Photoaffinity labeling of elongation factor-2 with 8-azido derivatives of GTP and ATP.
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.
Guillot
D
D
Institut de Bilogie et Chimie des Protéines du CNRS, Lyon, France.
Vard
C
C
Reboud
J P
JP
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D008027:Light; D008969:Molecular Sequence Data; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D010446:Peptide Fragments
C021961:8-azidoguanosine triphosphate; D000345:Affinity Labels; D001386:Azides; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D010446:Peptide Fragments; C017040:8-azidoadenosine 5'-triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate