Ribosome-inactivating and adenine polynucleotide glycosylase activities in Mirabilis jalapa L. tissues.
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.
Bolognesi
Andrea
A
Dipartimento di Patologia Sperimentale, Università di Bologna, Via San Giacomo 14, I-40126 Bologna, Italy. bolo@alma.unibo.it
Polito
Letizia
L
Lubelli
Chiara
C
Barbieri
Luigi
L
Parente
Augusto
A
Stirpe
Fiorenzo
F
D000225:Adenine / Q000378:metabolism; D000595:Amino Acid Sequence; D002460:Cell Line; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D019684:Magnoliopsida / Q000201:enzymology*; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D020033:Protein Isoforms; D011500:Protein Synthesis Inhibitors / Q000737:chemistry / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism; D054789:Ribosome Inactivating Proteins, Type 1; D054790:Ribosome Inactivating Proteins, Type 2; D012270:Ribosomes / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014018:Tissue Distribution; D014027:Tobacco Mosaic Virus / Q000378:metabolism
D010940:Plant Proteins; D020033:Protein Isoforms; D011500:Protein Synthesis Inhibitors; D012335:RNA, Ribosomal; D054789:Ribosome Inactivating Proteins, Type 1; D054790:Ribosome Inactivating Proteins, Type 2; D009699:N-Glycosyl Hydrolases; C437225:adenine glycosylase; C059351:MAP protein, Mirabilis jalapa; D000225:Adenine