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Adenine glycosylase activity in mammalian tissues: an equivalent of ribosome‐inactivating proteins
Author(s) -
Barbieri Luigi,
Valbonesi Paola,
Bondioli Marco,
Alvarez Marylin Lugo,
Dal Monte Paola,
Landini Maria Paola,
Stirpe Fiorenzo
Publication year - 2001
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(01)02789-2
Subject(s) - chemistry , dna glycosylase , ribosome , biochemistry , microbiology and biotechnology , biology , rna , gene , dna repair
Several plant tissues contain enzymes, provisionally called ribosome-inactivating proteins (RIPs), which damage ribosomes in an irreversible manner (reviewed in [1^3]) by removing a speci¢c adenine from the major rRNA. Subsequently it was observed that all RIPs remove adenine from DNA, and the denomination of polynucleotide:adenosine glycosidase was proposed for them [4], consistently with the o¤cial denomination of ricin (rRNA glycosidase, EC 3.2.2.22). In the present letter this denomination will be changed to adenine polynucleotide glycosylase (adenine glycosylase), by analogy with the denomination of DNA glycosylase used for similar enzymes. The presence of RIPs in diverse organisms led to the notion that equivalent proteins could exist in the animal kingdom [5]. We report now that partially puri¢ed preparations of some animal tissues remove adenine from DNA, and that their activity increases in stressed and virally infected cells. The crude extracts from several rat tissues did not exhibit any activity typical of RIPs; namely, they did not inhibit protein synthesis in a rabbit reticulocyte lysate nor caused any release of adenine from DNA. However, after the extracts were subjected to a chromatographic procedure on ProcionRed (Pharmacia) used to purify RIP, the extracts of rat spleens, lungs and brains exhibited a signi¢cant deadenylating activity (Table 1), still without any inhibitory activity on the reticulocyte lysate system at concentrations up to 100 Wg/ml of protein (results not shown). The rat spleens extracts were active also, in decreasing order, on poly(A), on rRNA (rat liver 28S+5S) and on tRNA. Much less or no activity at all was found in similarly treated extracts of mice, ox, hog, rabbit and human spleens. The activity was proportional to the concentration of extract present in the reaction mixture and had an optimum at pH 5.5^6.5 (optimum for plant RIPs acting on DNA is 4.0). When the semi-puri¢ed preparations were subjected to gel ¢ltration on a TSK-3000 column (Toso-Haas), the enzyme activity appeared in the fractions of the e¥uent corresponding to a molecular mass between 20 000 and 40 000. Also, the activity was bound to cation-exchange chromatography media in conditions of binding basic proteins (results not shown), suggestive of a protein with a high pI. Further puri¢cation of the enzyme activity from rat spleen extract was attempted on a micro-scale. After chromatography on Orange-matrix (Amicon), the activity was resolved in two peaks, although without a signi¢cant increase in speci¢c activity of either peak. Upon chromatography on mono-S ion exchange column (Pharmacia), the activity appeared widely distributed during a gradient elution, with a low yield, but with an increase of approx. 10-fold of speci¢c activity in selected fractions. These results indicate that these enzymes may be present in di¡erent isoforms, as it happens with plant RIPs. Fractions with higher speci¢c activity were subjected to analysis of reverse-phase chromatography, still showing a substantial heterogeneity of protein species. Rat spleen extracts enriched by Procion-Red chromatography had an activity on calf thymus DNA lower than on herring sperm-DNA, and had no activity at all on deoxy-adenosine or 3PdAMP and 5PdAMP (results not shown).