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Title: AN RIP-LIKE PROTEIN ISOLATED FROM NICOTIANA TABACCUM WITH DUAL ENZYMATIC ACTIVITY

Author
item NEELAM, SHARMA - COLORADO STATE UNIV.
item Savary, Brett
item SANG-WOOK, PARK - COLORADO STATE UNIV.
item RAMARAO, VAPACHEDU - COLORADO STATE UNIV.
item FIORENZO, STIRPE - UNIV. BOLOGNE, ITALY
item JORGE, VIVANCO - COLORADO STATE UNIV.

Submitted to: Journal of Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/30/2003
Publication Date: 2/20/2004
Citation: Sharma, N., Park, S.-W., Vepachedu, R., Barbieri, L. Ciani, M., Stirpe, F., Savary, B. J. Vivanco, J.M. 2004. Isolation and characterization of an RIP-like protein from Nicotiana tabacum with dual enzymatic activity. Plant Physiology 134:171-181.

Interpretive Summary: Ribosome-inactivating proteins (RIPs) are ribosomal N-glycosidases that remove a specific adenine from the sarcin/ricin (S/R) loop of the large subunit rRNA, thus arresting protein synthesis at the translocation step. Type-I RIPs are generally non-toxic monomeric proteins that are commonly consumed in foods such as grains. In contrast, type-II RIPs are highly-toxic dimeric proteins, and these are represented by the potent plant poison ricin. RIPs have generated considerable interest for their potential applications in medicine such as immunotoxins and in agriculture to improve disease resistance in plants. However, little information is known about the function of these enzymes in plants, particularly because no suitable model system exists. In this study, we isolated and characterized a new RIP from tobacco leaves. We discovered this RIP to also possess superoxide dismutase activity, thus indicating it is a rare bifunctional enzyme. These results now provide researchers a well-defined experimental system useful for investigating directly the biological function of RIPs in plants.

Technical Abstract: A ribosome-inactivating protein (TRIP) was purified from tobacco (Nicotiana tabaccum) leaves and purified using ion-exchange and gel filtration chromatography in combination with yeast ribosomal RNA depurination assays. TRIP was characterized as a basic 26 kDa monomeric protein, and it showed strong RNA N-glycosidase activity (0.1 ng/ml). The wheat germ translation system was inhibited more efficiently than rabbit reticulocytes, showing an ID50 at 30 nanograms. Antimicrobial assays using TRIP (50 g·ml-1) conducted against various fungi and bacterial pathogens showed the strongest inhibitory activity against Trichoderma reesei and Pseudomonas solancearum. A 15 amino acid internal polypeptide sequence from TRIP was identical with an internal sequence of the iron superoxide dismutase (Fe-SOD) from Nicotiana plumbaginifolia, Arabidopsis thaliana and Solanum tuberosum. Reciprocal assays indicated SOD activity by TRIP, and RIP activity by Escherichia coli Fe-SOD. These results indicate TRIP is a protein possessing dual enzymatic activities, and it may represent a new class of RIPs.