八棱丝瓜籽核糖体失活蛋白的分离纯化及其生化性质

通过盐溶液抽提、硫酸铵分级沉淀、CM 5 2纤维素阳离子交换层析、反相毛细管液相色谱 (re verse phasecapillaryliquidchromatography ,RP CLC)等步骤 ,从八棱丝瓜籽中分离到 2种单链核糖体失活蛋白luffaculin 1和luffaculin 2 .在SDS PAGE和IEF上均显示为单一条带 ,表观分子量均为 2 8kD ,其等电点分别为 8 86 (luffaculin 1)和 9 0 5 (luffaculin 2 ) .实验表明 ,它们具有RNAN 糖苷酶活性 .蛋白质合成抑制活性测试表明 ,它们对蛋白质合成具较强的抑制作用 .体外抑制肿瘤细胞生长活性检测表明 ,luffaculin 1和luffaculin 2对人白血病细胞株K5 6 2有较强的毒性 ,IC50 分别为 1 1×10 -6mol L和 2 0× 10 -7mol L .八棱丝瓜籽核糖体失活蛋白具有可以用于或构成免疫毒素治疗癌症的应用前景

Purification and Characterization of Two Luffaculins, Ribosome-inactivating Proteins from Seeds of Luffa acutangula

Two ribosome-inactivating proteins (RIPs) luffaculin-1 and luffaculin-2 were isolated and purified from the seeds of Luffa acutangula by extraction with NaCl solution, ammonium sulfate fractional precipitation, CM-52 cellulose ion exchange chromatography and reverse-phase capillary liquid chromatography (RP-CLC). The results of SDS-PAGE and IEF showed that the two RIPs were a homogeneous single band. The molecular mass of luffaculin-1 and luffaculin-2 was estimated to be about 28 kD by SDS-PAGE. luffaculin-1 and luffaculin-2 were basic proteins with pI of 8^86 and 9^05 by IEF analysis, respectively. N-glycosidase activities of luffaculin-1 and luffaculin-2 against animal rRNA were also confirmed. luffaculins could inhibit germinal vesicle broken-down (GVBD) of oocytes from Bufo gargarizans that showed strong inhibitory activities on protein biosynthesis. The cytotoxicities of luffaculin-1 and luffaculin-2 on human leukemia K562 cells were determined with IC 50 of 1.1×10 -6 mol/L and 2^0×10 -7 mol/L, respectively. It is promising that luffaculins can treat some cancers or may be used as an efficient moiety of immunotoxins.