Ribosome-inactivating proteins (RIP) are a family of plant enzymes for which a unique activity was determined: rRNAN-glycosidase at a specific universally conserved position, A4324in the case of rat ribosomes. Recently we have shown that the RIP from Saponaria officinalis have a much wider substrate specificity: they are actually polynucleotide:adenosine glycosidases. Here we extend studies on substrate specificity to most known RIP: 52 purified proteins, both type 1 (single-chain) and type 2 (two chain, an enzymatic chain and a lectin chain) were examined for adenine release on various substrates including RNAs from different sources, DNA, and poly(A). All RIP depurinated extensively DNA and some released adenine from all adenine-containing polynucleotides tested. From experimental evidence the entire class of plant proteins, up to now called ribosome-inactivating proteins, may be classified as polynucleotide:adenosine glycosidases. The newly identified substrates may be implicated in the biological role(s) of RIP.

No abstract

The ribosome-inactivating proteins (RIPs) from Hura crepitans and Phytolaeca americana release adenine from herring sperm DNA. Leaf extracts from these plants show the same enzymatic activities as the RIPs. The translation inhibitory activity and the activity on DNA are both increased in the leaves of both plants during senescence or when subjected to heat or osmotic stress. It is proposed that a physiological role of RIPs could be to intervene in the death of plant cells.

The ribosome-inactivating proteins (RIPs) are a family of plant enzymes for which a unique activity has been determined: rRNA N-glycosidase, which removes adenine at a specific universally conserved position (A4324 in the case of rat ribosomes). Here we report that saporin-L1, a RIP from the leaves of Saponaria officinalis, recognizes other substrates, including RNAs from different sources, DNA and poly(A). Saporin-L1 depurinated DNA extensively and released adenine from all adenine-containing polynucleotides tested. Adenine was the only base released from DNA or artificial polynucleotides. The characteristics of the reactions catalysed by saporin-L1 have been determined: optimal pH and temperature, ionic requirements, and the kinetic parameters Km and kcat. The reaction proceeded without cofactors, at low ionic strength, in the absence of Mg2+ and K+. Saporin-L1 had no activity towards various adenine-containing non-polynucleotide compounds (cytokinins, cofactors, nucleotides). This plant protein may now be classified as a polynucleotide:adenosine glycosidase.

Polynucleotide: adenosine glycosidases (PNAG) are a class of plant and bacterial enzymes commonly known as ribosome-inactivating proteins (RIP). They are presently classified as rRNA N-glycosidases in the enzyme nomenclature [EC 3.2.2.22]. Several activities on nucleic acids, other than depurination, have been attributed to PNAG: in particular modifications induced in circular plasmids, including linearisation and topological changes, and cleavage of guanidinic residues. Here we describe a chromatographic procedure to obtain nuclease-free PNAG by dye-chromatography onto Procion Red derivatized Sepharose((R)). Highly purified enzymes depurinate extensively pBR322 circular, supercoiled DNA at neutral pH and exhibit neither DNase nor DNA glycolyase activities, do not cause topological changes, and adenine is the only base released from DNA and rRNA, even at very high enzyme concentrations. A scanning force microscopy (SFM) study of pBR322 treated with saporin-S6 confirmed that (i) this PNAG binds extensively to the plasmid, (ii) the distribution of the bound saporin-S6 molecules along the DNA chain is markedly variable, (iii) plasmids already digested with saporin-S6 do not appear fragmented or topologically modified. The observations here described demonstrate that polynucleotide:adenosine glycosidase is the sole enzymatic activity of the four ribosome-inactivating proteins gelonin, momordin I, pokeweed antiviral protein from seeds and saporin-S6. These proteins belong to different families, suggesting that the findings here described may be generalized to all PNAG.