Isolation and characterization of an RIP (ribosome-inactivating protein)-like protein from tobacco with dual enzymatic activity.
Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a protein termed tobacco RIP (TRIP) was isolated from tobacco (Nicotiana tabacum) leaves and purified using ion exchange and gel filtration chromatography in combination with yeast ribosome depurination assays. TRIP has a molecular mass of 26 kD as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed strong N-glycosidase activity as manifested by the depurination of yeast rRNA. Purified TRIP showed immunoreactivity with antibodies of RIPs from Mirabilis expansa. TRIP released fewer amounts of adenine residues from ribosomal (Artemia sp. and rat ribosomes) and non-ribosomal substrates (herring sperm DNA, rRNA, and tRNA) compared with other RIPs. TRIP inhibited translation in wheat (Triticum aestivum) germ more efficiently than in rabbit reticulocytes, showing an IC50 at 30 ng in the former system. Antimicrobial assays using highly purified TRIP (50 microg mL(-1)) conducted against various fungi and bacterial pathogens showed the strongest inhibitory activity against Trichoderma reesei and Pseudomonas solancearum. A 15-amino acid internal polypeptide sequence of TRIP was identical with the internal sequences of the iron-superoxide dismutase (Fe-SOD) from wild tobacco (Nicotiana plumbaginifolia), Arabidopsis, and potato (Solanum tuberosum). Purified TRIP showed SOD activity, and Escherichia coli Fe-SOD was observed to have RIP activity too. Thus, TRIP may be considered a dual activity enzyme showing RIP-like activity and Fe-SOD characteristics.
Sharma
Neelam
N
Department of Horticulture and Landscape Architecture, Colorado State University, Fort Collins, Colorado, 80523, USA.
Park
Sang-Wook
SW
Vepachedu
Ramarao
R
Barbieri
Luigi
L
Ciani
Marialibera
M
Stirpe
Fiorenzo
F
Savary
Brett J
BJ
Vivanco
Jorge M
JM
D000225:Adenine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005658:Fungi / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D014176:Protein Biosynthesis; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013482:Superoxide Dismutase / Q000235:genetics; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
D000890:Anti-Infective Agents; D010940:Plant Proteins; D013482:Superoxide Dismutase; D009699:N-Glycosyl Hydrolases; D000225:Adenine