A Fluorescence-based Assay of Phospholipid Scramblase Activity.
A Fluorescence-based Assay of Phospholipid Scramblase Activity.
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DOI：
10.3791/54635
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发表时间：
2016-09-20
期刊：
影响因子：
--
通讯作者：
Menon AK
中科院分区：
文献类型：
--
作者：
Ploier B;Menon AK
乱序酶以ATP非依赖性方式将磷脂跨膜双分子层双向移位。第一个被鉴定和生物化学验证的乱序酶是视蛋白，光感受器视紫红质的脱辅基蛋白。视紫红质是位于视网膜的视杆细胞盘膜中的G蛋白偶联受体，其负责光的感知。视紫红质的乱序酶活性不依赖于其配体11-顺式-视黄醛，即，脱辅基蛋白视蛋白也具有作为乱序酶的活性。尽管组成型和调节型磷脂乱序在细胞生理学中起着重要作用，但迄今为止，除了视蛋白之外，只有少数磷脂乱序酶被鉴定。在这里，我们描述了一种基于荧光的测定视蛋白的scramblase活性。将视蛋白重构成由磷脂酰胆碱、磷脂酰甘油和痕量荧光NBD标记的PC（1-棕榈酰-2-{6-[7-硝基-2-1，3-苯并恶二唑-4-基）氨基]己酰基}-sn-甘油基-3-磷酸胆碱）组成的大单层脂质体。扰码酶活性通过测量位于囊泡内叶的NBD-PC分子能够进入外叶的程度来确定，在外叶中，它们的荧光被无法穿过膜的还原剂化学消除。我们所描述的方法具有普遍适用性，并可用于识别和表征其他膜蛋白的乱序酶活性。
Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin’s scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin’s scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.
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影响因子：
62.1
作者：
Ernst, Oliver P.;Lodowski, David T.;Elstner, Marcus;Hegemann, Peter;Brown, Leonid S.;Kandori, Hideki
通讯作者：
Kandori, Hideki
影响因子：
14.8
作者：
Geertsma, Eric R.;Mahmood, N. A. B. Nik;Poolman, Bert
通讯作者：
Poolman, Bert
DOI：
10.1073/pnas.1504967112
发表时间：
2015-05-19
影响因子：
11.1
作者：
Meeske, Alexander J.;Sham, Lok-To;Rudner, David Z.
通讯作者：
Rudner, David Z.
影响因子：
2.9
作者：
Romsicki, Y;Sharom, FJ
通讯作者：
Sharom, FJ
影响因子：
4.1
作者：
Eckford, PDW;Sharom, FJ
通讯作者：
Sharom, FJ