Detergent-insoluble glycosphingolipid/cholesterol microdomains of the myelin membrane
Detergent-insoluble glycosphingolipid/cholesterol microdomains of the myelin membrane
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DOI：
10.1046/j.1471-4159.2002.00884.x
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发表时间：
2002-06-01
影响因子：
4.7
通讯作者：
Pfeiffer, SE
中科院分区：
文献类型：
--
作者：
Taylor, CM;Coetzee, T;Pfeiffer, SE
鞘糖脂和胆固醇在生物膜内形成横向组装，或脂质“筏”。脂筏通常作为低密度、洗涤剂不溶性复合物（在4 ℃的非离子洗涤剂中; DIG，洗涤剂不溶性鞘糖脂/胆固醇微区）进行生物化学研究。最近的差异建议重新评估用于脂筏生化分析的条件。我们已经研究了洗涤剂不溶性的几个已知的蛋白质存在于鞘糖脂/胆固醇丰富的髓鞘膜，使用四种洗涤剂代表不同的化学类别（TX-100、CHAPS、Brij 96和TX-102），在四种条件下：在（i）4 ℃或（ii）37 ℃洗涤剂提取髓磷脂，或在用（iii）皂苷或（iv）甲基-β-环糊精（MbetaCD）预提取后在4 ℃下。每种去污剂溶解髓鞘蛋白的能力和产生的DIG的密度不同。Brij 96 DIG漂浮至比测试的其他去污剂更低的密度，可能代表髓磷脂中的DIG亚群。用皂苷预提取的DIG比用MbetaCD预提取的DIG更致密。此外，用MbetaCD预提取溶解了蛋白脂质蛋白（已知与胆固醇相关），而用皂苷预提取则没有，这表明皂苷作为胆固醇干扰剂不如MbetaCD有效。这些结果表明，通过不同去污剂分离的DIG不一定是可比的，并且这些去污剂特异性DIG可能代表不同的生物化学和可能的生理学实体，其基于特定脂质/蛋白质在每种类型去污剂中的溶解度。
Glycosphingolipids and cholesterol form lateral assemblies, or lipid 'rafts', within biological membranes. Lipid rafts are routinely studied biochemically as low-density, detergent-insoluble complexes (in non-ionic detergents at 4degreesC; DIGs, detergent-insoluble glycosphingolipid/cholesterol microdomains). Recent discrepancies recommended a re-evaluation of the conditions used for the biochemical analysis of lipid rafts. We have investigated the detergent insolubility of several known proteins present in the glycosphingolipid/cholesterol-rich myelin membrane, using four detergents representing different chemical classes (TX-100, CHAPS, Brij 96 and TX-102), under four conditions: detergent extraction of myelin either at (i) 4degreesC or (ii) 37degreesC, or at 4degreesC after pre-extraction with (iii) saponin or (iv) methyl-beta-cyclodextrin (MbetaCD). Each detergent was different in its ability to solubilize myelin proteins and in the density of the DIGs produced. Brij 96 DIGs floated to a lower density than other detergents tested, possibly representing a subpopulation of DIGs in myelin. DIGs pre-extracted with saponin were denser than DIGs pre-extracted with MbetaCD. Furthermore, pre-extraction with MbetaCD solubilized proteolipid protein (known to associate with cholesterol), whereas pre-extraction with saponin did not, suggesting that saponin is less effective as a cholesterol-perturbing agent than is MbetaCD. These results demonstrate that DIGs isolated by different detergents are not necessarily comparable, and that these detergent-specific DIGs may represent distinct biochemical, and possibly physiological, entities based on the solubilities of specific lipids/proteins in each type of detergent.