蓝圆鲹骨骼肌脯氨酸内肽酶的分离纯化及其对胶原肽的作用

以蓝圆鲹为研究对象,探讨脯氨酸内肽酶(Prolyl endopeptidase,PEP)对鱼类肌肉胶原蛋白的作用及其机理。通过硫酸铵分级盐析,DEAE-Sephacel阴离子交换,Phenyl-Sepharose疏水层析和Q-Sepharose阴离子交换,从蓝圆鲹骨骼肌中分离纯化得到一种脯氨酸内肽酶。SDS-PAGE结果显示,PEP的分子量为82 ku,肽质量指纹图谱分析得到16个肽片段,共169个氨基酸残基。片段序列与墨西哥鲷鱼(Neolamprologus brichardi)PEP的同源性达98.8%,证明纯化得到的酶是PEP。PEP的最适温度为35℃,但热稳定性较差;最适p H为6.0,在p H 5.0~7.5之间有较好的稳定性。将PEP与合成的鱼胶原蛋白小肽反应,利用反相高效液相色谱对产物进行分离,电喷雾质谱分析结果显示PEP的水解位点在脯氨酸残基的羧基端。以上结果表明,鱼类肌肉中的PEP能够协同金属蛋白酶,通过切割脯氨酸残基进一步降解胶原小肽从而参与到鱼肌肉胶原蛋白的新陈代谢中,是鱼死后参与胶原蛋白降解的重要酶类。

Purification and Characterization of a Prolyl Endopeptidase from Round Scad (Decapterus maruadsi) Skeletal Muscle and Its Role in Collagen Degradation

The action and mechanism of a prolyl endopeptidase (PEP) isolated from round scad was explored. The PEP was obtained from the skeletal muscle of blue scad via separation and purification by ammonium sulfate fractionation and a series of column chromatographies, including anion-exchange chromatography using a DEAE-Sephacel column, hydrophobic interaction chromatography using a Phenyl-Sepharose column, and anion-exchange chromatography using a Q-Sepharose column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the molecular weight of the PEP was 82 kDa. Peptide mass fingerprinting revealed that there were 16 peptide fragments, including a total of 169 amino acid residues. The sequence homology between the obtained peptide fragment and PEP from Neolamprologus brichardi was 98.8%, suggesting the purified enzyme was indeed a PEP. The PEP had an optimal temperature of 35°C, but showed poor thermal stability. The optimal pH of the purified enzyme was 6.0, and good stability was observed in the pH range of 5.0 to 7.5. Purified PEP was allowed to react with three synthetic fish collagen peptides, then the degraded peptides were further separated by reverse phase-high performance liquid chromatography (RP-HPLC), and electrospray ionization mass spectrometry (ESI/MS) results showed that the PEP hydrolysis site was at the carboxyl terminus of prolyl residues. The results indicate that PEP in fish muscle can collaborate with matrix metalloproteinases to further degrade collagen peptides by cleaving peptide bonds at the carboxyl side of prolyl residues, thus participating in the metabolism of fish muscle collagen. PEP is an important enzyme that participates in post-mortem fish collagen degradation.