Related Experiment Video
Updated: Aug 12, 2026
11:19
Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
Published on: February 25, 2011
A nonradioactive assay for ribosome-inactivating proteins
1Biotechnology Research and Information Network (BRAIN) GmbH, Zwingenberg, Germany.
Analytical Biochemistry
|December 1, 1996
Summary
A new in vitro assay accurately measures ribosome-inactivating protein (RIP) activity. This method reveals mistletoe lectin I is more potent than ricin, with its A-chain being highly toxic.
Area of Science:
- Biochemistry
- Molecular Biology
- Toxicology
Background:
- Ribosome-inactivating proteins (RIPs) are toxins that inhibit protein synthesis.
- Accurate and sensitive methods are needed to quantify RIP activity.
- Plant lectins like ricin and mistletoe lectin I (ML-I) are potent RIPs.
Purpose of the Study:
- To establish a sensitive, nonradioactive in vitro assay for determining RIP activity.
- To compare the RIP activities of ricin, ML-I, its A-chain, and recombinant ML-I A-chain.
Main Methods:
- Development of a combined transcription/translation in vitro assay.
- Determination of IC50 values for various RIPs using the established assay.
- Comparison of enzymatic activities of native and recombinant RIP components.
Main Results:
- ML-I exhibits approximately four times higher translation-inactivating activity than ricin.
- The ML-I A-chain (plant-derived) is more toxic than the holoprotein in a cell-free system.
- Recombinant ML-I A-chain showed lower activity compared to the plant-derived A-chain.
Conclusions:
- The developed in vitro assay is a convenient and sensitive method for RIP activity determination.
- The assay offers a 1000-fold lower detection limit than conventional RIP assays.
- ML-I and its A-chain demonstrate significant toxicity, with variations between native and recombinant forms.
