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.     

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 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.     

Antiproliferative and antimitogenic activities in a peptide from puffball mushroom Calvatia caelata.

A peptide with a molecular weight of 8 kDa and an N-terminal sequence closely resembling that of ubiquitin was isolated from fruiting bodies of the mosaic puffball mushroom Calvatia caelata. The peptide was purified using a protocol that involved ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and ion-exchange chromatography on Mono S. The peptide inhibited translation in the cell-free rabbit reticulocyte lysate system and exhibited N-glycosidase activity. It potently inhibited proliferation of spleen cells with an IC(50) of about 100 nM as indicated by the suppression of [methyl-(3)H]thymidine uptake. The viability of breast cancer cells was reduced to half at a ubiquitin concentration of about 100 nM.     

A napin-like polypeptide with translation-inhibitory, trypsin-inhibitory, antiproliferative and antibacterial activities from kale seeds.

A heterodimeric napin-like polypeptide with translation-inhibiting and antibacterial activities has been isolated from kale seeds. The purification procedure entailed ion-exchange chromatography on dielthylaminoethyl (DEAE)-cellulose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S, and gel filtration by FPLC on Superdex 75. The napin-like polypeptide was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and Mono S. Its 7-kDa large subunit differs in N-terminal amino acid sequence from the 4-kDa small subunit. The polypeptide inhibited translation in the rabbit reticulocyte lysate system with an IC50 of 37.5 nM. This activity was preserved between pH 5 and pH 11, and between 10 and 40 degrees C. It fell to a low level at pH 3 and pH 13 and at 70 degrees C. Antibacterial activity against Bacillus, Megabacterium, and Pseudomonas species and antiproliferative activity against leukemia L1210 cells were observed. However, the polypeptide did not exert antifungal, ribonuclease, or protease activity.     

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.     

First isolation of an antifungal lipid transfer peptide from seeds of a Brassica species

An antifungal peptide with a molecular mass of 9412 and an N-terminal sequence exhibiting notable homology to those of lipid transfer proteins was isolated from seeds of the vegetable Brassica campestris. The purification protocol entailed ion exchange chromatography on Q-Sepharose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S, and gel filtration by FPLC on a Superdex peptide column. The antifungal peptide was adsorbed on Affi-gel blue gel and Mono S. It inhibited mycelial growth in Fusarium oxysporum and Mycosphaerella arachidicola with an IC(50) value of 8.3 microM and 4.5 microM, respectively. It exhibited dose-dependent binding to lyso-alpha-lauroyl phosphatidylcholine. The present findings constitute the first report on a non-specific lipid transfer protein from the seeds of a Brassica species.     

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.     

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.     

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.     

Adustin, a small translation-inhibiting polypeptide from fruiting bodies of the wild mushroom Polyporus adusta

A polypeptide, with a molecular mass of 16.5 kDa as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has been isolated from the mushroom Polyporus adusta. The polypeptide, designated as adustin, inhibited translation in a cell-free rabbit reticulocyte lysate system with an IC50 of 0.34 microM. It was isolated using a protocol that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-Sepharose and fast protein liquid chromatography-gel filtration on Superdex 75. Adustin was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and CM-Sepharose.