Authors: Andrea D Hoffman Ina L Urbatsch Pia D Vogel
Publish Date: 2010/06/19
Volume: 29, Issue: 5, Pages: 373-379
Abstract
We used a spinlabeled ATP analog SLATP to study nucleotide binding to highly purified human multidrug resistance protein 3 MRP3 which had been expressed in the yeast Pichia pastoris SLATP was shown to be a good substrate analog and is hydrolyzed by MRP3 at about 10 of the Vmax for normal ATP ESR titrations showed that 2 mol of SLATP readily bound per mole of MRP3 with a dissociation constant of about 100 μM in the presence of Mg2+ ions The binding curve was easily fitted for a hyperbolic binding relationship SLATP also bound readily to MRP3 in the absence of divalent ions and presence of EDTA The resulting binding curve however could not be satisfactorily fitted using the equation for hyperbola Analysis showed that a good fit was only obtained with the Hill equation using a Hill coefficient of 4 or close to 4 Lower Hill coefficients resulted in lower goodness of the fit Such cooperative binding may be explained by a dimerization event triggered in the absence of divalent ions and a close communication of nucleotide binding sites of the interacting dimers These findings may be of great importance for the overall mechanism and regulation of multidrug resistance proteinsThis work was supported by a grant from the SMU University Research Council to PDV and a grant from the Jasper and Jack Wilson Foundation to ILU The authors wish to thank J G Wise for helpful discussions The authors wish to thank Dr Jürgen Zirkel Lipoid GmbH Germany for providing us with 100 pure lysophosphatidyl cholin
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