一种凋亡相关蚯蚓丝氨酸蛋白酶的纯化、活性鉴定及部分性质研究

Purification, Identification and Partial Characterization of an Apoptosis-related Serine Protease From Earthworm

通过多级柱层析 ,从赤子爱胜蚓抽提物 (一组抗肿瘤活性蛋白成分 )中纯化得到凋亡相关丝氨酸蛋白酶 1(apoptosis relatedserineprotease 1,ARSP1) ,SDS 聚丙烯酰胺凝胶电泳 (SDS PAGE)测得其表观分子质量为2 8ku .ARSP1非变性PAGE图谱为相连的多条带 ,质谱图为多头峰 ,MALDI TOF MS测得各主峰相对分子质量为 2 464 5 ,2 5 0 5 2和 2 5 2 81,等电聚焦电泳测得等电点 pI <3 8.测得ARSP1N端 2 5个氨基酸序列为 :I(V)IGGT(S)N(D)ASPGEFPWQLSQTRGGSHS ,N端序列比较结果显示其与丝氨酸蛋白酶类高度同源 .体外实验中 ,不仅通过凋亡细胞的相差显微观察验证了ARSP1的细胞杀伤活性 ,而且进一步通过荧光抗体技术对其直接杀伤细胞活性进行了定位研究 .Schiff s试剂糖蛋白染色法鉴定ARSP1为糖蛋白 (或糖肽 ) ,纤维蛋白平板法测得ARSP1同时具有纤溶酶和纤溶酶原激活酶活性 ,进一步通过苯甲磺酰氟 (PMSF)对其纤溶酶活性的抑制实验 。

An apoptosis-related serine protease (ARSP1) was purified from Eisenia fetida extract (mainly a group of anti-tumor protein components) by hydrophobic interaction chromatography and ion exchange chromatography. The molecular mass assayed by SDS-PAGE and isoelectric point of ARSP1 were 28 ku and less than 3.8, separately. However, several coterminous bands could be observed by PAGE of natural ARSP1 and several coterminous peaks of ARSP1 were also detected with MALDI-TOF-MS when the relative molecular mass of three main peaks are 24 645, 25 052 and 25 281, separately. The N-term amino acid sequence of ARSP1 was assayed as following: I(V)IGGT(S)N(D)ASPGEFPWQLSQTRGGSHS. And, a result that ARSP1 is highly homologous with serine proteases was concluded by the comparison of N-term amino acid sequences. In vitro, the cytotoxicity of ARSP1 was not only identified by phase-contrast microscopy observation of apoptotic cells, but also studied further by the localization of fluorescent antibodies. By Schiff's staining, ARSP1 was identified to be glycoprotein or glycopeptide. By fibrin plate assay, ARSP1 was identified to be a plasmin and also a plasminogen activator, and the fibrinolytic activity was inhibited by. PMSF (an inhibitor of serine proteases).