Continuous measurement of galactolipid hydrolysis by pancreatic lipolytic enzymes using the pH-stat technique and a medium chain monogalactosyl diglyceride as substrate
Continuous measurement of galactolipid hydrolysis by pancreatic lipolytic enzymes using the pH-stat technique and a medium chain monogalactosyl diglyceride as substrate
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DOI：
10.1016/j.bbalip.2009.05.002
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发表时间：
2009-10-01
影响因子：
4.8
通讯作者：
De Caro, Alain
中科院分区：
文献类型：
--
作者：
Amara, Sawsan;Lafont, Dominique;De Caro, Alain
半乳糖脂是植物中的主要脂类，半乳糖脂酶在其代谢过程中起着重要作用。然而，到目前为止，对这些酶的研究还很少，只开发了几种分析方法。以重组人(RHPLRP2)和豚鼠(RGPLRP2)胰腺脂肪酶相关蛋白2(RGPLRP2)为模型酶，以人工合成的中链单半乳糖二酰甘油(MGDG)为底物，建立了一种特异、连续的半乳糖脂酶检测方法。PLRP2s是胃肠道中半乳糖脂消化的主要酶。单半乳糖二辛酰甘油与胆盐溶液通过超声波混合形成胶束底物，然后启动检测。胆盐的性质和胆盐与MGDG的比例显著影响rHPLRP2和rGPLRP2对MGDG的水解率。在脱氧胆酸钠(NADC)、脱氧胆酸钠与MGDG之比为1.33、碱性pH值为8.0~9.0的条件下，两种酶的半乳糖脂酶活性均达到最大。当MGDG的浓度为10(-2)M时，获得了最大的水解率，并且发现氯化钙不是获得最大活性所必需的。在此条件下，rGPLRP2和rHPLRP2在NaDC/MGDG混合胶束上的最大转化率分别为8000+/-500和2800+/-60mU/min/mg(U/mg)。这些活性与脂肪酶甘油三酯上的活性具有相同的数量级，是迄今报道的半乳糖酶的最高比活性。为了便于比较，胆盐/MGDG混合胶束对MGDG的水解酶活性也进行了比较，发现只有天然和重组人羧酸酯水解酶对MGDG的活性较低(分别为240+/-17和432+/-62U/mg)。(C)2009爱思唯尔B.V.保留所有权利。
Galactolipids are the main lipids from plants and galactolipases play a major role in their metabolism. These enzymes were however poorly studied so far and only few assays have been developed. A specific and continuous galactolipase assay using synthetic medium chain monogalactosyl diacylglycerol (MGDG) as substrate was developed using the pH-stat technique and recombinant human (rHPLRP2) and guinea pig (rGPLRP2) pancreatic lipase-related protein 2 as model enzymes. PLRP2s are the main enzymes involved in the digestion of galactolipids in the gastrointestinal tract. Monogalactosyl di-octanoylglycerol was mixed with bile salt solutions by sonication to form a micellar substrate before launching the assay. The nature of the bile salt and the bile salt to MGDG ratio were found to significantly affect the rate of MGDG hydrolysis by rHPLRP2 and rGPLRP2. The maximum galactolipase activity of both enzymes was recorded with sodium deoxycholate (NaDC) and at a NaDC to MGDG ratio of 1.33 and at basic pH values (8.0-9.0). The maximum rates of hydrolysis were obtained using a MGDG concentration of 10(-2) M and calcium chloride was found to be not necessary to obtain the maximum of activity. Under these conditions, the maximum turnovers of rGPLRP2 and rHPLRP2 on mixed NaDC/MGDG micelles were found to be 8000 +/- 500 and 2800 +/- 60 mu mol/min/mg (U/mg), respectively. These activities are in the same order of magnitude as the activities on triglycerides of lipases and they are the highest specific activities ever reported for galactolipases. For the sake of comparison, the hydrolysis of mixed bile salt/MGDG micelles was also tested using other pancreatic lipolytic enzymes and only native and recombinant human carboxyl ester hydrolase were found to display significant but lower activities (240 +/- 17 and 432 +/- 62 U/mg, respectively) on MGDG. (C) 2009 Elsevier B.V. All rights reserved.