A sensitive test system for toxin-treated ribosomes was worked out by treating rabbit reticulocyte ribosomes with abrin A-chain, ricin A-chain or ricinus agglutinin A-chain, adding neutralizing amounts of specific antitoxins and testing for polyphenylalanine-synthesizing activity in a system where the concentration of elongation factors and ribosomes were varied. The strongest inhibition was obtained in the presence of low concentrations of elongation factor 2 (EF-2).The activity of the ribosomes decreased with time of incubation with the toxin A-chains. Addition of anti-toxins stopped further inactivation. In systems containing untreated and toxin-treated ribosomes the ability to polymerize phenylalanine was proportional to the concentration of untreated ribosomes.There was a linear relationship between toxin A-chain concentration and the number of ribosomes inactivated per minute. The inactivation rate increased with temperature, and the estimated activation energy was 10.6 kcal(44.3 kJ). Linewaver-Burk plots of the data obtained by incubating various ribosome concentrations with toxins indicated a molecular activity of about 1500 ribosomes/minute for abrin and rich A-chains and 100 ribosomes/minute for ricinus agglutinin A-chain. The apparent Michaelis constant was 0.1 -0.2 pM for all three A-chains. The activity of the A-chains in the intact cell is discussed.

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The effects of ricin on the different steps of the elongation cycle of protein synthesis in a rabbit reticulocyte cell-free system are studied in this paper. The toxin most probably acts by catalytically inactivating the ribosomes, since a single molecule of the toxin can inactivate 300 ribosomes for poly(U)-directed phenylalanine incorporation. The effect of the toxin on the ribosome is irreversible.Ricin specifically inhibits elongation-factor-1-dependent aminoacyl-tRNA binding to ribosomes but has no effect on the non-enzymic binding of aminoacyl-tRNA. Ricin also inhibits formation of the complex elongation-factor-2 . ribosome . nucleotide with GTP, GDP or GMP-P(CH,)P.However, the toxin has no effect on translocation. These apparently conflicting results are discussed in this study.Ricin and abrin are two closely related proteins isolated from seeds of Ricinus communis and Abrus precatorius respectively [l, 21. These proteins are very toxic to mammals [3] and display antitumour activity [4]. Ricin and abrin appear to have a similar mode of action, although a number of differences have been reported concerning their primary structure and physicochemical properties [5-81. Abrin is composed of two subunits [9,10] the A chain or effectomer ( M , 30000) and the B chain or haptomer ( M , 35 000) [l 11. A and B chains very similar to those of abrin are present in the ricin molecule [7,8,11]. The B chain of ricin attaches to galactose-containing receptors on the cell surface [lo, 111 and only then is the A chain able to enter the cell, possibly by pinocytosis, being finally released in the cytoplasm. The A chain is then a very active inhibitor of protein synthesis [1,12] but cannot penetrate into the cell in the absence of the B chain. Hybrids of the A chain of ricin and B chain of abrin, and vice versa, are very active inhibitors of mammalian cells [8].Studies on cell-free systems from mammalian cells have shown that ricin is a potent inhibitor of protein synthesis [l, 12-141 by inactivating the ribosome [15] at the level of the 60-S subunit [16]. Ricin is a strong inhibitor of the elongation phase of protein synthesis but no clear-cut effect of the toxin was observed in the individual steps of this process [14].

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The inactivation of rabbit reticulocyte ribosomes by abrin and ricin A-chains was studied by incubating ribosomes with the A-chains and testing, after various periods of time, aliquots of the ribosomes for their ability to polymerize phenylalanine.The presence of elongation factor 2 (EF-2) reduced the rate of inactivation of ribosomes by the A-chains. The protective effect of EF-2 was strongly enhanced by GTP and, to a lesser extent, also by GDP or dGTP. Other nucleotides had no demonstrable effect.Much less protection was found after binding of Phe-tRNA to ribosomes in the presence of EF-1 (enzymic binding) or in the presence of high Mg2 + concentration (non-enzymic binding). The data indicate that when EF-2 binds to the ribosomes it completely or partially covers the target site for abrin and ricin A-chains. The possibility that EF-1 also binds to this site is discussed.The two plant toxins abrin and ricin both consist of two polypeptide chains, one of which, the B-chain or 'haptomer', binds the toxins to cell surface receptors, whereas the other one, the A-chain or 'effectomer', penetrates into the cytoplasm and inhibits protein synthesis by inactivating irreversibly the 60-S ribosomal subunits by an enzymic process [l-41. After A-chain treatment the ribosomes show reduced ability to hydrolyze GTP in the presence of the elongation factors EF-1 and EF-2 [4,5]. Furthermore their ability to bind EF-2 in the presence of GTP ([5,6] and R. Nolan, personal communication) and to bind enzymically aminoacyl-tRNA [6] is strongly reduced.In a previous paper [7] we have described the kinetics of the A-chain-catalyzed ribosome inactivation. In the present report we demonstrate that EF-2 in the presence of GTP is able to protect ribosomes against the action of abrin and ricin A-chains. MATERIALS AND METHODS Toxins and Anti-toxinsAbrin and ricin A-chains were prepared as earlier described [8,9]. Anti-abrin and anti-ricin were produced by immunizing rabbits with toxoids prepared Abbreviation. EF-1, EF-2, elongation factors 1 and 2 by formaldehyde treatment of the toxins [8,9], and the anti-A-chain antibodies were isolated by passing the sera through columns containing the isolated abrin and ricin A-chains, and eluting them with lowpH buffer as earlier described [lo]. The anti-A-chain antibodies were quantitated as earlier described [lo]. 1 pl of anti-abrin-A-chain solution here used inactivated 1 pg abrin A-chain, and 1 pl of anti-ricin-A-chain inactivated 0.3 pg rich A-chain. RibosomesRabbit reticulocyte ribosomes were prepared as earlier described [6]. The ribosomes were washed with 0.5 M KC1, and resuspended in 50 mM Tris-HCI (pH 7.4), 25 mM KC1, 5 mM MgC1, and 1 mM dithiothreitol and stored in small aliquots in liquid nitrogen. Elongation FactorsElongation factors EF-1 and EF-2 were prepared from the reticulocyte S-100 fraction (supernatant from 1OOOOOxg spin) and purified by ammonium sulphate fractionation and gel filtration on Sephadex G-200. EF-1 was further purified by chromatography on hydroxyapatite, and EF-2 by ch...

The mode and site of action of inhibitors of translation (initiation, elongation and termination of protein synthesis) in eukaryotic systems is reviewed. The isolation and characterization of a factor is described that binds Ac-Phe-tRNA to form a complex made up of binding factors, Ac-Phe-tRNA, and ribosome. The binding of Ac-Phe-tRNA probably occurs at the ribosomal site involved in the binding of the initiator substrate Met-tRNAF. The effect of inhibitors of the intitiation phase of protein synthesis on the nonenzymic Ac-Phe-tRNA binding to ribosomes is investigated. The two sites translocation model for translation in eukaryotic cells is presented and the effects of inhibitors on the various steps of protein synthesis are determined empirically. The site of action of inhibitors of peptide bond formation at the ribosomal peptidyl transferase center is elucidated. The action of inhibitors of translocation is sutdied in model cell-free systems from human cells. In addition, a number of methylxanthines are shown to enhance the elongation phase in polypeptide synthesis by stimulating the enzymic binding of aminoacyl-tRNA. The effect of caffeine, theophylline and its derivatives are shown to be fairly specific and dependent on the ribosome concentration. Aminophylline is shown to have a similar effect but also enhances aminoacyl-tRNA synthetase activity at low Mg++ concentrations, probably displacing the optimal concentration of Mg++ in the reaction. This second effect of aminophylline appears to be due to the ethylenediamine moiety of aminophylline since it is also observed in the presence of different polyamines but not in the presence of caffeine or theophylline.

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