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Photoaffinity Labeling of Elongation Factor-2 with 8-Azido Derivatives of GTP and ATP

Content Provider	Scilit
Author	Guillot, Dominique Yard, Cecile Reboud, Jean-Paul
Copyright Year	1996
Description	Journal: Journal of Biological Inorganic Chemistry Elongation factor 2 (eEF-2) can interact not only with guanylic nucleotides but also with adenylic ones, as was shown by intrinsic fluorescence quenching studies [Sontag, B., Reboud, A.M., Divita, G., Di Pietro, A., Guillot, D. & Reboud, J.P. (1993) Biochemistry 32, 1976-1980]. Here we studied sites of these interactions by using photoactivable 8-azido-[gamma-32P]GTP and 8-azido-[gamma-32P]ATP. Photoincorporation of the radioactive GTP derivative into eEF-2 was prevented by the previous addition of GTP and GDP. The addition of adenylic nucleotides (ATP, ADP) and some adenylic derivatives [NAD+, NADH,poly(A)] decreased the photoincorporation by only 40% at most. However, photoincorporation of the radioactive ATP derivative was prevented by the previous addition not only of adenylic compounds [ATP, ADP, NAD+, NADH, poly(A)] but also of GTP and GDP. Photoincorporation of radioactive nucleotide derivatives was not decreased by the addition of other nucleotidic compounds [UTP, poly(U), ITP, NADP+, NADPH]. ATP and GTP acted as non-competitive inhibitors of the photoincorporation of 8-azido-[gamma-32P]GTP and 8-azido-[gamma-32P]ATP, respectively. eEF-2 photolabeled with these radioactive nucleotide derivatives was submitted to trypsin digestion under different conditions and the labeled peptidic fragments identified after HPLC purification and gel electrophoresis by N-terminal sequencing. An octapeptide, Y264FDPANGK271, was the only peptide photolabeled with 8-azido-[gamma-32P]GTP whereas a N-terminal fragment of about 7 kDa was the only one photolabeled with 8-azido-[gamma-32P]ATP. The different results support the hypothesis that guanylic and adenylic nucleotides do not interact with the same site of eEF-2.
Related Links	http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1996.00149.x/pdf
Ending Page	154
Page Count	6
Starting Page	149
ISSN	09498257
e-ISSN	14321327
DOI	10.1111/j.1432-1033.1996.00149.x
Journal	Journal of Biological Inorganic Chemistry
Issue Number	1
Volume Number	236
Language	English
Publisher	Wiley-Blackwell
Publisher Date	1996-02-15
Access Restriction	Open
Subject Keyword	Journal: Journal of Biological Inorganic Chemistry Biochemistry and Molecular Biology Photoaffinity Labeling Elongation Factor‐2
Content Type	Text
Resource Type	Article
Subject	Biochemistry Inorganic Chemistry

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