Effect of modeccin on the steps of peptide-chain elongation

Abstract

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.

Bryodin, a ribosome-inactivating protein from the roots of Bryonia dioica L. (white bryony)

“…This makes bryodin suitable for the preparation of immunotoxins or Vol. 240 ]phenylalanyl-tRNA and 20 pmol of rabbit reticulocyte ribosomes, calculated as described by Montanaro et al (1978). After incubation at 24°C for 30 min, 0.25 ml of 10% (w/v) trichloroacetic acid was added, and the acid-insoluble radioactivity was determined as described by Montanaro et al (1978).…”

Enzymatic activity of toxic and non‐toxic type 2 ribosome‐inactivating proteins

“…Rat liver ribosomes were prepared essentially as described elsewhere [24] in RNase‐free conditions. Their concentration was determined by the A 260 as described in [25], assuming that 12.5 absorbance units/ml was equivalent to 1 mg/ml and that 1 mg contained 250 pmol of ribosomes. Ribosomes were stored in aliquots at 80°C.…”

Isolation and partial characterization of a new ribosome-inactivating protein from Petrocoptis glaucifolia (Lag.) Boiss

“…The homogenate was centrifuged 10 min at 10000 9 g at 2 ~ C. The supernatant was centrifuged for 15 min at 30000 9 g at 2 ~ C. The supernatant was diluted 1:1 with cold homogenization buffer containing 2 % (w/v) Triton X-100 and 1% (w/v) sodium deoxicholate, and then centrifuged 3 h at 105000 "g at 2 ~ C. The pellet was resuspended in 24 ml of homogenization buffer containing 0.5 M KC1, and was centrifuged 10 min at 20000. g at 2 ~ C. The supernatant was centrifuged on a cushion of 3 ml of homogenization buffer containing 60% (w/v) sucrose. A 1-mg aliquot of ribosomes was assumed to contain 250 pmol of ribosomes (Montanaro et al 1978). …”