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J Biol Chem. 2001 Mar 30;276(13):10000-9. Epub 2000 Dec 14.

Maximal rate and nucleotide dependence of rhodopsin-catalyzed transducin activation: initial rate analysis based on a double displacement mechanism.

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1
Institut für Medizinische Physik und Biophysik, Humboldt-Universität zu Berlin, Universitätsklinikum Charité, Schumannstrasse 20-21, 10098 Berlin, Germany. martin.heck@charite.de

Abstract

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, G(t), 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 G(t)GDP substrate; (ii) to titrate G(t)GDP, 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 K(m) = 2200 G(t)/microm(2) for G(t)GDP and K(m) = 230 microm for GTP; dissociation constants are K(d) = 530 G(t)/microm(2) for R*-G(t)GDP dissociation and K(d) = 270 microm for GDP release from R*G(t)GDP, once formed. Maximal catalytic rates per photoexcited rhodopsin are 600 G(t)/s at 22 degrees C and 1300 G(t)/s at 34 degrees 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.

PMID:
11116153
DOI:
10.1074/jbc.M009475200
[Indexed for MEDLINE]
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