Yakymyshyn L M, Walker K, Thomson A B
Biochim Biophys Acta. 1982 Sep 9;690(2):269-81. doi: 10.1016/0005-2736(82)90331-5.
(1) Intestinal absorption is altered under a variety of circumstances in health and disease and to determine a possible relationship between intestinal absorptive function and intestinal brush border membrane composition, we undertook the isolation and purification of rabbit jejunal and ileal brush borders, to allow further studies of their lipid composition under varied experimental conditions. (2) A modification of an established method (Schmitz, J., Preiser, H., Maestracci, D., Ghosh, B.K., Cerda, J.J. and Crane, R.K. (1973) Biochim. Biophys. Acta 323, 98-112) utilized CaCl2 aggregation and sequential centrifugation followed by purification of the brush border pellet (P2) at 27,000 X g on a PercollTM (Pharmacia) self-forming gradient. The PercollTM was removed by ultracentrifugation for 30 min at 100 000 X g, utilizing a batch rotor in the Beckman airfugeTM. (3) Pure brush border membrane vesicles were obtained and characterized by specific marker analysis and electron microscopy. Comparative marker analyses performed on P2 and final PercollTM preparations from animals showed that the purification achieved was 8-11-fold greater when compared to the original homogenates. Verification of purity was also demonstrated by the absence of DNA and very low levels of Beta-gluconridase and (Na+ + K+)-ATPase in the PercollTM preparations. (4) Comparative lipid analyses of P2 and final PercollTM preparations showed that levels of total phospholipid and free fatty acids were several-fold higher in the PercollTM preparations on a per mg protein basis. (5) A comparison of the activity of enzyme markers and the levels of total free fatty acids in P2 pellets obtained after Cacl2 and MgCl2 aggregation showed that CaCl2 aggregation gave the more consistently reproducible results. (6) Although standard procedures of membrane preparations not involving density gradient separation provide membranes of reasonable purity for the estimation of lipid components, we consider the final purification step of density gradient separation using PercollTM is essential for determining small quantitative changes which might occur in the membrane lipid composition under experimental conditions were intestinal absorptive function is altered.
（1）在健康和疾病的各种情况下，肠道吸收都会发生改变。为了确定肠道吸收功能与肠道刷状缘膜组成之间的可能关系，我们对兔空肠和回肠刷状缘进行了分离和纯化，以便在不同实验条件下进一步研究其脂质组成。（2）对一种既定方法（施密茨，J.，普雷泽尔，H.，马埃斯特拉奇，D.，戈什，B.K.，塞尔达，J.J.和克兰，R.K.（1973年）《生物化学与生物物理学报》323卷，第98 - 112页）进行了改进，利用氯化钙聚集和连续离心，随后在27,000×g的条件下在PercollTM（法玛西亚公司）自形成梯度上对刷状缘沉淀（P2）进行纯化。通过在贝克曼空气离心机TM中使用批量转子，以100,000×g的转速超速离心30分钟来去除PercollTM。（3）获得了纯的刷状缘膜囊泡，并通过特异性标记分析和电子显微镜进行了表征。对动物的P2和最终PercollTM制剂进行的比较标记分析表明，与原始匀浆相比，纯化程度提高了8 - 11倍。PercollTM制剂中不存在DNA以及β - 葡萄糖醛酸酶和（钠 + 钾）- ATP酶水平极低也证明了纯度的验证。（4）对P2和最终PercollTM制剂的脂质进行比较分析表明，以每毫克蛋白质计，PercollTM制剂中总磷脂和游离脂肪酸的水平高出几倍。（5）对氯化钙和氯化镁聚集后获得的P2沉淀中酶标记物的活性和总游离脂肪酸水平进行比较，结果表明氯化钙聚集产生的结果更具一致性和可重复性。（6）尽管不涉及密度梯度分离的标准膜制备程序可为脂质成分的估计提供纯度合理的膜，但我们认为使用PercollTM进行密度梯度分离的最终纯化步骤对于确定在肠道吸收功能改变的实验条件下膜脂质组成可能发生的微小定量变化至关重要。
