鱿鱼肝脏含有丰富的蛋白酶,为利用其内源蛋白酶进行可控的酶解,本研究以鲤鱼肌原纤维蛋白为底物对鱿鱼肝脏内源蛋白酶的种类和性质进行了研究.反应体系中添加E-64、1,10-菲啰啉和苯甲基磺酰氟(phenylmethylsulfonyl fluoride,PMSF)后,肌球蛋白重链(myosin heavy chain,MHC)的降解得到了显著抑制,确定了鱿鱼肝脏含有金属类、半胱氨酸类、丝氨酸类3类蛋白酶.半胱氨酸类蛋白酶热稳定性最好,在50℃以上仍然具有较大活性,可将肌原纤维蛋白酶解成小分子质量的降解产物.利用特异性底物对半胱氨酸蛋白酶种类进行鉴定发现,该酶只酶解Z-Phe-Arg-MCA,添加亮抑酶肽后相对酶活性为0%,添加E-64相对酶活性仅存0.6%,初步确定鱿鱼肝脏中的半胱氨酸蛋白酶主要为组织蛋白酶L.最后,通过硫酸铵沉淀、离子交换层析、凝胶过滤对组织蛋白酶L进行分离纯化,在电泳上得到了分子质量约为25kD单一条带.%In order to understand the applicability of endogenous proteases from squid liver as a rich source of proteases, the endogenous proteases of squid liver were identified and characterized with common carp myofibrillar protein as substrate by adding a variety of protease inhibitors. It was found that the degradation of myosin heavy chain (MHC) was significantly inhibited by addition of E-64, 1,10-phenanthroline and PMSF to the reaction system indicating that squid liver contained three kinds of proteases, metalloprotease, cysteine protease and serine protease. The cysteine protease had the best thermal stability and highest activity, which still maintained a high activity at temperatures higher than 50 ℃, and it could hydrolyze myofibrillar proteins into small molecules. Substrate specificity analysis indicated that the cysteine protease could only hydrolyze Z-phe-Arg-MCA, and was inhibited 100% by leupeptin and 99.4% by E-64, indicating that the enzyme consisted mainly of cathepsin L. At last, cathepsin L was purified to homogeneity by ammonium sulfate precipitation, ion exchange and gel filtration chromatography. The purified enzyme showed a molecular weight of about 25 kD.
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