Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog
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 stabi...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Journal article |
| Language: | English |
| Published: |
2008
|
| _version_ | 1826266469023875072 |
|---|---|
| author | Moll, D Prinz, A Brendel, C Berrera, M Guske, K Zaccolo, M Genieser, H Herberg, F |
| author_facet | Moll, D Prinz, A Brendel, C Berrera, M Guske, K Zaccolo, M Genieser, H Herberg, F |
| author_sort | Moll, D |
| collection | OXFORD |
| description | 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. © 2008 Moll et al; licensee BioMed Central Ltd. |
| first_indexed | 2024-03-06T20:39:25Z |
| format | Journal article |
| id | oxford-uuid:33c35f31-7689-4e66-b675-1a4361105d75 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T20:39:25Z |
| publishDate | 2008 |
| record_format | dspace |
| spelling | oxford-uuid:33c35f31-7689-4e66-b675-1a4361105d752022-03-26T13:22:07ZBiochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analogJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:33c35f31-7689-4e66-b675-1a4361105d75EnglishSymplectic Elements at Oxford2008Moll, DPrinz, ABrendel, CBerrera, MGuske, KZaccolo, MGenieser, HHerberg, FBackground. 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. © 2008 Moll et al; licensee BioMed Central Ltd. |
| spellingShingle | Moll, D Prinz, A Brendel, C Berrera, M Guske, K Zaccolo, M Genieser, H Herberg, F Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog |
| title | Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog |
| title_full | Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog |
| title_fullStr | Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog |
| title_full_unstemmed | Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog |
| title_short | Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog |
| title_sort | biochemical characterization and cellular imaging of a novel membrane permeable fluorescent camp analog |
| work_keys_str_mv | AT molld biochemicalcharacterizationandcellularimagingofanovelmembranepermeablefluorescentcampanalog AT prinza biochemicalcharacterizationandcellularimagingofanovelmembranepermeablefluorescentcampanalog AT brendelc biochemicalcharacterizationandcellularimagingofanovelmembranepermeablefluorescentcampanalog AT berreram biochemicalcharacterizationandcellularimagingofanovelmembranepermeablefluorescentcampanalog AT guskek biochemicalcharacterizationandcellularimagingofanovelmembranepermeablefluorescentcampanalog AT zaccolom biochemicalcharacterizationandcellularimagingofanovelmembranepermeablefluorescentcampanalog AT genieserh biochemicalcharacterizationandcellularimagingofanovelmembranepermeablefluorescentcampanalog AT herbergf biochemicalcharacterizationandcellularimagingofanovelmembranepermeablefluorescentcampanalog |