Interactions of adenosine diphosphate-ribosylated elongation factor 2 with ribosomes.
The binding of adenosine diphosphate-ribosylated elongation factor 2 (ADPRib-EF-2) to ribosomes was inhibited both in the presence and absence of GTP in proportion to the amounts of unmodified EF-2 added. Concomitant with this inhibition, an increase in the activity of ribosome-bound EF-2 in polyphenylalanine synthesis was observed. On the other hand, the addition of ADPRib-EF-2 reduced the rate of poly(Phe) synthesis observed in the presence of a saturating amount of EF-2 and increased the amount of EF-2 required for the half-maximal rate of poly(Phe) synthesis. Phe-tRNA, nonenzymatically bound to the ribosome in the presence of poly(U), inhibited the subsequent binding of ADPHRib-EF-2. The same ribosomal population appeared to preferentially bind either aminoacyl-tRNA or ADPRib-EF-2. The Scatchard plot of the binding of ADPRib-EF-2 to the ribosome in the presence of GTP revealed the presence of two ribosomal binding sites (or ribosomal populations) with apparent different affinities for the modified factor (K371 degrees d,1 = 6.6 nM and K37 degrees d,2 = 126 nM). At saturating concentrations of ADPRib-EF-2, a maximum of about 1 molecule of the factor was bound per ribosome. The binding of ADPRib-EF-2 to the ribosome was stimulated by GTP. The binding of radioactive GTP to the ribosome was observed concomitantly with the binding of ADPRib-EF-2. One mole of GTP was bound per mole of ADPRib-EF-2. No significant difference could be found in the binding of GTP to ribosome required in the presence of either EF-2 or ADPRib-EF-2. The binding of ADPRib-EF-2 to the ribosome required the presence of Mg2+ and reached a maximum at 5 mM. The binding was greatest at K+ concentrations below 20 mM. ADPRib-EF-2 was bound primarily to the large ribosomal subunit. A slight, but reproducible binding to the 40 S subunit was also observed. The addition of 40 S to 60 S subunits stimulated the binding of ADPRib-EF-2. GTP displayed a stimulatory effect on the binding only in the presence of recombined subunits. Human ADPRib-EF-2 was bound to rat liver ribosomes as efficiently as to human tonsil ribosomes, while the binding to Escherichia coli ribosomes was insignificant.
D000247:Adenosine Diphosphate Sugars / Q000378:metabolism*; D000818:Animals; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D014066:Palatine Tonsil / Q000378:metabolism; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects; D010445:Peptide Elongation Factors; D010649:Phenylalanine; D011485:Protein Binding; D012343:RNA, Transfer / Q000378:metabolism; D051381:Rats; D012266:Ribose / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*
D000247:Adenosine Diphosphate Sugars; D009702:Nucleoside Diphosphate Sugars; D010445:Peptide Elongation Factors; D010649:Phenylalanine; D012266:Ribose; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D008274:Magnesium