Ribosome-inactivating and Adenine Polynucleotide Glycosylase Activities in Mirabilis jalapa L. Tissues

Abstract

Several tissues of Mirabilis jalapa L. (Nyctaginaceae)were assayed for inhibition of translation by a rabbit reticulocyte lysate (as a signal of ribosome-inactivating activity) and for adenine DNA glycosylase activity, activities that are both due to the presence of a class of enzymes called ribosome-inactivating proteins (RIPs), currently classified as rRNA N-glycosylases (EC 3.2.2.22). These activities were highest in seed; intermediate in flower bud, immature seed, sepal ؉ gynoecium, leaf, and root; and very low in all other tissues. By cation-exchange chromatography, four protein peaks with inhibitory activity on cell-free translation were identified in extracts from seeds, and two proteins were isolated from peaks 1 and 4, all of which have the properties of single-chain type 1 RIP. One is Mirabilis antiviral protein (MAP), so far purified only from roots. The second is a new protein that we propose to call MAP-4. The distribution of MAP and MAP-4 in several tissues was determined with a novel experimental approach based on liquid chromatography/mass spectrometry. The direct enzymatic activity of MAP on several substrates is described here for the first time. MAP depurinated not only rRNA in intact ribosomes, thus inhibiting protein synthesis, but also other polynucleotides such as poly(A), DNA, and tobacco mosaic virus RNA. Autologous DNA was depurinated more extensively than other polynucleotides. Therefore, the enzymatic activity of this protein may be better described as adenine polynucleotide glycosylase activity rather than rRNA N-glycosylase activity. Finally, MAP does not cross-react immunologically with other commonly utilized RIPs.

Novel antibodies as anticancer agents

“…Type 1 RIPs and the A subunit of type 2 RIPs is an RNA Nglycosidase that cleaves adenine molecules from ribosomal RNA, thus inactivating the ribosome in an irreversible manner. In addition, they remove adenine residues from the DNA (Barbieri et al, 2000;Bolognesi et al, 2002).…”

The conjugate Rituximab/saporin-S6 completely inhibits clonogenic growth of CD20-expressing cells and produces a synergistic toxic effect with Fludarabine

“…Saporin-S6 was revealed by incubation overnight with a rabbit anti-saporin-S6 serum. 15 Detection was carried out with a horseradish peroxidase-conjugated goat anti-(rabbit IgG)Ig and with 4-chloro-naphtol (Sigma).…”

“…They also remove adenine residues from DNA, hence the denomination of adenine polynucleotide glycosylase proposed for RIPs. 15 To date, several clinical trials have been conducted with immunotoxins, and some of them are still ongoing, to evaluate their antitumor efficacy. The response rates observed in phase I/II trials have often been higher than those reported for some of the conventional antiblastic drugs.…”

Molecular characterization and post-transcriptional regulation of ME1, a type-I ribosome-inactivating protein from Mirabilis expansa

“…Depurination of the S/R loop prevents binding of elongation factor 2 to the ribosome, and results in protein synthesis inhibition (Stirpe et al 1992). Apart from the depurination of rRNA, some RIPs exhibit the ability to depurinate DNA, poly(A) and viral RNA (Barbieri et al 1994(Barbieri et al , 1997Bolognesi et al 2002). Many RIPs possess antimicrobial and antiviral activities.…”