms of safety and labor.

Original languageEnglish (US)
Pages (from-to)1913-1920
Number of pages8
JournalELECTROPHORESIS
Volume28
Issue number12
DOIs
StatePublished - Jun 1 2007

Keywords

  • Adenylyl cyclase
  • CE
  • Enzyme assay
  • G protein-coupled receptor

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

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