Characterization of two novel type I ribosome-inactivating proteins from the storage roots of the Andean crop Mirabilis expansa

Two novel type I ribossme-inactivating proteins (RIPs) were found in the st orage roots of Mirabilis expansa, an underutilized Andean root crop. The tw o RIPs, named ME1 and ME2, were purified to homogeneity by ammonium sulfate precipitation, cation-exchange perfusion chromatography, and C-4 reverse-p hase chromatography. The two proteins were found to be similar in size (27 and 27.5 kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their isoelectric points were determined to be greater than pH 10.0. Am ino acid N-terminal sequencing revealed that both ME1 and ME2 had conserved residues characteristic of RIPs. Amino acid composition and western-blot a nalysis further suggested a structural similarity between ME1 and ME2. ME2 showed high similarity to the Mirabilis jalapa antiviral protein, a type I RIP. Depurination of yeast 26S rRNA by ME1 and ME2 demonstrated their ribos ome-inactivating activity. Because these two proteins were isolated from ro ots, their antimicrobial activity was tested against root-rot microorganism s, among others. ME1 and ME2 were active against several fungi, including P ythium irregulare, Fusarium oxysporum solani, Alternaria solani, Trichoderm a reesei, and Trichoderma harzianum, and an additive antifungal effect of M E1 and ME2 was observed. Antibacterial activity of both ME1 and ME2 was obs erved against Pseudomonas syringae, Agrobacterium tumefaciens, Agrobacteriu m radiobacter, and others.