Isolation of alliumin, a novel protein with antimicrobial and antiproliferative activities from multiple-cloved garlic bulbs

A protein designated alliumin, with a molecular mass of 13 kDa and an N-terminal sequence similar to a partial sequence of glucanase, and demonstrating antifungal activity against Mycosphaerella arachidicola, but not against Fusarium oxysporum, was isolated from multiple-cloved garlic (Allium sativum) bulbs. The protein, designated as alliumin, was purified using ion exchange chromatography on DEAE-cellulose, CM-cellulose and Mono S, affinity chromatography on Affi-gel blue gel, and gel filtration on Superdex 75. Alliumin was unadsorbed on DEAE-cellulose, but was adsorbed on Affi-gel blue gel, CM-cellulose and Mono S. Its antifungal activity was retained after boiling for 1 h and also after treatment with trypsin or chymotrypsin (1:1, w/w) for 30 min at room temperature. Alliumin was inhibitory to the bacterium Pseudomonas fluorescens and exerted antiproliferative activity toward leukemia L1210 cells. However, it was devoid of ribonuclease activity, protease activity, mitogenic activity toward mouse splenocytes, and antiproliferative activity toward hepatoma Hep G2 cells.     

Isolation and characterization of luffacylin,a ribosome inactivating peptide with anti-fungal activity from sponge gourd (Luffa cylindrica) seeds

A purification scheme involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and ion exchange chromatography on CM-Sepharose and Mono S was employed to isolate a peptide with a molecular weight of 7.8kDa from sponge gourd seeds. The peptide, which was designated luffacylin, exhibited an N-terminal sequence with pronounced resemblance to that of the 6.5kDa arginine-glutamate rich polypeptide previously isolated from sponge gourd seeds. Luffacylin inhibited translation in a rabbit reticulocyte lysate system with an IC(50) of 140pM and reacted positively in the N-glycosidase assay for ribosome inactivating proteins. Luffacylin exerted anti-fungal activity against Mycosphaerella arachidicola and Fusarium oxysporum.     

Purification and characterization of a ubiquitin-like peptide with macrophage stimulating,antiproliferative and ribonuclease activities from the mushroom Agrocybe cylindracea

A peptide, with a molecular mass of 9.5kDa and demonstrating an N-terminal sequence similar to ubiquitin, was isolated from fruiting bodies of the mushroom Agrocybe cylindracea. The peptide was isolated with a purification protocol involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, FPLC-ion exchange chromatography on Mono S and FPLC-gel filtration on Superdex 75. The peptide was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and Mono S. It showed antiproliferative activity on leukemia cell line (M1) and hepatoma cell line (HepG2), and enhanced nitric oxide production in murine peritoneal macrophages with a potency comparable to that of lipopolysaccharide. A pH of 6.0 was required for optimal RNase activity. Its RNase activity was stable over the temperature range of 0-60 degrees C. It exerted ribonucleolytic activity preferentially on polyC, much lower activity on polyU, and negligible activity on polyA and polyG.     

A novel alkaline protease with antiproliferative activity from fresh fruiting bodies of the toxic wild mushroom Amanita farinosa

A novel protease with a molecular mass of 15 kDa was purified from fresh fruiting bodies of the wild mushroom Amanita farinosa. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and SP-Sepharose. It demonstrated a single 15-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and a 15-kDa peak in gel filtration. The optimal pH and optimal temperature of the protease were pH 8.0 and 65 °C, respectively. Proliferation of human hepatoma HepG2 cells was inhibited by the protease with an IC(50) of 25 μM. The protease did not have antifungal or ribonuclease activity.     

A mannose-specific tetrameric lectin with mitogenic and antibacterial activities from the ovary of a teleost,the cobia (<Emphasis Type="Italic">Rachycentron canadum</Emphasis>)

A tetrameric lectin, with hemagglutinating activity toward rabbit erythrocytes and with specificity toward d-mannosamine and d(+)-mannose, was isolated from the ovaries of a teleost, the cobia Rachycentron canadum. The isolation protocol comprised ion exchange chromatography on CM-cellulose and Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q, and finally gel filtration by FPLC on Superose 12. The lectin was adsorbed on all ion exchangers used. It exhibited a molecular mass of 180 kDa in gel filtration on Superose 12 and a single 45-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is a tetrameric protein. The hemagglutinating activity of the lectin was stable up to 40°C and between pH 4 and pH 10. All hemagglutinating activity disappeared at 60°C and at pH 1 and pH 13. The hemagglutinating activity was doubled in the presence of 0.1 μM FeCl₃. The lectin exerted antibacterial activity against Escherichia coli with 50% inhibition at 250 μg. There was no antifungal activity toward Coprinus comatus, Fusarium oxysporum, Mycosphaerella arachidicola, and Rhizoctonia solani at a dose of 300 μg. The lectin exhibited maximal mitogenic response from mouse splenocytes at a concentration of 14 μM.     

A ubiquitin-like peptide from the mushroom Pleurotus sajor-caju exhibits relatively potent translation-inhibitory and ribonuclease activities

The fruiting bodies of the edible mushroom Pleurotus sajor-caju were extracted with an aqueous buffer and then subjected to affinity chromatography on Affi-gel Blue gel, ion exchange chromatography on DEAE-cellulose and gel filtration on Superdex 75. From the fraction of the extract adsorbed on Affi-gel Blue gel and unadsorbed on DEAE-cellulose, a 9.5 kDa peptide with an N-terminal sequence similar to ubiquitin was isolated with a yield of 0.25 mg/kg mushroom. The peptide inhibited cell-free translation with an IC(50) of 30 nM. It exhibited a ribonuclease activity of 450 U/mg toward yeast transfer RNA. The activities were substantially more potent than those of previously isolated mushroom ubiquitin-like protein and peptide.