A robust cysteine-deficient chitinase-like antifungal protein from inner shoots of the edible chive Allium tuberosum.
From the inner shoots of the chive Allium tuberosum, a single-chained protein with a molecular weight of 36 kDa and an N-terminal sequence manifesting resemblance to chitinases but lacking in cysteine residues characteristic of a cysteine-rich domain present in chitinases of other Allium species, was purified. The isolation procedure entailed affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on DEAE-cellulose and Mono S, and gel filtration on Superdex 75. The protein was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and Mono S. It exhibited antifungal activity against Rhizoctonia solani, Fusarium oxysporum, Coprinus comatus, Mycosphaerella arachidicola, and Botrytis cinerea. The IC(50) for its antifungal effect against Botrytis cinerea was 0.2 microM. The antifungal activity was stable after 1 h at pH 1.6 and 12.3, and up to 60 degrees C for 5 min. Incubation of the protein with trypsin or chymotrypsin at an enzyme:substrate ratio of 1:100 and pH 7.6 up to 150 min did not affect its antifungal activity. The protein did not exhibit antibacterial activity. The protein inhibited cell-free translation in a rabbit reticulocyte system with an IC(50) of 0.8 microM, but did not affect the proliferation of mouse splenocytes. It exerted some cytotoxic effect on breast cancer cells and was inhibitory toward HIV-1 reverse transcriptase.
Lam
Y W
YW
Department of Biochemistry, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories, China.
Wang
H X
HX
Ng
T B
TB
D000490:Allium / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002688:Chitinases / Q000737:chemistry*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003545:Cysteine / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D010940:Plant Proteins; D002688:Chitinases; D003545:Cysteine