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J. Biol. Chem., Vol. 276, Issue 13, 10000-10009, March 30, 2001
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From the Institut für Medizinische Physik und Biophysik,
Humboldt-Universität zu Berlin, Universitätsklinikum
Charité, Schumannstrasse 20-21, 10098 Berlin, Germany
Despite the growing structural information on
receptors and G proteins, the information on affinities and kinetics of
protein-protein and protein-nucleotide interactions is still not
complete. In this study on photoactivated rhodopsin (R*) and the rod G
protein, Gt, we have used kinetic light scattering,
backed by direct biochemical assays, to follow G protein activation.
Our protocol includes the following: (i) to measure initial rates on
the background of rapid depletion of the GtGDP substrate;
(ii) to titrate GtGDP, GTP, and GDP; and (iii) to apply a
double displacement reaction scheme to describe the results. All data
are simultaneously fitted by one and the same set of parameters. We
obtain values of Km = 2200 Gt/µm2 for GtGDP and
Km = 230 µM for GTP; dissociation
constants are Kd = 530 Gt/µm2 for R*-GtGDP dissociation
and Kd = 270 µM for GDP release from
R*GtGDP, once formed. Maximal catalytic rates per
photoexcited rhodopsin are 600 Gt/s at 22 °C and 1300 Gt/s at 34 °C. The analysis provides a tool to allocate
and quantify better the effects of chemical or mutational protein
modifications to individual steps in signal transduction.
Maximal Rate and Nucleotide Dependence of Rhodopsin-catalyzed
Transducin Activation
INITIAL RATE ANALYSIS BASED ON A DOUBLE DISPLACEMENT
MECHANISM*
and
*
This work was supported by Grant SFB 366 from the Deutsche
Forschungsgemeinschaft (to K. P. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence may be addressed: Institut für
Medizinische Physik und Biophysik, Humboldt-Universität zu
Berlin, Universitätsklinikum Charité, Schumannstrasse
20-21, 10098 Berlin, Germany. Tel.: 49-30-450-524111; Fax:
49-30-450-524952; E-mail: martin.heck@charite.de.
§
To whom correspondence may be addressed: Institut für
Medizinische Physik und Biophysik, Humboldt-Universität zu
Berlin, Universitätsklinikum Charité, Schumannstrasse
20-21, 10098 Berlin, Germany. Tel.: 49-30-450-524111; Fax:
49-30-450-524952; E-mail: kph@charite.de.
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