Studies on isolation and characterization of fungal, plant and animal defense proteins (lectins, ribosome-inactivating protein and antifungal protein).

摘要

Defense proteins are present in various plants and animals. Three kinds of defense proteins: lectin, ribosome-inactivating protein (RIP) and antifungal protein were isolated by different chromatographic techniques, involving ion exchange chromatography, affinity chromatography and gel filtration chromatography and characterized in terms of their N-terminal amino acid sequences, biological activities, and effects on normal and tumor cells.; A rhamnose-specific lectin and a mannose-specific lectin were isolated from grass carp (Ctenopharyngodon idellus) and chive ( Allium tuberosum). The grass carp lectin possesses a molecular weight of 190 kDa. It is a hexamer composed of six subunits each possessing a molecular weight of 35 kDa. The amino acid sequence of the grass carp showed similarity to that of other fish species with 20–30% amino acid identity. It is mitogenic towards murine splenocytes and peritoneal exduate cells.; The mannose-binding lectin was isolated from the inner shoots of the chive Allium tuberosum. The lectin is a single-chained protein with a molecular weight of 13 kDa. N-terminal sequence analysis revealed its remarkable homology to Allium cepa lectin and a lesser extent of similarity to lectins from members of the Amaryllidaceae, Orchidaceae and Liliaceae families. The lectin manifested mitogenic activity in murine splenocytes and inhibitory activity against human immunodeficiency virus type 1 reverse transcriptase.; An RIP, designated calvatin, and an ubiquitin-like protein (CULP) were isolated from the mushroom, Calvatia caelata. Calvatin, possesses two subunits with a molecular weight of 19 kDa and 20 kDa, respectively. It inhibited cell-free translation with an IC₅₀ value of 3 nM and exhibited a ribonuclease activity of 1.58 U/mg toward yeast tRNA. On the other hand, the 8 kDa CULP isolated from the same mushroom inhibited cell-free protein synthesis by 30% at 11 uM and expressed a ribonuclease activity towards yeast tRNA of 1 U/mg. Calvatin and CULP inhibited mitogenic response in murine splenocytes, with IC₅₀ values of 5.5 uM and 0.12 uM, respectively. They exerted an antiproliferative action on breast tumor cells with an ED₅₀ value of 2.4 uM and 120 nM, respectively.; From the inner shoots of the chive Allium tuberosum, a single-chained antifungal protein with a molecular weight of 36 kDa and an N-terminal sequence manifesting resemblance to chitinases but lacking cysteine residues which are characteristic of a cysteine-rich domain present in chitinases of other Allium species, was purified. It exhibited antifungal activity against Rhizoctonia solani, Fusarium oxysporum, Coprinus comatus, Mycosphaerella arachidicola and Botrytis cinerea . The IC₅₀ for its antifungal effect against Botrytis cinerea was 0.2 μM. The protein inhibited cell-free translation in a rabbit reticulocyte system with an IC₅₀ of 0.8 μM, 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.; The results indicate that structurally different defense proteins with some overlap in their spectra of biological activities are elaborated by phylogenetically remote organisms comprising a teleost fish, a monocotyledonons plant and a mushroom.