Inhibition of phospholipase A1, lipase and galactolipase activities of pancreatic lipase-related protein 2 by methyl arachidonyl fluorophosphonate (MAFP)
Inhibition of phospholipase A1, lipase and galactolipase activities of pancreatic lipase-related protein 2 by methyl arachidonyl fluorophosphonate (MAFP)
复制标题
DOI：
10.1016/j.bbalip.2012.07.014
复制
发表时间：
2012-11-01
影响因子：
4.8
通讯作者：
Carriere, Frederic
中科院分区：
文献类型：
--
作者：
Amara, Sawsan;Delorme, Vincent;Carriere, Frederic
花生四烯基氟膦酸甲酯 (MAFP) 是一种已知的胞质磷脂酶 A2 和其他一些丝氨酸酶的抑制剂。此处发现 MAFP 是人胰腺脂肪酶相关蛋白 2 (HPLRP2) 的不可逆抑制剂，HPLRP2 是一种具有脂肪酶、磷脂酶 A1 和半乳糖脂肪酶活性的酶。在 MAFP 存在的情况下，HPLRP2 的质谱分析显示质量增加了 351 Da，表明 MAFP 与活性位点丝氨酸残基共价结合。当在测量残余活性之前将 HPLRP2 与 MAFP 预孵育时，发生了 HPLRP2 的直接抑制，证实 HPLRP2 具有可自由接触溶剂的活性位点，并且与溶液中的大多数脂肪酶不同。 HPLRP2 对三丁酸甘油酯 (TC4)、磷脂酰胆碱 (PC) 和单半乳糖基二辛酰甘油 (C8-MGDG) 的活性在这些条件下同样受到抑制。胆汁盐不需要触发抑制，但它们显着增加了 HPLRP2 的抑制率，可能是因为 MAFP 胶束溶解。由于 HPLRP2 在各种底物上都有活性，这些底物在水的存在下会以不同的方式自组织。在脂解反应过程中添加 MAFP 后，在这些底物存在的情况下测试 MAFP 对 HPLRP2 的抑制作用。在这种情况下，脂肪酶、磷脂酶 A1 和半乳糖脂肪酶活性的抑制率并不相等（甘油三酯 > PC > MGDG），表明水相和脂质聚集体之间的酶/抑制剂分配不同。 MAFP 对胰液和人单核细胞中存在的一种已明确确定的磷脂酶 A1 (HPLRP2) 的抑制表明 MAFP 不能用于在细胞水平上区分磷脂酶 A2 和 A1 活性。 (C) 2012 Elsevier B.V. 保留所有权利。
Methyl arachidonyl fluorophosphonate (MAFP) is a known inhibitor of cytosolic phospholipase A2 and some other serine enzymes. MAFP was found here to be an irreversible inhibitor of human pancreatic lipase-related protein 2 (HPLRP2), an enzyme displaying lipase, phospholipase A1 and galactolipase activities. In the presence of MAFP, mass spectrometry analysis of HPLRP2 revealed a mass increase of 351 Da, suggesting a covalent binding of MAFP to the active site serine residue. When HPLRP2 was pre-incubated with MAFP before measuring residual activity, a direct inhibition of HPLRP2 occurred, confirming that HPLRP2 has an active site freely accessible to solvent and differs from most lipases in solution. HPLRP2 activities on tributyrin (TC4), phosphatidylcholine (PC) and monogalactosyl dioctanoylglycerol (C8-MGDG) were equally inhibited under these conditions. Bile salts were not required to trigger the inhibition, but they significantly increased the rate of HPLRP2 inhibition, probably because of MAFP micellar solubilization. Since HPLRP2 is active on various substrates that self-organize differently in the presence of water. HPLRP2 inhibition by MAFP was tested in the presence of these substrates after adding MAFP in the course of the lipolysis reaction. In this case, the rates of inhibition of lipase, phospholipase A1 and galactolipase activities were not equivalent (triglycerides>PC>MGDG), suggesting different enzyme/inhibitor partitioning between the aqueous phase and lipid aggregates. The inhibition by MAFP of a well identified phospholipase A1 (HPLRP2), present in pancreatic juice and also in human monocytes, indicates that MAFP cannot be used for discriminating phospholipase A2 from A1 activities at the cellular level. (C) 2012 Elsevier B.V. All rights reserved.