Ribosome inactivation by ricin A chain: A sensitive method to assess the activity of wild-type and mutant polypeptides

M. J. May, M. R. Hartley, L. M. Roberts, Paul A Krieg, R. W. Osborn, J. M. Lord

Research output: Contribution to journalArticle

78 Citations (Scopus)

Abstract

When recombinant ricin A chain transcripts are translated in a rabbit reticulocyte lysate the ribosomes are rapidly inactivated as shown by their inability to support translation of yeast preproalpha factor or chicken lysozyme transcripts added subsequently. In contrast, ribosomes which have translated transcripts encoding non-toxic polypeptides such as ricin B chain, readily translate the second transcript under identical conditions. Ribosome inactivation is accompanied by a highly specific modification of 28S rRNA which occurs at the same position as the N-glycosidic cleavage of an adenine residue and which is thought to cause inactivation of the ribosomes. Protein synthesis by wheat germ ribosomes was not inhibited under the conditions which inhibit reticulocyte ribosomes confirming earlier observations that plant cytoplasmic ribosomes are much less sensitive to inhibition by ricin A chain than are mammalian ribosomes. Using the same assay we have shown that deleting an internal hexapeptide, which shares homology with hamster elongation factor-2, completely abolishes catalytic activity. Deleting a second pentapeptide conserved between ricin A chain and the ribosome-inactivating plant toxin trichosanthin, had no effect. Deleting the first nine residues from the N-terminus of A chain did not affect toxicity whereas deleting a further three residues inactivated the polypeptide. Point mutations which individually converted arginine 48 and arginine 56 of ricin A chain to alanine residues or which deleted arginine 56 were also without effect on the catalytic activity of the toxin.

Original languageEnglish (US)
Pages (from-to)301-308
Number of pages8
JournalEMBO Journal
Volume8
Issue number1
StatePublished - 1989
Externally publishedYes

Fingerprint

Ricin
Ribosomes
Peptides
Arginine
Catalyst activity
Trichosanthin
Peptide Elongation Factor 2
Reticulocytes
Adenine
Muramidase
Alanine
Yeast
Toxicity
Assays
Point Mutation
Cricetinae
Triticum
Chickens
Yeasts
Proteins

ASJC Scopus subject areas

  • Cell Biology
  • Genetics

Cite this

May, M. J., Hartley, M. R., Roberts, L. M., Krieg, P. A., Osborn, R. W., & Lord, J. M. (1989). Ribosome inactivation by ricin A chain: A sensitive method to assess the activity of wild-type and mutant polypeptides. EMBO Journal, 8(1), 301-308.

Ribosome inactivation by ricin A chain : A sensitive method to assess the activity of wild-type and mutant polypeptides. / May, M. J.; Hartley, M. R.; Roberts, L. M.; Krieg, Paul A; Osborn, R. W.; Lord, J. M.

In: EMBO Journal, Vol. 8, No. 1, 1989, p. 301-308.

Research output: Contribution to journalArticle

May, MJ, Hartley, MR, Roberts, LM, Krieg, PA, Osborn, RW & Lord, JM 1989, 'Ribosome inactivation by ricin A chain: A sensitive method to assess the activity of wild-type and mutant polypeptides', EMBO Journal, vol. 8, no. 1, pp. 301-308.
May, M. J. ; Hartley, M. R. ; Roberts, L. M. ; Krieg, Paul A ; Osborn, R. W. ; Lord, J. M. / Ribosome inactivation by ricin A chain : A sensitive method to assess the activity of wild-type and mutant polypeptides. In: EMBO Journal. 1989 ; Vol. 8, No. 1. pp. 301-308.
@article{2723ea7a75a5411d840db740d7e4cc5f,
title = "Ribosome inactivation by ricin A chain: A sensitive method to assess the activity of wild-type and mutant polypeptides",
abstract = "When recombinant ricin A chain transcripts are translated in a rabbit reticulocyte lysate the ribosomes are rapidly inactivated as shown by their inability to support translation of yeast preproalpha factor or chicken lysozyme transcripts added subsequently. In contrast, ribosomes which have translated transcripts encoding non-toxic polypeptides such as ricin B chain, readily translate the second transcript under identical conditions. Ribosome inactivation is accompanied by a highly specific modification of 28S rRNA which occurs at the same position as the N-glycosidic cleavage of an adenine residue and which is thought to cause inactivation of the ribosomes. Protein synthesis by wheat germ ribosomes was not inhibited under the conditions which inhibit reticulocyte ribosomes confirming earlier observations that plant cytoplasmic ribosomes are much less sensitive to inhibition by ricin A chain than are mammalian ribosomes. Using the same assay we have shown that deleting an internal hexapeptide, which shares homology with hamster elongation factor-2, completely abolishes catalytic activity. Deleting a second pentapeptide conserved between ricin A chain and the ribosome-inactivating plant toxin trichosanthin, had no effect. Deleting the first nine residues from the N-terminus of A chain did not affect toxicity whereas deleting a further three residues inactivated the polypeptide. Point mutations which individually converted arginine 48 and arginine 56 of ricin A chain to alanine residues or which deleted arginine 56 were also without effect on the catalytic activity of the toxin.",
author = "May, {M. J.} and Hartley, {M. R.} and Roberts, {L. M.} and Krieg, {Paul A} and Osborn, {R. W.} and Lord, {J. M.}",
year = "1989",
language = "English (US)",
volume = "8",
pages = "301--308",
journal = "EMBO Journal",
issn = "0261-4189",
publisher = "Nature Publishing Group",
number = "1",

}

TY - JOUR

T1 - Ribosome inactivation by ricin A chain

T2 - A sensitive method to assess the activity of wild-type and mutant polypeptides

AU - May, M. J.

AU - Hartley, M. R.

AU - Roberts, L. M.

AU - Krieg, Paul A

AU - Osborn, R. W.

AU - Lord, J. M.

PY - 1989

Y1 - 1989

N2 - When recombinant ricin A chain transcripts are translated in a rabbit reticulocyte lysate the ribosomes are rapidly inactivated as shown by their inability to support translation of yeast preproalpha factor or chicken lysozyme transcripts added subsequently. In contrast, ribosomes which have translated transcripts encoding non-toxic polypeptides such as ricin B chain, readily translate the second transcript under identical conditions. Ribosome inactivation is accompanied by a highly specific modification of 28S rRNA which occurs at the same position as the N-glycosidic cleavage of an adenine residue and which is thought to cause inactivation of the ribosomes. Protein synthesis by wheat germ ribosomes was not inhibited under the conditions which inhibit reticulocyte ribosomes confirming earlier observations that plant cytoplasmic ribosomes are much less sensitive to inhibition by ricin A chain than are mammalian ribosomes. Using the same assay we have shown that deleting an internal hexapeptide, which shares homology with hamster elongation factor-2, completely abolishes catalytic activity. Deleting a second pentapeptide conserved between ricin A chain and the ribosome-inactivating plant toxin trichosanthin, had no effect. Deleting the first nine residues from the N-terminus of A chain did not affect toxicity whereas deleting a further three residues inactivated the polypeptide. Point mutations which individually converted arginine 48 and arginine 56 of ricin A chain to alanine residues or which deleted arginine 56 were also without effect on the catalytic activity of the toxin.

AB - When recombinant ricin A chain transcripts are translated in a rabbit reticulocyte lysate the ribosomes are rapidly inactivated as shown by their inability to support translation of yeast preproalpha factor or chicken lysozyme transcripts added subsequently. In contrast, ribosomes which have translated transcripts encoding non-toxic polypeptides such as ricin B chain, readily translate the second transcript under identical conditions. Ribosome inactivation is accompanied by a highly specific modification of 28S rRNA which occurs at the same position as the N-glycosidic cleavage of an adenine residue and which is thought to cause inactivation of the ribosomes. Protein synthesis by wheat germ ribosomes was not inhibited under the conditions which inhibit reticulocyte ribosomes confirming earlier observations that plant cytoplasmic ribosomes are much less sensitive to inhibition by ricin A chain than are mammalian ribosomes. Using the same assay we have shown that deleting an internal hexapeptide, which shares homology with hamster elongation factor-2, completely abolishes catalytic activity. Deleting a second pentapeptide conserved between ricin A chain and the ribosome-inactivating plant toxin trichosanthin, had no effect. Deleting the first nine residues from the N-terminus of A chain did not affect toxicity whereas deleting a further three residues inactivated the polypeptide. Point mutations which individually converted arginine 48 and arginine 56 of ricin A chain to alanine residues or which deleted arginine 56 were also without effect on the catalytic activity of the toxin.

UR - http://www.scopus.com/inward/record.url?scp=0024446196&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024446196&partnerID=8YFLogxK

M3 - Article

C2 - 2714255

AN - SCOPUS:0024446196

VL - 8

SP - 301

EP - 308

JO - EMBO Journal

JF - EMBO Journal

SN - 0261-4189

IS - 1

ER -