A nonradioactive assay for ribosome-inactivating proteins

Journal/Book: Anal-Biochem 243 (1), 150-153. 1996;

Abstract: A sensitive nonradioactive method to determine the activity ofribosome-inactivating proteins (RIPs) based on a combinedtranscription/translation in vitro assay was established. Using thisassay we investigated the RIP activities of the heterodimeric toxicplant lectins ricin and mistletoe lectin I (ML-I). The enzymaticactivities of the holoproteins were compared to that of the RIP-activechain of ML-I (ML-I A-chain) and recombinant ML-I A-chain expressed inEscherichia coli. The IC50 values determined for the plant toxins showedthat the translation-inactivating activity of ML-I (39.8 pM) is aboutfour times higher than that of ricin (176.0 pM). The plant-derived ML-IA-chain is more toxic (3.4 pM) in the cell-free translation system thanthe respective holoprotein. The recombinant ML-I A-chain was found to beabout three times less active (IC50 10.6 pM) than the A-chain fromplant. The in vitro assay described here is a convenient method for thefast determination of RIP activity with a 1000-fold lower detectionlimit than that of commonly used RIP assays. Author.

Keyword(s): ANIMAL

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