Ribosome-inactivating proteins (RIPs) display adenine polynucleotide glycosylase activity on di¡erent nucleic acid substrates, which at the ribosomal level is responsible for the arrest of protein synthesis. Some type 2 RIPs, namely ricin and related proteins, are extremely toxic to mammalian cells and animals whilst other type 2 RIPs (non-toxic type 2 RIPs) display three to four logs less toxicity. We studied whether a correlation exists between toxicity on cells and enzymatic activity on nucleic acids. All type 2 RIPs di¡er in their depurinating activity on the di¡erent substrates with di¡erences of up to one to two logs. The toxicity of type 2 RIPs is independent of their enzymatic activity on nucleic acids or on ribosomes.

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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It has been known that ribosome-inactivating proteins (RIPs) from plants damage ribosomes by removing adenine from a precise position of rRNA. Subsequently it was observed that all tested RIPs depurinate DNA, and some of them also non-ribosomal RNAs and poly(A), hence the denomination of adenine polynucleotide glycosylases was proposed. We report now that ricin, saporin-L2, saporin-S6, gelonin and momordin depurinate also poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase (auto modi¢ed enzyme), an enzyme involved in DNA repair. We observed also that all RIPs but gelonin induce transformation of ¢broblasts, possibly as a consequence of damage to DNA and of the altered DNA repair system. ß

Although the type-2 ribosome-inactivating proteins (SNA-I, SNA-V, SNLRP) from elderberry (Sambucus nigra L.) are all devoid of rRNA N-glycosylase activity towards plant ribosomes, some of them clearly show polynucleotideadenosine glycosylase activity towards tobacco mosaic virus RNA. This particular substrate specificity was exploited to further unravel the mechanism underlying the in planta antiviral activity of ribosome-inactivating proteins. Transgenic tobacco (Nicotiana tabacum L. cv Samsun NN) plants expressing the elderberry ribosome-inactivating proteins were generated and challenged with tobacco mosaic virus in order to analyze their antiviral properties. Although some transgenic plants clearly showed antiviral activity, no clear correlation was observed between in planta antiviral activity of transgenic tobacco lines expressing the different ribosomeinactivating proteins and the in vitro polynucleotideadenosine glycosylase activity of the respective proteins towards tobacco mosaic virus genomic RNA. However, our results suggest that the in planta antiviral activity of some ribosome-inactivating proteins may rely on a direct mechanism on the virus. In addition, it is evident that the working mechanism proposed for pokeweed antiviral protein cannot be extrapolated to elderberry ribosome-inactivating proteins because the expression of SNA-V is not accompanied by induction of pathogenesis-related proteins.Keywords: elderberry; polynucleotide-adenosine glycosylase activity; ribosome-inactivating protein; Sambucus nigra; viral protection.Ribosome-inactivating proteins (RIPs; EC 3.2.2.22) are a heterogeneous family of structurally and evolutionary related plant proteins sharing a common functional domain that catalytically removes a specific adenine residue from a highly conserved, surface-exposed stem-loop structure found in the large rRNA of prokaryotic and eukaryotic ribosomes [1,2]. At present, they are subdivided on the basis of the structure of the genes and the corresponding proteins into holo-RIPs and chimero-RIPs [3]. Whereas holo-RIPs consist exclusively of a single catalytically active protomer of either one (classical type-1 RIPs) or two smaller polypeptide chains (e.g. maize RIP b-32), chimero-RIPs are built up of chimeric protomers with an N-terminal catalytically active domain arranged in tandem with a structurally and functionally unrelated C-terminal domain (classical type-2 and type-3 RIPs).Biochemical and molecular studies have shown that the elderberry tree expresses a complex mixture of type-2 RIPs and/or lectins in virtually all tissues. In agreement with the chronological order of their discovery, these Sambucus nigra agglutinins (SNAs) are numbered SNA-I to SNA-V. The first elderberry lectin was identified in bark tissue and described as a NeuAc(a-2,6)Gal/GalNAc-specific agglutinin (called SNA-I) [4,5]. Although already discovered in 1984, SNA-I was recognized as a type-2 RIP only when the corresponding gene was cloned in 1996 [6]. Besides SNA-I, elderberry bark contains a second NeuAc(...