Alveolarin, a novel antifungal polypeptide from the wild mushroom Polyporus alveolaris

An antifungal polypeptide, with a molecular mass of 28 kDa as judged by gel filtration and appearing as a single band with a molecular mass of 14 kDa in sodium dodecyl suflate-polyacrylamide gel electrophoresis, was isolated from fresh fruiting bodies of the mushroom Polyporus alveolaris. The antifungal polypeptide, designated as alveolarin, demonstrated an inhibitory action on mycelial growth in Botrytis cinerea, Fusarium oxysporum, Mycosphaerella arachidicola and Physalospora piricola. Alveolarin was isolated with a procedure that entailed ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and gel filtration on Superdex 75 by fast protein liquid chromatography.     

Isolation of a napin-like polypeptide with potent translation-inhibitory activity from Chinese cabbage (Brassica parachinensis cv green-stalked) seeds.

A heterodimeric napin-like polypeptide was isolated from Brassica parachinensis seeds with a procedure 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 N-terminal sequence of the 5 kDa subunit of the polypeptide (PAGPFRIPKKRKKEE) showed high homology with other 2S storage proteins like napins and albumins. The polypeptide potently inhibited translation in a cell free system with an IC50 of 6.2 nM. The translation-inhibiting activity of the polypeptide was relatively stable in the pH range 6-11 and in the temperature range 10-50 degrees C.     

Isolation of allicepin, a novel antifungal peptide from onion (Allium cepa) bulbs.

From the bulbs of the onion Allium cepa, a novel antifungal peptide distinct from the antimicrobial peptide previously reported from onion seeds was isolated. The antifungal peptide, designated allicepin, was purified with a procedure that involved aqueous extraction, ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and FPLC-gel filtration on Superdex 75. Allicepin was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel. The molecular weight of allicepin was estimated to be 10 K by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on Superdex 75. Allicepin exerted an inhibitory activity on mycelial growth in several fungal species including Botrytis cinerea, Fusarium oxysporum, Mycosphaerella arachidicola and Physalospora piricola.     

Cicerarin,a novel antifungal peptide from the green chickpea

A peptide designated cicerarin, with an N-terminal amino acid sequence VKSTGRADDDLAVKTKYLPP dissimilar from known proteins and peptides and a molecular mass of 8kDa, was isolated from seeds of the green chickpea Cicer arietinum cv green chickpea. Cicerarin was isolated with a procedure that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and gel filtration by fast protein liquid chromatography on Superdex 75. Cicerarin was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel in 10mM Tris-HCl buffer (pH 7.3). Cicerarin exerted antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola, and Physalospora piricola. The antifungal activity was preserved after exposure to 100 degrees C for 15min.     

Pleurostrin, an antifungal peptide from the oyster mushroom

A 7kDa peptide, with inhibitory activity on mycelial growth in the fungi Fusaerium oxysporum, Mycosphaerella arachidicola and Physalospora piricola, was isolated from fresh fruiting bodies of the oyster mushroom. The isolation procedure entailed extraction with an aqueous buffer, ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and gel filtration by fast protein liquid chromatography on Superdex 75. The protein was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel. It demonstrated an N-terminal sequence different from known antifungal proteins and peptides.     

The trypsin-inhibitory, immunostimulatory and antiproliferative activities of a napin-like polypeptide from Chinese cabbage seeds.

A heterodimeric 13.8 kDa napin-like polypeptide has previously been isolated from Chinese cabbage (Brassica parachinensis) seeds with a procedure 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. In the present study the N-terminal sequence of the 8.8 kDa subunit of the polypeptide (PQGPQQRPPKLLQQQTNEEHE) was found to have pronounced homology to napins, albumins and trypsin inhibitors, but demonstrated little similarity to the 5 kDa subunit. The polypeptide stimulated nitrite production by mouse peritoneal macrophages and reduced the viability of leukaemia (L1210) cells. It inhibited trypsin with a higher potency than it inhibited chymotrypsin, but was devoid of ribonuclease and antifungal activities.     

Purification of allivin,a novel antifungal protein from bulbs of the round-cloved garlic

A novel antifungal protein, designated allivin, was isolated from bulbs of the round-cloved garlic Allium sativum var. round clove with a procedure involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-Sepharose and FPLC-gel filtration on Superdex 75. Allivin possessed an N-terminal sequence demonstrating very little similarity to sequences of Allium sativum chitinases and ribosome inactivating proteins. Allivin exhibited a molecular weight of 13 kDa in gel filtration and SDS-polyacrylamide gel electrophoresis. It displayed antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola and Physalospora piricola. It inhibited translation in a cell-free rabbit reticulocyte system with an IC50 of 1.6 microM.     

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.     

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.     

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.