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Resonance energy transfer as a direct monitor of GTP-binding protein-effector interactions: activated .alpha.-transducin binding to the cGMP phosphodiesterase in the bovine phototransduction cascade
- Source :
- Biochemistry. 30:7112-7118
- Publication Year :
- 1991
- Publisher :
- American Chemical Society (ACS), 1991.
-
Abstract
- Resonance energy-transfer approaches have been used to directly monitor the interactions of the GTP gamma S-bound alpha subunit of transducin (alpha T GTP gamma S) with the retinal cyclic GMP phosphodiesterase (PDE). The PDE was labeled with 5-(iodoacetamido) fluorescein (IAF-PDE) and served as the fluorescence donor in these experiments while the alpha T GTP gamma S was labeled with eosin-5-isothiocyanate (EITC-alpha T GTP gamma S) and served as the energy acceptor. The EITC-alpha T GTP gamma S species was able to quench a significant percentage of the IAF-PDE fluorescence (typically greater than or equal to 30%) due to resonance energy transfer between the IAF and EITC moieties. The quenching by the EITC-alpha T GTP gamma S species was dose-dependent, saturable (Kd = 21 nM), and specific for the GTP gamma S-bound form of the alpha T subunit. Limited trypsin treatment of the IAF-PDE, which selectively removes a fluorescein-labeled gamma subunit (gamma PDE), completely eliminates the quenching of the IAF fluorescence by the EITC-alpha T GTP gamma S complex. Although the EITC-alpha T GTP gamma S complex competes with the unlabeled alpha T GTP gamma S for a binding site on the IAF-PDE, as well as for a site on the native PDE, it is not able to stimulate PDE activity. Thus, the modification of a single EITC-reactive residue on the alpha T GTP gamma S complex prevents this subunit from eliciting a key activation event within the retinal effector enzyme.
- Subjects :
- GTP'
Photochemistry
G protein
Protein subunit
In Vitro Techniques
Biochemistry
3',5'-Cyclic-GMP Phosphodiesterases
GTP-Binding Proteins
Animals
Trypsin
heterocyclic compounds
Transducin
Binding site
G alpha subunit
Chemistry
Hydrolysis
Phosphodiesterase
Rod Cell Outer Segment
Enzyme Activation
Spectrometry, Fluorescence
Energy Transfer
Guanosine 5'-O-(3-Thiotriphosphate)
Eosine Yellowish-(YS)
Cattle
sense organs
Gamma subunit
Subjects
Details
- ISSN :
- 15204995 and 00062960
- Volume :
- 30
- Database :
- OpenAIRE
- Journal :
- Biochemistry
- Accession number :
- edsair.doi.dedup.....6560c3802b3193b4ff34b25f637bad83