A nonradioactive assay for ribosome-inactivating proteins.
A sensitive nonradioactive method to determine the activity of ribosome-inactivating proteins (RIPs) based on a combined transcription/translation in vitro assay was established. Using this assay we investigated the RIP activities of the heterodimeric toxic plant lectins ricin and mistletoe lectin I (ML-I). The enzymatic activities of the holoproteins were compared to that of the RIP-active chain of ML-I (ML-I A-chain) and recombinant ML-I A-chain expressed in Escherichia coli. The IC50 values determined for the plant toxins showed that the translation-inactivating activity of ML-I (39.8 pM) is about four times higher than that of ricin (176.0 pM). The plant-derived ML-I A-chain is more toxic (3.4 pM) in the cell-free translation system than the respective holoprotein. The recombinant ML-I A-chain was found to be about three times less active (IC50 10.6 pM) than the A-chain from plant. The in vitro assay described here is a convenient method for the fast determination of RIP activity with a 1000-fold lower detection limit than that of commonly used RIP assays.
Langer
M
M
Biotechnology Research and Information Network (BRAIN) GmbH, Zwingenberg, Germany.
Rothe
M
M
Eck
J
J
Möckel
B
B
Zinke
H
H
D000818:Animals; D004305:Dose-Response Relationship, Drug; D005573:Formycins / Q000494:pharmacology; D037102:Lectins / Q000032:analysis; D014784:Mistletoe; D037121:Plant Lectins; D028321:Plant Preparations; D010940:Plant Proteins; D010946:Plants, Medicinal; D011817:Rabbits; D011994:Recombinant Proteins / Q000032:analysis; D012265:Ribonucleotides / Q000494:pharmacology; D012269:Ribosomal Proteins / Q000032:analysis*; D054790:Ribosome Inactivating Proteins, Type 2; D012276:Ricin / Q000032:analysis; D014118:Toxins, Biological / Q000032:analysis
D005573:Formycins; D037102:Lectins; D037121:Plant Lectins; D028321:Plant Preparations; D010940:Plant Proteins; D011994:Recombinant Proteins; D012265:Ribonucleotides; D012269:Ribosomal Proteins; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological; C488862:mistletoe lectin I; C009674:ribosome inactivating protein, Viscum; C021683:formycin 5'-phosphate; D012276:Ricin