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Preparation and characterization of nucleotide-free and metal ion-free p21 "apoprotein"

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Feuerstein,  Jürgen
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Goody,  Roger S.
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Wittinghofer,  Alfred
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Citation

Feuerstein, J., Goody, R. S., & Wittinghofer, A. (1987). Preparation and characterization of nucleotide-free and metal ion-free p21 "apoprotein". The Journal of Biological Chemistry, 262(18), 8455-8458. Retrieved from https://pubmed.ncbi.nlm.nih.gov/3298232/.


Cite as: http://hdl.handle.net/21.11116/0000-0007-9FBF-0
Abstract
p21 isolated under nondenaturing conditions is obtained as a complex with guanosine nucleotides and magnesium ions. We have developed a high performance liquid chromatography method which removes greater than 95% of bound nucleotide and the metal ion very rapidly under mild conditions. At the same time, p21 is purified from minor protein impurities. The protein thus prepared is thermally much less stable than the complexed p21, but can be used for studying its interaction with nucleotides and metal ions at low temperatures. The association rate constant for p21 and GDP is 1.47 X 10(6) M-1 s-1 and for GTP is 2.9 X 10(6) M-1 s-1 at 0 degree C. By using appropriately determined dissociation rate constants we have determined the binding constant for p21.GDP and p21.GTP in the presence of excess Mg2+ to be 5.7 X 10(10) M-1 and 6.0 X 10(10) M-1, respectively, at 0 degree C.