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 ). 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.
Highlights
Ribosome-inactivating proteins (RIPs)1 from plants may be classified as type 1 or 2 according to their single- or doublechain structure
The enzymatic activity of ribosome-inactivating proteins (RIPs) was defined as polynucleotide:adenosine glycosidase [6], which we propose to change to adenine polynucleotide glycosylase (APG) in analogy with the EC nomenclature of nucleic acid glycosylases
The first eluted protein, which is identical to Mirabilis antiviral protein (MAP) from roots, and the fourth protein (MAP-4) appeared to be Ͼ98% homogeneous upon reverse-phase HPLC (Fig. 2), and both gave a single band upon SDS-PAGE
Summary
Ribosome-inactivating proteins (RIPs) from plants may be classified as type 1 or 2 according to their single- or doublechain structure (reviewed in Refs. 1– 4). The mechanism of action of this class of proteins became clearer when it was found that ricin and subsequently all RIPs tested release a single adenine residue from ribosomes in a precise position (A4324 in the case of rat liver ribosomes) of a universally conserved GAGA sequence in a peculiar stem-loop structure (review in Ref. 2) They were classified as rRNA N-glycosidases (EC 3.2.2.22). From the roots of M. jalapa, a protein was purified that was highly effective in preventing viral infection caused by contact-transmitted virus [11] This protein, named Mirabilis antiviral protein (MAP), was later identified as a RIP [12, 13] for its activity on the major rRNA in intact ribosomes. We describe the distribution of both translation inhibitory activity
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