Interaction of phosphorylated elongation factor EF-2 with nucleotides and ribosomes.
The intrinsic fluorescence emission spectrum of elongation factor EF-2 due to the 7 Trp residues was not modified after complete phosphorylation of the factor by the specific Ca2+/Calmodulin-dependent kinase III. The effect of nucleotide binding on this fluorescence revealed differences between phosphorylated and unmodified EF-2. Low concentrations of GTP had a smaller quenching effect on the fluorescence of phosphorylated EF-2 than on the fluorescence of unmodified EF-2, whereas GDP had exactly the same quenching effect on the fluorescence of both samples. These results suggest that phosphorylation of EF-2 decreased its affinity for GTP but not for GDP. Ability of phosphorylated EF-2 to form a ternary complex with ribosomes in the presence of a non-hydrolysable GTP analog and its ability to protect ribosomes against ricin-inactivation were both decreased to the same extent. The lower affinity of phosphorylated EF-2 for GTP could be responsible for a weaker and/or incorrect interaction of the factor with the ribosome, in particular with the ricin-site of the 28-S rRNA assumed to be involved in translocation initiation.
Dumont-Miscopein
A
A
Laboratoire de Biochimie Médicale, Institut de Biologie et Chimie des Protéines, UPR CNRS 412, 7, Lyon, France.
Lavergne
J P
JP
Guillot
D
D
Sontag
B
B
Reboud
J P
JP
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D012270:Ribosomes / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D009711:Nucleotides; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D010750:Phosphoproteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D017871:Calcium-Calmodulin-Dependent Protein Kinases