Bonnin Stéphanie, El Kirat Karim, Becchi Michel, Dubois Madeleine, Grangeasse Christophe, Giraud Claire, Prigent Annie-France, Lagarde Michel, Roux Bernard, Besson Françoise
Laboratoire de Physico-Chimie Biologique, UMR-CNRS 5013, Université Claude Bernard-Lyon I, Bat. 303, 43, Boulevard du 11 novembre 1918, 69622 Villeurbanne cedex, France.
Biochimie. 2003 Dec;85(12):1237-44. doi: 10.1016/j.biochi.2003.11.004.
Detergent-resistant membranes (DRM) were prepared from bovine kidney cortex. The criterion used to test their purification was the increase in the activity of a GPI membrane-anchored protein, the alkaline phosphatase. Its association with specific proteins and lipids was tested. Two successive Triton X-100 treatments followed by purification on sucrose gradient at 4 degrees C were necessary to obtain DRM with a maximum of alkaline phosphatase activity and a typical protein pattern. A third Triton treatment did not alter this DRM composition. Among the enriched protein, we identified, by mass spectrometry, a microsomal dipeptidase, which was GPI membrane-anchored. Protein-kinase activities, mainly serine-kinase, were enriched during the DRM purification. Using the typical FTIR olefinic =C-H bands of the acyl chains, a global decrease in the unsaturation level of DRM lipids was observed as compared with total membranes. Three main phospholipids were identified in DRM. Their fatty acid compositions were determined by gas chromatography and compared with those of total membranes. The most enriched saturated fatty acid was palmitic acid (+44% for phosphatidylethanolamine, +52% for phosphatidylcholine and +49% for sphingomyelin), agreeing with a selection of specific phospholipids among the saturated ones during the DRM purification.
去污剂抗性膜（DRM）由牛肾皮质制备。用于测试其纯化的标准是糖基磷脂酰肌醇（GPI）膜锚定蛋白碱性磷酸酶的活性增加。测试了其与特定蛋白质和脂质的结合。需要在4℃下进行两次连续的Triton X-100处理，然后在蔗糖梯度上进行纯化，以获得具有最大碱性磷酸酶活性和典型蛋白质模式的DRM。第三次Triton处理并未改变这种DRM组成。在富集的蛋白质中，我们通过质谱鉴定出一种微粒体二肽酶，它是GPI膜锚定的。在DRM纯化过程中，蛋白激酶活性（主要是丝氨酸激酶）得到了富集。利用酰基链典型的傅里叶变换红外光谱（FTIR）烯烃=C-H谱带，观察到与总膜相比，DRM脂质的不饱和水平总体下降。在DRM中鉴定出三种主要的磷脂。通过气相色谱法测定了它们的脂肪酸组成，并与总膜的脂肪酸组成进行了比较。最富集的饱和脂肪酸是棕榈酸（磷脂酰乙醇胺增加44%，磷脂酰胆碱增加52%，鞘磷脂增加49%），这与DRM纯化过程中在饱和磷脂中选择特定磷脂的情况一致。
