Isolation and Characterization of an RIP (Ribosome-Inactivating Protein)-Like Protein from Tobacco with Dual Enzymatic Activity

Abstract

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 IC 50 at 30 ng in the former system. Antimicrobial assays using highly purified TRIP (50 g mL Ϫ1 ) 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.Various plants contain enzymes called ribosomeinactivating proteins (RIPs), officially called rRNA N-glycosidases (EC 3.2.2.22), which catalytically inactivate eukaryotic as well as prokaryotic ribosomes (Barbieri et al., 1993) by removing single adenine residues from the large rRNA (A 4324 from rat liver rRNA). RIPs selectively cleave an adenine from the universally conserved sarcin/ricin loop of the large rRNA (Endo and Tsurugi, 1987, Endo et al., 1987; Wool et al., 1992; Barbieri et al.,1993), thus interrupting the interaction of elongation factors I and II with the ribosomes and ultimately arresting protein synthesis at the translocation step (for reviews, see Stirpe et al., 1992;Mehta and Boston, 1998; Tumer et al., 1999;Nielsen and Boston, 2001; Van Damme et al., 2001). In addition to its N-glycosidase activity, some RIPs have DNAse, DNA glycosylase, and apurinic pyrimidinic lyase activities (Li et al., 1991; Roncuzzi and Gasperi-Gampani, 1996;Nicolas et al., 1997Nicolas et al., , 1998Nicolas et al., , 2000 Hudak et al., 2000). It has been suggested, however, that the sporadically observed nuclease activities are due to contamination by other enzymes (Day et al., 1998; Barbieri et al., 2000; Van Damme et al., 2001), whereas all RIPs tested remove adenine from DNA and some of them also from poly(A) (Barbieri et al., 1997).Dif...

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