Methodology article

Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog

Daniela Moll^{* 1} , Anke Prinz^{* 1} , Cornelia M Brendel² , Marco Berrera³ , Katrin Guske¹ , Manuela Zaccolo³ , Hans-Gottfried Genieser⁴ and Friedrich W Herberg¹

¹University of Kassel, Department of Biochemistry, Heinrich-Plett-Strasse 40, 34132 Kassel, Germany

²University of Kassel, Department of Physical Chemistry, Heinrich-Plett-Strasse 40, 34132 Kassel, Germany

³University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK

⁴BIOLOG Life Science Institute, Flughafendamm 9a, P.O. Box 107125, Bremen, Germany

author email corresponding author email* Contributed equally

BMC Biochemistry 2008, 9:18doi:10.1186/1471-2091-9-18

Published:	25 June 2008

Abstract

Background

A novel fluorescent cAMP analog (8-[Pharos-575]- adenosine-3', 5'-cyclic monophosphate) was characterized with respect to its spectral properties, its ability to bind to and activate three main isoenzymes of the cAMP-dependent protein kinase (PKA-Iα, PKA-IIα, PKA-IIβ) in vitro, its stability towards phosphodiesterase and its ability to permeate into cultured eukaryotic cells using resonance energy transfer based indicators, and conventional fluorescence imaging.

Results

The Pharos fluorophore is characterized by a Stokes shift of 42 nm with an absorption maximum at 575 nm and the emission peaking at 617 nm. The quantum yield is 30%. Incubation of the compound to RIIα and RIIβ subunits increases the amplitude of excitation and absorption maxima significantly; no major change was observed with RIα. In vitro binding of the compound to RIα subunit and activation of the PKA-Iα holoenzyme was essentially equivalent to cAMP; RII subunits bound the fluorescent analog up to ten times less efficiently, resulting in about two times reduced apparent activation constants of the holoenzymes compared to cAMP. The cellular uptake of the fluorescent analog was investigated by cAMP indicators. It was estimated that about 7 μM of the fluorescent cAMP analog is available to the indicator after one hour of incubation and that about 600 μM of the compound had to be added to intact cells to half-maximally dissociate a PKA type IIα sensor.

Conclusion

The novel analog combines good membrane permeability- comparable to 8-Br-cAMP – with superior spectral properties of a modern, red-shifted fluorophore. GFP-tagged regulatory subunits of PKA and the analog co-localized. Furthermore, it is a potent, PDE-resistant activator of PKA-I and -II, suitable for in vitro applications and spatial distribution evaluations in living cells.