The mechanism of protein synthesis inhibition by the toxic lectins, abrin and ricin, has been studied in crude and in purified cell-free systems from rabbit reticulocytes and Krebs 11 ascites cells.In crude systems abrin and rich strongly inhibited protein synthesis from added aminoacyl-tRNA, demonstrating that the toxins act at some point after the charging of tRNA.Supernatant factors and polysomes washed free of elongation factors were treated separately with the toxins and then neutralizing amounts of anti-toxins were added. Recombination experiments between toxin-treated ribosomes and untreated supernatant factors and vice versa showed that the toxin-treated ribosomes had lost most of their ability to support polyphenylalanine synthesis, whereas treatment of the supernatant factors with the toxins did not inhibit polypeptide synthesis.Recombination experiments between toxin-treated isolated 40-S subunits and untreated 60-S subunits and vice versa showed that only when the 60-S subunits had been treated with the toxins was protein synthesis inhibited in the reconstituted system. The incorporation of [3H]puromycin into nascent peptide chains was unaffected by the toxins, indicating that the peptidyl transferase is not inhibited.Both the EF-1-catalyzed and the EF-2-catalyzed ability of the ribosomes to hydrolyze [ Y -~~P I G T P was inhibited by abrin and ricin.An 8-S complex released from the 6 0 4 subunit by EDTA treatment possessed both GTPase and ATPase activity, while the particle remaining after the EDTA treatment had lost most of its GTPase activity. Both enzyme activities of the 8-S complex were inhibited by abrin and ricin.The present data indicate that there is a common site on the 6 0 4 subunits for EF-1-and EF-2-stimulated GTPase activity and they suggest that abrin and ricin inhibit protein synthesis by modifying this site.In previous papers we have reported that the toxic plant proteins abrin and ricin inhibit protein synthesis in eucaryotic cells by interfering with peptide chain elongation [1,2]. Both abrin and ricin consist of two polypeptide chains, the A-chain or 'effectomer' and the B-chain or 'haptomer' [3]. The function of the B-chains is to bind the toxins to the cell surface and it is, therefore, necessary for the cellular uptake of the toxin. The ability of the toxins to inhibit protein synthesis in cell-free systems is associated exclusively with the A-chains [4,5]. Experiments in our laboratory [6] as well as by Montanaro et al. [7] indicate that the ribosomes are the targets for abrin and ricin. In contrast to many antibiotics, which inhibit protein synthesis by being bound to the ribosomes [8], abrin and ricin inactivate the ribosomes by a catalytic mechanism ([6] and unpublished data).In the present study experiments were performed to identify the step in peptide chain elongation which is inhibited by abrin and ricin and to elucidate in more detail the mechanism of action of these toxins. MATERIALS AND METHODS ToxinsAbrin and ricin and their A-chains were prepared as earlier desc...

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Binding of g~nosine nucIeotid~ to puriikd native and ADP-~~syiat~ wheat germ EF-2 was measured. Both forms of EF-2 hound t3aGDP to the same extent.[3H]GDP binding to native but not to ADP-ribosylated EF-2 was reduced in the presence of GTF and ribosomes. Binding of [Y-~~P]GTP to EF-2 was significantly reduced upon ADP-ribosylation. ADP-ribosylation almost abolished both the stimulatory effect of ribosomes on GTP binding to EF-2 and the ability of EF-2 to form a high-affinity complex with GuoPP(CH&P and ribosomes. Low-affinity complex formation between EF-2. GDP and ribosomes was not influenced by ADP-ribosylation. The results indicate that the inhibition of the elongation process caused by the toxin is probably due to the inability of modified EF-2 to exchange GDP with GTP.

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Cell‐free systems from wheat germ were preincubated before and after addition of immunoglobulin mRNA to generate systems dependent and independent on peptide chain initiation, respectively. When abrin A chain was added, the system dependent on peptide chain initiation was much more strongly inhibited than the system measuring primarily chain elongation. Sucrose density gradient analysis showed that abrin A chain did not affect the binding of labelled methionine or labelled mRNA to the 40‐S subunit. However, it caused a strong reduction in the amount of labelled mRNA present in the 80‐S and polysome region. The results indicate that abrin A chain interferes with initiation of protein synthesis by inhibiting the binding of the 40‐S initiation complex to the 60‐S subunit to form the 80‐S initiation complex. The inhibiting effect of abrin A chain on poly(U)‐directed synthesis of polyphenylalanine was partly overcome by increasing the Mg2+ concentration. A similar effect of Mg2+ was found also in a cell‐free system from Krebs II ascites cells under conditions where primarily chain elongation was measured. Experiments with ribosomes treated with abrin A chain at different concentrations of MgCl2 and then tested for their ability to support polymerization of phenylalanine showed that high magnesium concentrations do not protect the ribosomes against the attack of abrin A chain. The data suggest that high Mg2+ concentrations tend to reverse toxin‐induced conformation changes in the ribosomes.