血管紧张素转化酶(angiotensin-converting enzyme,ACE)在血压调控系统中起到关键作用。该文通过优化盐析、离子交换层析和超滤等步骤从猪肺中分离纯化ACE并研究其酶学性质。以食源ACE抑制肽Ala-Gly-Pro(AGP)为对照抑制剂,研究了还原型谷胱甘肽(reduced glutathione,GSH)对猪肺ACE活性的影响。当猪肺匀浆液蛋白质量浓度在30 mg/mL时,在35 ℃的条件下进行离子交换层析纯化ACE,酶活回收率分别达75.6%和61.9%,超滤液流速为5.0 mL/min进行超滤,最终ACE的比活为1.9 U/mg,纯化倍数为475倍,酶活回收率为43.4%。在不增加成本的基础上减少了ACE分离纯化过程中的酶损失,提高ACE分离纯化效率,凝胶电泳结果显示猪肺ACE分子质量为160 kDa,酶学性质分析表明纯化后ACE的最适反应温度为37 ℃,最适反应pH值为7.5,米氏常数Km为2.05 mmol/L,最大反应速率Vmax为4.64 nmol/L。体外活性实验表明AGP和GSH均能够抑制猪肺ACE的活性,且AGP和GSH对ACE的IC50值分别为564.0、26.2 μmol/L,对其抑制类型分别为竞争型抑制和非竞争性抑制,抵制常数Ki分别为127.0、15.5 μmol/L。该研究为从抗氧化物质中筛选可能对预防高血压有效的降血压肽提供了可能。
Angiotensin-Converting Enzyme (ACE) plays a key role in the blood pressure regulation system.ACE was purified from pig lung by optimized salt fractionation, ion exchange chromatography and ultrafiltration, and its enzymatic properties were studied.The effect of reduced glutathione (GSH) on ACE activity was investigated by using the food-derived ACE inhibitory peptide Ala-Gly-Pro (AGP) as a control inhibitor.The recovery of ACE activity of salt fractionation (the protein mass concentration of pig lung homogenate was 30 mg/mL) and ion exchange chromatography (at 35 ℃) were 75.6% and 61.9%, respectively.Then the final purity (475-fold) was achieved by ultrafiltration and generated a specific activity of 1.9 U/mg and at 43.4% recovery of ACE activity.The enzyme loss during isolation and purification processes was reduced without increasing the cost, and the purification efficiency was improved.The molecular mass of the purified ACE determined from SDS-PAGE analysis was 160 kDa.Moreover, the enzyme was optimally active at pH 7.5 and 37 ℃ with Km value of 2.05 mmol/L and Vmax of 4.64 nmol/L.In vitro activity test indicated that both AGP and GSH exhibited the ACE (from pig lung) inhibitory activity.Moreover, AGP was a competitive ACE inhibitor (IC50 values of 564.0 μmol/L) and GSH was a non-competitive ACE inhibitor (IC50 values of 26.2 μmol/L).This study provides a possibility for screening effective antihypertensive peptides from antioxidant substances for the prevention of hypertension.
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