蓝圆鲹骨骼肌内源性脯氨酸内肽酶抑制剂的纯化及其抑制机理

Purification and Mechanism of Action of a Prolyl Endopeptidase Inhibitor from the Skeletal Muscle of Blue Scad(Decapterus maruadsi)

对蓝圆鲹(Decapterus maruadsi)骨骼肌采用热处理、硫酸铵分级沉淀、DEAE-Sepharose弱阴离子交换柱层析、Sephacryl S-200 HR凝胶过滤层析和HiTrap Q HP强阴离子交换柱层析相结合方法,分离、纯化出一种内源性脯氨酸内肽酶抑制剂(prolyl endopeptidase inhibitor,PEPI)。研究了PEPI对脯氨酸内肽酶(prolyl endopeptidase,PEP)活性的影响及抑制机理。Tricine-十二烷基硫酸钠-聚丙烯酰氨凝胶电泳分析表明纯化的PEPI分子质量约为10 kDa,具有较强的热稳定性和酸碱耐受性,其为丝氨酸蛋白酶类PEP的专一性、可逆竞争型抑制剂,抑制常数Ki为0.34 μmol/L。PEPI与PEP形成复合物后,α-螺旋、无规卷曲比例增加,β-折叠比例减少,PEP活性中心构象的改变是酶活力被抑制的主要原因。

In the present study, an endogenous prolyl endopeptidase inhibitor (PEPI) was purified from the skeletal muscle of the marine fish blue scad (Decapterus maruadsi) by thermal precipitation, ammonium sulfate fractionation and a series of column chromatographies on DEAE-Sepharose, Sephacryl S-200 HRand HiTrap Q HP. Tricine-sodium dodecyl sulfate- polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) showed that the molecular mass of PEPI was about 10 kDa. It was thermally stable and displayed tolerance to pH change. PEPI was a specific inhibitor to serine proteinase PEP and it worked in a reversible competitive manner with an inhibitory constant (Ki) of 0.34 μmol/L. A significant increase in α-helix and random coil and decrease in β-sheet were detected after formation of the PEP-PEPI complex. Quite possibly, the conformational change of PEP was the major reason for enzymatic activity inhibition.