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Isolation and Characterization of an RIP (Ribosome-Inactivating Protein)-Like Protein from Tobacco with Dual Enzymatic Activity^1,[w]

Department of Horticulture and Landscape Architecture (N.S., S.-W.P., R.V., J.M.V.), and Cellular and Molecular Biology Graduate Program (J.M.V.), Colorado State University, Fort Collins, Colorado, 80523; Dipartimento di Patologia sperimentale dell'Università degli Studi di Bologna, Via San Giacomo 14, I–40126, Bologna, Italy (L.B., M.C., F.S.); and United States Department of Agriculture, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038 (B.J.S.)

Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a protein termed tobacco RIP (TRIP) was isolated from tobacco (Nicotiana tabacum) leaves and purified using ion exchange and gel filtration chromatography in combination with yeast ribosome depurination assays. TRIP has a molecular mass of 26 kD as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed strong N-glycosidase activity as manifested by the depurination of yeast rRNA. Purified TRIP showed immunoreactivity with antibodies of RIPs from Mirabilis expansa. TRIP released fewer amounts of adenine residues from ribosomal (Artemia sp. and rat ribosomes) and non-ribosomal substrates (herring sperm DNA, rRNA, and tRNA) compared with other RIPs. TRIP inhibited translation in wheat (Triticum aestivum) germ more efficiently than in rabbit reticulocytes, showing an IC₅₀ at 30 ng in the former system. Antimicrobial assays using highly purified TRIP (50 µg 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 of TRIP was identical with the internal sequences of the iron-superoxide dismutase (Fe-SOD) from wild tobacco (Nicotiana plumbaginifolia), Arabidopsis, and potato (Solanum tuberosum). Purified TRIP showed SOD activity, and Escherichia coli Fe-SOD was observed to have RIP activity too. Thus, TRIP may be considered a dual activity enzyme showing RIP-like activity and Fe-SOD characteristics.

¹ This work was supported by a CAREER award from the National Science Foundation (grant no. MCB-0093014 to J.M.V.) and by the Colorado State University Agricultural Experiment Station (to J.M.V.). The work done in Bologna was supported by grants from the University of Bologna (the Ministero dell'Istruzione), Università e Ricerca (the Ministero della Salute). and the Pallotti's Legacy for Cancer Research.

^[w] The online version of this article contains Web-only data.

^* Corresponding author; e-mail jvivanco{at}lamar.colostate.edu; fax 970–491–7745.

Received July 21, 2003; returned for revision September 10, 2003; accepted September 22, 2003.

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