Detection of adenylyl cyclase activity using a fluorescent ATP substrate and capillary electrophoresis

Analytical Chemistry
Jennifer M CunliffeRobert T Kennedy

Abstract

A capillary electrophoresis laser-induced fluorescence (CE-LIF) assay was developed for detection of adenylyl cyclase (AC) activity using BODIPY FL ATP (BATP) as substrate. In the assay, BATP was incubated with AC and the resulting mixture of BATP and enzyme product (BODIPY cyclic AMP, BcAMP) separated in 5 min by CE-LIF. Substrate depletion and product accumulation were simultaneously monitored during the course of the reaction. The rate of product formation depended upon the presence of AC activators forskolin or Galpha(s)-GTPgammaS as evidenced by a more rapid BATP turnover to BcAMP compared to basal levels. The CE-LIF assay detected EC50 values for forskolin and Galpha(s)-GTPgammaS of 27 +/- 6 microM and 317 +/- 56 nM, respectively. These EC50 values compared well to those previously reported using [alpha-32P]ATP as substrate. When AC was concurrently activated with 2.5 microM forskolin and 25 nM Galpha(s)-GTPgammaS, the amount of BcAMP formed was 3.4 times higher than the additive amounts of each activator alone indicating a positively cooperative activation by these compounds in agreement with previous assays using radiolabeled substrate. Inhibition of AC activity was also demonstrated using the AC inhibitor 2'-(or-3')-O-...Continue Reading

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Citations

Jul 11, 2007·Proceedings of the National Academy of Sciences of the United States of America·François O LaforgeMichael V Mirkin
Sep 2, 2006·Analytical Chemistry·James R KralyNorman J Dovichi
Jul 8, 2008·Analytical Biochemistry·Corinna M SpanglerMichael Schäferling
Mar 17, 2007·Electrophoresis·Feliciano Priego Capote, María Dolores Luque de Castro
May 8, 2007·Electrophoresis·Jennifer M CunliffeRobert T Kennedy
Sep 4, 2009·Electrophoresis·Garry D SiskDamien W M Arrigan
Dec 22, 2009·Bioorganic & Medicinal Chemistry Letters·Hyun-Woo RheeJong-In Hong

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