Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP an
June 28th, 2008 | by admin |Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog.
ABSTRACT: BACKGROUND: A novel, fluorescent cAMP analog, 8- [Pharos-575]- adenosine- 3\’, 5\’- cyclic monophosphate was characterized in its spectral, biochemical and cellular properties. The ability of the analog to interact and activate different isoforms of the cAMP-dependent protein kinase (PKA-I and -II) was demonstrated using fluorescence polarization and spectrophotometric assays, respectively. Finally, resonance energy transfer based indicators were utilized to visualize its permeability into living cells. RESULTS: The Pharos-575 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 8-Pharos-575-cAMP with PKA-II regulatory (R) subunits increases the amplitude of excitation and absorption maxima significantly; however, no major change was observed with PKA-I. In vitro binding of the compound to RI subunit and activation of the PKA-I holoenzyme was essentially identical to cAMP. In contrast, the RII subunits bound the fluorescent analog up to ten times less efficiently, resulting in about two times reduced apparent activation constants of the respective holoenzymes. The cellular uptake of the fluorescent analog was monitored side by side with an already well established cAMP indicator, Sp-5,6-DCI-cBIMPS. Finally it was demonstrated, that regulatory subunits of PKA-I and -II tagged with green fluorescent protein co-localized with the fluorescent cAMP analog in living cells. CONCLUSIONS: The novel cAMP analog 8-Pharos-575-cAMP combines good membrane permeability- comparable to the commonly used cAMP analog 8-Br-cAMP - with superior spectral properties of a modern, red-shifted fluorophore. It is a potent, PDE-resistant activator of PKA-I and aII, suitable for in vitro applications and spatial distribution evaluations in living cells.
Moll D, Prinz A, Brendel CM, Berrera M, Guske K, Zaccolo M, Genieser HG, Herberg FW.