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 with respect to its spectral properties, its ability to bind to and activate three main isoenzymes of the cAMP-dependent protein kinase (PKA-Ialpha, PKA-IIalpha, PKA-IIbeta) 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 RIIalpha and RIIbeta subunits increases the amplitude of excitation and absorption maxima significantly; no major change was observed with RIalpha. In vitro binding of the compound to RIalpha subunit and activation of the PKA-Ialpha 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 muM of the fluorescent cAMP analog is available to the indicator after one hour of incubation and that about 600 muM of the compound had to be added to intact cells to half-maximally dissociate a PKA type IIalpha sensor.…
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