The high fluorescence of adenine-containing compounds after reaction with chloroacetaldehyde was used to measure the adenine released from rat liver and Artemia salina ribosomes by the action of ricin A chain and gelonin, two ribosome-inactivating proteins (RIPs) that share the same mechanism of action, consisting in the hydrolysis of the N-glycosidic bond of A-4324 of 28 S rRNA. Two methods were employed: (i) h.p.l.c. of the chloroacetaldehyde-reactive material released by RIPs; h.p.l.c. associated with a fluorescence detector allows the identification of adenine and its dosage at quantities as low as 2 ng; (ii) the direct fluorimetric measurement of the material that had reacted with chloroacetaldehyde. The amount of adenine released increases when ribosomes are pretreated in conditions that lead to their dissociation into subunits. Adenine protects ribosomes from the inhibition by ricin A-chain. When ribosomes were incubated with ricin A-chain in the presence of [14C]adenine no incorporation of radioisotope in ribosomes was observed, indicating that neither exchange nor reversal reactions occurred. A binding of [14C]adenine to ricin A chain was not detected by equilibrium dialysis.

alpha-Sarcin from Aspergillus giganteus and the ribosome-inactivating proteins (RIPs) from higher plants inactivate the 60 S ribosomal subunit. The former is an RNAase, whereas RIPs are N-glycosidases. The site of cleavage of RNA and that of N-glycosidic depurinization are at one nucleotide distance in 28 S rRNA [Endo & Tsurugi (1987) J. Biol. Chem. 262, 8128-8130]. The effect of alpha-sarcin and that of RIPs on the interaction of elongation factors with Artemia salina (brine shrimp) ribosomes have been investigated. alpha-Sarcin inhibits both the EF1 (elongation factor 1)-dependent binding of aminoacyl-tRNA and the GTP-dependent binding of EF2 (elongation factor 2) to ribosomes, whereas two of the RIPs tested, ricin from Ricinus communis (castor bean) and volkensin from Adenia volkensii (kilyambiti), inhibit only the latter reaction. EF2 protects ribosomes from inactivation by both alpha-sarcin and ricin. The EF1-binding site is affected only by alpha-sarcin. The sensitivity of this site to alpha-sarcin is increased by pretreatment of ribosomes with ricin. A. salina ribosomes were highly resistant to the third RIP tested, namely gelonin from Gelonium multiflorum. All four proteins tested have, however, a comparable activity on the rabbit reticulocyte-lysate system.

1. A haemagglutinating lectin was purified from the seeds of Momordica charantia by affinity chromatography on Sepharose 4B and on acid-treated Sepharose 6B. It has mol.wt. 115 000 and consists of four subunits, of mol.wts. 30 500, 29 000, 28 500 and 27 000. 2. The lectin inhibits protein synthesis by a rabbit reticulocyte lysate with an ID50 (concentration giving 50% inhibition) of approx. 5 micrograms/ml. Protein synthesis by Yoshida ascites cells is partially inhibited by the lectin at a concentration of 100 micrograms/ml. 3. From the same seeds another protein was purified which has mol.wt. 23 000 and is a very potent inhibitor of protein synthesis in the lysate system, with an ID50 of 1.8 ng/ml. This inhibitor has no effect on protein synthesis by Yoshida cells, and has no haemagglutinating properties. 4. Artemia salina ribosomes preincubated with the lectin or with the inhibitor lose their capacity to perform protein synthesis. The proteins seem to act catalytically, since they inactivate a molar excess of ribosomes. 5. The lectin and the inhibitor are somewhat toxic to mice, the LD50 being 316 and 340 micrograms/100 g body wt. respectively.

Modeccin inhibits polypeptide-chain elongation catalysed by Artemia salina (brine shrimp) ribosomes by inactivating the 60 S ribosomal subunit. Among the individual steps of elongation, peptide-bond formation, catalysed by 60S peptidyltransferase, is unaffected by the toxin, whereas the binding of EF 2 (elongation factor 2) to ribosomes is strongly inhibited. Modeccin does not affect the poly(U)-dependent non-enzymic binding of either deacylated tRNAPhC or phenylalanyl-tRNA to ribosomes. The inhibitory effect of modeccin on the EF 1 (elongation factor 1)-dependent binding of phenylalanyltRNA is discussed, since it is decreased by tRNAPhC, which stimulates the binding reaction. The analysis of the distribution of ribosome-bound radioactivity during protein synthesis shows that modeccin consistently inhibits the radioactivity bound as longchain peptides, but, depending on the experimental conditions, can leave unchanged or even greatly stimulates the radioactivity bound as phenylalanyl-tRNA and/or shortchain peptides. It is concluded that, during the complete elongation cycle, modeccin does not affect the binding of the first aminoacyl-tRNA to ribosomes, but inhibits some step in the subsequent repetitive activity of either EF 1 or EF 2. The results obtained indicate that the mechanism of action of modeccin is very similar to that of ricin and related plant toxins such as abrin and crotin.

Inactivation of Artemia salina and rabbit ribosomes by gelonin requires ATP and a high-Mr factor present in the rabbit reticulocyte-lysate post-ribosomal supernatant. The kinetic constants of the gelonin-catalysed release of adenine from A. salina ribosomes are Km = 4.35 microM and Kcat. = 0.1 min-1 in the absence of cofactors, and Km = 1.15 microM and Kcat. = 108 min-1 in their presence. The last two values are similar to those measured for ricin A chain in the absence of cofactors (Km = 2.02 microM and Kcat. = 317 min-1).