Etemadi A H
Biochim Biophys Acta. 1980 Dec 31;604(3):423-75. doi: 10.1016/0005-2736(80)90579-9.
In the companion paper, I have reviewed the techniques employed for assessment of the asymmetric distribution and orientation of membrane proteins. This article deals with methods applicable to the investigation of the unequal distribution of lipids between the two membrane leaflets. Among the techniques I will discuss are the use of immunological techniques and lectins, chemical reagents, enzymatic isotopic labeling and degradation of membrane lipids, exchange proteins and physical techniques. Whenever appropriate, problems of crypticity and non-availability of lipids to interact with the appropriate ligands, reagents, modifying enzymes or exchange proteins have been envisaged. It appears that in many case, highly discordant results, sometimes with the same biological material, have been obtained. Some of the difficulties encountered presumably stem from the reported existence of non-bilayer arrangements and isotropic movement of lipids as evidenced by freeze-fracture and NMR studies. Other problems may be related to the induction of such arrangements, especially the inverted micellar arrangement, by the modifying agents, particularly degradation enzymes or exchange proteins when they cause severe unilateral modification of the lipids of the exposed leaflet. In addition, the situation is complicated by the role of the induced increase in the flip-flop rate under different experimental conditions and by modification of the rearrangement of lipid molecules as a result of the metabolic state of the cell or ghost preparation and of the reactivity of lipids as a consequence of temperature changes. Here, more so than with proteins, one must be cautious in interpreting experimental results. Moreover, it would appear that the use of different techniques in conjunction and the consequent comparison of results should be recommended. It has been emphasized that 'general rules' do not hold and that each new material should be assay again. To give one example, it is not pertinent to state that proteins enhance the flip-flop rate in lipid vesicles (and hence in membranes). This holds true for glycophorin from erythrocyte membrane, but could not be proved when mitochondrial cytochrome oxidase was used. There seems to be no rule for the distribution of lipids between the two leaflets of different membranes. For example, even for different strains of the same bacterial species, highly divergent results have been reported. It is generally (and probably under the influence of different studies with erythrocytes) believed that in mammalian plasma membranes, choline phospholipids are enriched in the outer leaflet and aminophospholipids in the inner leaflet. Though this contention may prove to be correct, different instances of contradictory results have been given in the text. This shows that if rules do exist, they remain to be discovered or established...
在配套论文中，我回顾了用于评估膜蛋白不对称分布和取向的技术。本文讨论适用于研究脂质在两个膜小叶间分布不均的方法。我将讨论的技术包括免疫技术和凝集素的使用、化学试剂、酶促同位素标记和膜脂质的降解、交换蛋白以及物理技术。在适当的时候，还考虑了脂质与合适的配体、试剂、修饰酶或交换蛋白相互作用时的隐蔽性和不可用性问题。似乎在许多情况下，即使使用相同的生物材料，也会得到高度不一致的结果。遇到的一些困难可能源于据报道存在的非双层排列以及脂质的各向同性运动，冷冻断裂和核磁共振研究证明了这一点。其他问题可能与修饰剂诱导的这种排列有关，特别是反相胶束排列，当修饰剂，尤其是降解酶或交换蛋白导致暴露小叶脂质的严重单侧修饰时。此外，不同实验条件下诱导的翻转速率增加的作用以及细胞或空壳制备的代谢状态导致的脂质分子重排的改变和温度变化导致的脂质反应性的改变使情况变得复杂。在这里，与蛋白质相比，人们在解释实验结果时必须更加谨慎。此外，似乎应该推荐结合使用不同技术并随后比较结果。有人强调“一般规则”并不适用，每种新材料都应该重新进行测定。举个例子，说蛋白质会提高脂质囊泡（进而在膜中）的翻转速率是不恰当的。这对于红细胞膜上的血型糖蛋白是成立的，但当使用线粒体细胞色素氧化酶时却无法证明。不同膜的两个小叶间脂质的分布似乎没有规律。例如，即使对于同一细菌物种的不同菌株，也报道了高度不同的结果。一般来说（可能受对红细胞的不同研究影响），人们认为在哺乳动物质膜中，胆碱磷脂在外小叶中富集，氨基磷脂在内小叶中富集。尽管这一论点可能被证明是正确的，但文中给出了不同的矛盾结果实例。这表明如果确实存在规则，它们仍有待发现或确立……
