Name: a fluorescence-based assay of phospholipid scramblase activity
Uploaded: 2016-09-20T08:00:00.000+00:00
Duration: 9 min 52 s
Description: Watch this Scientific Journal Video about A Fluorescence-based Assay of Phospholipid Scramblase Activity at JoVE.com

This study was supported by the Velux Stiftung (A.K.M.), NIH grant EY024207 (A.K.M.) and the Austrian Science Fund (FWF) project J3686 (B.P.).

1.脂质体和蛋白脂质体的制备<ol><li>脂质体形成<ol><li>用玻璃注射器，添加1435微升POPC（25毫克/毫升，在氯仿中）和160微升POPG（25毫克/毫升，在氯仿中）到一个圆底烧瓶中，以获得在POPC的摩尔比52.5微摩尔脂质:POPG = 9:1。 </li><li>使用旋转蒸发器在145 rpm的转速（需要此体积的溶剂没有水浴）干燥30分钟的脂质，然后将烧瓶转移到真空干燥器为至少3小时，或过夜，在室温下（RT）。 </li><li>水合干燥的脂质膜用10ml的50mM HEPES pH 7.4的，100毫摩尔NaCl的通过轻轻摇动烧瓶直至均匀，混浊悬浮液的结果（以下称为缓冲液A）; 注:在此阶段的脂质浓度预期为5.25毫米，因为没有损失应该迄今发生。 </li><li>超声处理水浴中的悬浮液10分钟，在室温下用一个频率ö˚F40千赫。该解决方案将看起来有点清晰。 </li><li>使用挤出机，通过膜传递悬挂10次以400纳米的孔径，随后用4个道次挤出的第二循环通过具有200nm的孔尺寸的膜。 注:将所得的LUV的平均直径为〜175纳米。如果需要的话，的LUV的大小和均匀性，可以通过动态光散射，根据制造商的说明检查。 </li><li>量化LUV悬浮液的磷脂浓度为1.2节所述）。 注意:由于挤压时的损失，该浓度通常是大约3.6毫米。 </li><li>存储的LUV在4℃下约2周，如果不立即使用。 </li></ol></li><li>量化磷脂 注意:要确定用于重构的LUV悬浮液的磷脂浓度，以及该被最终生成的脂蛋白的，样品的等分试样是subje由高氯酸反恐执行氧化。此过程分解磷脂释放无机磷酸盐，然后由一比色测 ​​定中使用的标准23比较定量。 <ol><li>制备在去离子蒸馏水磷酸钠的40mM的储备溶液（钠2 HPO 4）。 </li><li>稀释原液用去离子蒸馏水，得到4毫米和0.4毫米的工作的解决方案，将作为校准标准。 </li><li>使用工作溶液，在13×100毫米2玻璃管从0到在50微升的最终体积80磷酸纳摩尔钠制备的标准。 </li><li>采取10微升的每个LUV和脂蛋白体样本进行量化，并与在13×100毫米2玻璃管DDH 2 O的40微升稀释。 注:由于的LUV和蛋白脂质体的脂质浓度范围为2.5-5微米，在10微升样品的应含有25-50纳摩尔脂磷。</li><li>在145℃下在加热块添加300μl的高氯酸到每个标准品和样品并加热1小时。沽上管道弹珠防止蒸发。 </li><li>让管冷却至室温，并加入1 ml的DDH 2 O的</li><li>添加400μl的每个新鲜编写了12克/升钼酸铵和50g / L的抗坏血酸钠和涡流的混合。 </li><li>热，在100℃10分钟以上的管的顶部大理石。从加热块中取出管，并让他们冷却到室温。 </li><li>衡量用分光计空白（含有0纳摩尔磷酸钠标准样品）的样品的吸光度的797纳米的波长。 </li><li>确定样品对校准标准曲线的磷酸盐含量。 </li></ol></li><li>视蛋白的重建 注:有必要确定用于重建最佳膨胀的条件，因为这些依赖于洗涤剂的性质，以及作为的LUV的脂质组成和浓度。如的LUV更改溶胀过程中的光散射性质可以通过测量由里戈和利维24和​​Geertsma 等人 25审查吸光度（ 图2）来监测。 <ol><li>吸移管800微升的LUV（来自1.1部分;挤压后的脂质回收率为70％，的LUV的预期浓度是3.6毫磷脂）到2ml微量离心管中。 </li><li>添加缓冲液A 5.3微升和34.7微升的10％（重量/体积）DDM溶解在缓冲液A </li><li>孵育在室温下与最终过端搅拌3小时。 </li><li>同时准备聚苯乙烯珠粒: <ol><li>使用400毫克的小珠每个样品并称量出来在玻璃烧杯中。 </li><li>用甲醇与水用缓冲液A洗两次，三次和一次对于每个洗涤步骤使用溶于5ml液体并缓慢搅拌10分钟。 注:建议准备聚苯乙烯对几个样品一次， 例如珠，称量6克小珠，并用75毫升的液体洗净。过量的珠子可以储存在冰箱中数天。 </li></ol></li><li>期间囊泡不稳定的最后30分钟，干燥在螺丝帽玻璃管NBD标记的磷脂（"重建玻璃管&#39;）:每样品，9.5微升NBD-PC（1毫克/毫升的氯仿，得到0.4％（摩尔）总磷脂的）都是在氮气流中的玻璃管干燥，并随后溶解于0.1​​％的45微升（重量/体积）在缓冲液A DDM </li><li>后囊泡不稳定的3小时添加溶解-NBD标记的磷脂，所述的DDM溶解的蛋白质和缓冲液A，使得1毫升的最终体积中含有0.36％（重量/体积）， 即 ，7毫米，DDM。 <ol><li>因此，为了产生无蛋白质的脂质体（用作对照样品）添加45微升NBD-PC（溶解在0.1％DDM）的，60微升0.1％DDM和55微升缓冲液A;对于脂蛋白增加，对于考试PLE蛋白质的40微升（从〜110毫微克/微升的典型原液）0.1％DDM，45微升NBD-PC的（溶于0.1％DDM），0.1％DDM 20微升和55微升缓冲液A 。 注:加入顺序应列出。 </li></ol></li><li>混合样品在室温下额外小时结束以上结束。 </li><li>加80毫克的制备聚苯乙烯珠粒孵育与在RT 1小时结束过端混合的样品。 </li><li>接下来，添加一个额外的160毫克的聚苯乙烯珠，并在室温再2小时结束了高端混合培养。 </li><li>样品（离开废聚苯乙烯珠后面）转移到含有160毫克的新鲜聚苯乙烯珠的玻璃螺丝帽管中并在4℃混合过夜。 注:以传输样品和避免吸干珠的最简单的方法是使用被压入到玻璃管的底部用少许正压巴斯德吸管。一旦吸管尖是牢固的管的底部，则该样品可容易地在不脱离珠粒干扰撤回。 </li><li>第二天早上，将样品转移到离心管不结转珠置于冰上，准备scramblase活性测定。 </li></ol></li></ol> 2. Scramblase活性测定注意:用缓冲液A稀释的脂质体或脂蛋白体的荧光强度随时间后在荧光光谱仪在加入连二的监测。为了获得稳定的起始强度，荧光是加入连二亚到一个不断搅拌样品前记录，至少为50秒（或直到一个稳定的信号时实现），然后接着进行至少500秒加入连二亚之后。 <ol><li>添加1,950微升缓冲液A含迷你搅拌棒的塑料杯中。 </li><li>加入50μl准备PROTEO的（脂质体），并让在荧光分光样品平衡计在恒定搅拌下几秒钟。 </li><li>与此同时，在0.5M的缓冲三（ 例如，对于两个样品称出20毫克的连二亚硫酸盐在离心管中，并在114溶解准备1M的连二亚硫酸钠溶液微升冰冷的0.5M的Tris直接使用前保持在冰上下一个样品）。 注:连二亚溶液必须是新制备的，并在制备后不应该使用多于20分钟如果许多测量是制成，连二亚硫酸盐的等分试样可以预先称出并在使用前右溶解。 </li><li>启动荧光检测（激发波长470，发射530纳米，狭缝宽度0.5纳米）。 </li><li>添加40微升的1M二亚溶液到试管50秒开始的荧光记录（使用隔膜中的反应杯腔室如果可能的盖子）后，继续记录荧光再400-600秒。 </li><li>如在第3节中所述分析数据。 </li></ol> 3。数据分析<ol><li>扰动力学<ol><li>通过定义初始荧光表征由scramblase活性测定得到的各样品的荧光微量， 的 F i，之前加入连二亚硫酸盐，以及终点荧光，F到达后&gt; 400秒。 的 F i是对每个样品荧光的用于加入连二亚硫酸盐之前的30秒期间的平均值来确定。 </li><li>确定对应于荧光减少的程度，R = 100的结束点数据•F / 岛我们使用术语R L为无蛋白质的脂质体和R P代表含视蛋白-蛋白脂质体。 </li></ol></li><li>确定功能复溶Scramblase的分子量<ol><li>根据下列等式转换荧光减少数据: ρ（≥1scramblase）=（; R P - R L）/（R 最大 - R L）（等式1） 注意:当R max为当足够的蛋白质复原，使得样品中的所有囊泡具有至少一个官能scramblase使得获得最大限度的降低，而p是一个重构样品中特定囊泡&#39;scramblase活性"的概率， 即 ，它具有至少一个官能scramblase。对于R L中的值通常为45％3而读取最大典型地82.5％3，而不是预期的100％（R 最大可用于与&gt; 1毫克/毫摩尔的PPR视蛋白脂蛋白体通过实验确定）。作为R 最大 &lt;100％，假设囊泡的亚群是难治重建。对于R 最大值 = 82.5％，这是无法接受蛋白质囊泡的分数为0.35。 </li><li>描述如下泊松统计P（≥1scramblase）和PPR（毫克蛋白/毫摩尔磷脂）之间的关系: P（≥1scramblase）= 1 - 电子<s向上&gt; -m = 1 - EXP（-PPR /α）（式2） 注:其中M =毫克蛋白质/毫摩尔磷脂个单位囊泡和α=单指数拟合常数scramblases的数量。 </li><li>由于囊泡的分数并不即使在高PPR扰码（见讨论）贡献，修改公式: ρ（≥1scramblase）= 1 - EXP（-PPR * /α）（公式3） 注:在哪里PPR * = PPR /（1 F），其中f是囊泡的耐火人口或，在这种情况下，PPR * = PPR / 0.65（等式4）。 注:拟合常数α通过用单指数函数拟合P（≥1scramblase）与PPR *的曲线图确定的。如果视蛋白（分子量41.7 kDa）的功能重新构造成纳米直径175囊泡（各泡囊有28万磷脂26），为单体，α= 0.187毫克毫摩尔-1。如果视蛋白二聚化前复溶3产生scramblase活跃的囊泡，则α= 0.37毫克毫摩尔-1。如果PPR而非PPR *分别被用于分析，那么相应的α值将是0.122和0.244毫克毫摩尔-1。对于α这些预测值假设所有的视蛋白分子在功能上主管。如果仅分子的一小部分是主管加扰脂质，则α的对应值将更大。 </li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+vesicle+formation+and+transmembrane+protein+incorporation+using+octyl+glucoside.">Phospholipid vesicle formation and transmembrane protein incorporation using octyl glucoside.</a> Biochemistry. 20, 833-840 (1981).</li><li>Malvezzi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2+-dependent+phospholipid+scrambling+by+a+reconstituted+TMEM16+ion+channel.">Ca2+-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel.</a> Nat. Commun. 4, 2367 (2013).</li><li>Eckford, P. D. W., Sharom, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reconstituted+P-glycoprotein+multidrug+transporter+is+a+flippase+for+glucosylceramide+and+other+simple+glycosphingolipids.">The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids.</a> Biochem. J. 389, 517-526 (2005).</li><li>Marek, M., G&#252;nther-Pomorski, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+Flippase+Activity+in+Proteoliposomes+Using+Fluorescent+Lipid+Derivatives.">Assay of Flippase Activity in Proteoliposomes Using Fluorescent Lipid Derivatives.</a> Methods Mol. Biol. 1377, 181-191 (2016).</li><li>Coleman, J. A., Kwok, M. C. M., Molday, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+purification,+and+functional+reconstitution+of+the+P4-ATPase+Atp8a2,+a+phosphatidylserine+flippase+in+photoreceptor+disc+membranes.">Localization purification, and functional reconstitution of the P4-ATPase Atp8a2, a phosphatidylserine flippase in photoreceptor disc membranes.</a> J. Biol. Chem. 284, 32670-32679 (2009).</li><li>Coleman, J. A., Quazi, F., Molday, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+P4-ATPases+and+ABC+transporters+and+their+role+in+phospholipid+transport.">Mammalian P4-ATPases and ABC transporters and their role in phospholipid transport.</a> Biochim. Biophys. Acta. 1831, 555-574 (2013).</li><li>Romsicki, Y., Sharom, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+flippase+activity+of+the+reconstituted+P-glycoprotein+multidrug+transporter.">Phospholipid flippase activity of the reconstituted P-glycoprotein multidrug transporter.</a> Biochemistry. 40, 6937-6947 (2001).</li><li>Zhou, X., Graham, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+phospholipid+translocase+activity+with+purified+Drs2p,+a+type-IV+P-type+ATPase+from+budding+yeast.">Reconstitution of phospholipid translocase activity with purified Drs2p, a type-IV P-type ATPase from budding yeast.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 16586-16591 (2009).</li></ol>

作者什么都没有透露。

该scramblase活性测定，使我们原来确定的视蛋白具有磷脂scramblase活动2。该测定法还允许我们通过测试特异性表征视蛋白的scramblase活性（我们采用了各种NBD标记的报告脂质如NBD-磷脂酰乙醇胺，如图所示为NBD-PC在图1A中，或在上的酰基链与NBD标记的首基，NBD-鞘磷脂或NBD-磷脂2），囊泡脂质组合物（的效应， 例如 ，在不同量的作为囊泡组分胆固醇），和是否视蛋白?...

感光体视紫红质，一个典型的G蛋白偶联受体（参考文献1中综述例如），要被识别的第一磷脂scramblase和生物化学验证2,3。 Scramblases是磷脂转运会增加transbilayer磷脂运动的本质慢的速度在双向生理上适当水平，ATP无关的方式4-6。他们的行动的实例可于内质网和细菌胞质膜，其中需要对膜稳态和生长构加扰，以及针对各种糖基化途径5中找到。扰码是需要调节磷脂以暴露凋亡细胞的表面上的磷脂酰丝氨酸（PS），其中它充当一个"吃我" -信号为巨噬细胞7和对活化血小板提供一种促凝表面以催化生产蛋白质因子的š所需血液凝固。在感光体光盘的膜，视紫红质的扰频活动已经提出以抵消由ATP依赖，单向脂质产生的双层的两个薄膜小叶之间的磷脂失调翻转酶ABCA4 4,8,9 10-12。 尽管scramblases的生理重要性，它们的身份仍然难以捉摸直到视紫红质被报告为在感光体圆盘一个scramblase 2，TMEM16蛋白家族的成员被确定为钙离子需要在质膜PS暴露依赖性scramblases（在参考审查13），并作为脂类II scramblase所需肽合成14提出的细菌蛋白质FtsW。这些发现是基于纯化的蛋白质的脂质体中使用的方法describ重构和scramblase活性示范在所得脂蛋白体这里编。其他潜在scramblases 15-21 -的MurJ和AMJ蛋白肽聚糖的生物合成牵连，WzxE和相关蛋白牵涉于扰O-抗原的前体，MPRF蛋白需要穿过细菌胞质膜易位氨基酰化磷脂，并且已经提出Xkr8家族成员以暴露的PS凋亡细胞的表面上 - 有待生化测试。这凸显了一个强大的分析的重要性，以确定和识别scramblase活动。 这里，我们描述了纯化的视蛋白的在使用基于荧光的测定法所得到的蛋白脂质体的重建，感光体的视紫红质的脱辅基蛋白，成大单层囊泡（的LUV）和scramblase活性随后的分析。有在文献中用于异源表达和视蛋白的纯化可用几个充分描述的协议，因此，我们将不描述它在这个协议;我们使用这产生FLAG标签，热稳定视蛋白在约100纳克在戈伦等 3所述的协议/微升的0.1％（重量/体积）十二烷基麦芽糖（DDM）。 重构是通过使得它们溶胀但不溶于治疗与足够洗涤剂的LUV实现。在这些条件下，膜蛋白 - 蛋白质 - 去污剂胶束的形式提供 - 将整合入脂质体和成为重建成在洗涤剂除去脂质体膜，导致脂蛋白体。重构视蛋白（作为纯化蛋白得到的0.1％（重量/体积），DDM），的LUV由（1- POPC（1-棕榈酰-2-油酰- SN -glycero -3-磷酸胆碱）和POPG的混合物制备棕榈酰-2-油酰- SN -glycero -3- [磷酸外消旋- （1-甘油）]）和添加视蛋白和NBD-PC之前，用DDM饱和。洗涤剂然后通过用聚苯乙烯珠粒的样品中除去。 小姑娘="jove_content"&gt;的基于荧光的测定法的基本原理示于图1B。的LUV对称用NBD-PC或其他NBD标记的荧光磷脂记者（ 图1A）的微量重构。上加入连二亚硫酸盐，膜不通透性二价阴离子，在的LUV的外部小叶NBD-PC分子呈现非荧光作为硝基组NBD的被还原成非荧光氨基 - 基团。既不NBD-PC分子也不连二是能够在时间尺度的实验（&lt;10分钟）的遍历膜，这导致荧光信号的减少50％。然而，如果脂质体与scramblase重构，在内小叶NBD-PC分子可以迅速加扰向外部在那里它们被降低。这导致荧光的在理想的情况下（ 图1C）的总损耗。 ES / ftp_upload / 54635 / 54635fig1.jpg"/&gt; 图1:scramblase活性测定的示意图的测定使用荧光NBD标记的报告脂质; NBD-PC如图（A）。大单层囊泡重构与NBD-PC的痕量。重组产生对称囊泡，以在外部和内部小叶均匀分布NBD-PC。连二亚硫酸盐（S 2 O 4 2-）化学还原硝基组NBD到一个非荧光氨基-基团。用连二亚硫酸盐（B，上图）无蛋白质的脂质体处理引起，因为只有在该外部小叶的NBD-PC分子荧光的减少50％的降低:连二亚硫酸盐被带负电荷的，并且不能穿过膜与NBD-PC分子反应在内部小叶。含有视蛋白，脂蛋白（B，下），连二亚的治疗即 scramblase活性脂蛋白，导致的F 100％的损失luorescence如视蛋白促进内层和外层小叶之间的NBD-PC的移动。 （C）表示对治疗无蛋白质的脂质体和含有视蛋白-蛋白脂质体与连二亚得到理想化荧光痕迹。荧光损失率是在两种情况下，指示由连二亚化学还原NBD的是限速，并且加扰出现在比化学反应的速率等于或大于一个速率是相同的。从实际实验中获得痕迹如图3所示。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54635/54635fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 我们描述的方法可以被用来重建和测定等纯化的蛋白质，以及获得，例如，通过用洗涤剂22提取微粒的膜蛋白的混合物。

<table><tbody><tr><td>1-palmitoyl-2-oleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine</td><td>Avanti Polar Lipids</td><td>850457C</td><td>POPC</td></tr><tr><td>1-Palmitoyl-2-oleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phospho-rac-(1-glycerol) (sodium salt)</td><td>Avanti Polar Lipids</td><td>840457C</td><td>POPG</td></tr><tr><td>1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine</td><td>Avanti Polar Lipids</td><td>810130C</td><td>NBD-PC</td></tr><tr><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid</td><td>VWR Scientific</td><td>EM-5330</td><td>HEPES</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653-1KG</td><td>NaCl</td></tr><tr><td>Dodecyl-&amp;beta;-D-maltoside</td><td>Anatrace</td><td>D310 5 GM</td><td>DDM</td></tr><tr><td>Fluorimeter cuvettes</td><td>sigma</td><td>C0918-100EA</td><td>cuvettes</td></tr><tr><td>Spectrofluorometer</td><td>Photon Technology International, Inc.</td><td>fluorimeter</td><td></td></tr><tr><td>Sodium hydrosulfite technical grade, 85%</td><td>Sigma</td><td>157953-5G</td><td>dithionite</td></tr><tr><td>GraphPad Prism 5 software</td><td></td><td></td><td>Prism</td></tr><tr><td>Tris Base</td><td>VWR</td><td>JTX171-3</td><td>Tris</td></tr><tr><td>LIPEX 10 ml extruder&amp;nbsp;</td><td>Northern Lipids, Inc.</td><td></td><td>Extruder</td></tr><tr><td>Whatman, Drain disc, PE, 25 mm</td><td>Sigma</td><td>28156-243</td><td>Disc support</td></tr><tr><td>Whatman Nuclepore Track-Etched Membranes, 0.4 &amp;micro;m, 25 mm diameter</td><td>Sigma</td><td>WHA110607</td><td>400 nm membrane</td></tr><tr><td>Whatman Nucleopore Track-Etched Membranes, 0.2 &amp;micro;m, 25 mm diameter</td><td>Sigma</td><td>WHA110606</td><td>200 nm membrane</td></tr><tr><td>sodium phosphate</td><td>Sigma</td><td>S3264-500G</td><td></td></tr><tr><td>VWR Culture Tubes, Disposable, Borosilicate Glass, 13 x 100 mm</td><td>VWR Scientific</td><td>47729-572</td><td>glass tubes</td></tr><tr><td>Perchloric acid</td><td>Sigma</td><td>30755-500ML</td><td></td></tr><tr><td>Ammonium Molybdate Tetrahydrate</td><td>Sigma</td><td>A-7302</td><td>ammonium molybdate</td></tr><tr><td>(+)-Sodium L-ascorbate</td><td>Sigma</td><td>A7631-25G</td><td>sodium ascorbate</td></tr><tr><td>Bio-Beads SM2 adsorbents</td><td>Bio Rad</td><td>1523920</td><td>polystyrene beads</td></tr><tr><td>&amp;nbsp;2.0 ml Microtubes clear</td><td>VWR Scientific</td><td>10011-742</td><td>Reconstitution tubes</td></tr><tr><td>Reconstitution glass tube</td><td>VWR Scientific</td><td>53283-800</td><td>Reconstitution glass tubes</td></tr><tr><td>Zetasizer&amp;nbsp;</td><td>Malvern&amp;nbsp;</td><td></td><td>DLS</td></tr></tbody></table>

a fluorescence-based assay of phospholipid scramblase activity

Scramblases易位穿过膜双层磷脂双向在一个ATP无关的方式。第一scramblase被识别和生化核实被视蛋白，感光体的视紫红质的脱辅基蛋白。视紫红质是在它负责的光的感知的视网膜视杆圆盘膜局部的G蛋白偶合受体。视紫红质的scramblase活动并不取决于其配体11- 顺 -retinal， 即载脂蛋白视蛋白还积极为scramblase。虽然组成型和调节磷脂扰在细胞生理学中起重要作用，只有少数磷脂scramblases迄今已除了视蛋白鉴定。这里，我们描述视蛋白的scramblase活性的基于荧光的测定法。视蛋白被重组到磷脂酰胆碱，磷脂酰甘油和荧光NBD标记的PC（1-p的微量组成的大单层脂质体almitoyl -2- {6- [7-硝基2-1,3苯并恶-4-基）氨基]己酰基} - SN -glycero -3-磷酸胆碱）。 Scramblase活性通过测量到位于囊泡的内小叶NBD-PC分子能够访问外部小叶，他们的荧光化学由不能越过膜还原剂消除的程度来确定。我们描述的方法具有普遍的适用性，并可以用于鉴定和表征其他膜蛋白的scramblase活动。

我们描述视蛋白的重建成的LUV使用基于荧光的测定法来表征其scramblase活性。我们分析结果，以放置在视蛋白介导的磷脂加扰的速率的下限，并确定在哪个视蛋白功能上重新构造成囊泡的寡聚状态。 以确定最佳的重组的条件下，有必要确定凭经验必须使用溶胀的LUV使它们接受蛋白质插入洗涤剂的量。这样的实验是在图2A</s...

Watch this Scientific Journal Video about A Fluorescence-based Assay of Phospholipid Scramblase Activity at JoVE.com

A Fluorescence-based Assay of Phospholipid Scramblase Activity

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

磷脂Scramblase活动的基于荧光的测定法

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

a-fluorescence-based-assay-of-phospholipid-scramblase-activity

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

13.8K 次观看.  Weill Cornell Medical College. We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin. 

视频: A Fluorescence-based Assay of Phospholipid Scramblase Activity

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a determinant phosphoinositide of the plasma membrane (PM), and it is required to modulate ion channels, actin dynamics, exocytosis, endocytosis, intracellular signaling, and many other cellular processes. However, the spatial organization of PI(4,5)P2 in the PM is controversial, and its nanoscale distribution is poorly understood due to the technical limitations of research approaches. Here by utilizing single molecule localization microscopy and the Pleckstrin Homology (PH) domain based dual color fluorescent probes, we describe a novel method to visualize the nanoscale distribution of PI(4,5)P2 in the PM in fixed membrane sheets as well as live cells.

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale.

在质膜用新型荧光探针磷脂酰-4,5-二磷酸的单分子超分辨率成像

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

科研

生物化学

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. Alternatively, other (phospho)lipases may be physically associated with the producing organism causing a turnover of intrinsic lipids and frequently giving rise to a remodeling of the cellular membranes. Although potential (phospho)lipases can be predicted with a number of algorithms when the gene/protein sequence is available, experimental proof of the enzyme activities, substrate specificities, and potential physiological functions has frequently not been obtained. This manuscript describes the optimization of assay conditions for prospective (phospho)lipases with unknown substrate specificities and how to employ these optimized conditions in the search for the natural substrate of a respective (phospho)lipase. Using artificial chromogenic substrates, such as p-nitrophenyl derivatives, may help to detect a minor enzymatic activity for a predicted (phospho)lipase under standard conditions. Having encountered such a minor enzymatic activity, the distinct parameters of an enzyme assay can be varied in order to obtain a more efficient hydrolysis of the artificial substrate. After having determined the conditions under which an enzyme works well, a variety of potential natural substrates should be assayed for their degradation, a process that can be followed employing distinct chromatographic methods. The definition of substrate specificities for new enzymes, often provides hypotheses for a potential physiological role of these enzymes, which then can be tested experimentally. Following these guidelines, we were able to identify a phospholipase C (SMc00171) that degrades phosphatidylcholine to phosphocholine and diacylglycerol, in a crucial step for the remodeling of membranes in the bacterium Sinorhizobium meliloti upon phosphorus-limiting conditions of growth. For two predicted patatin-like phospholipases (SMc00930 and SMc01003) of the same organism, we could redefine their substrate specificities and clarify that SMc01003 is a diacylglycerol lipase.

Many predicted (phospho)lipases are poorly characterized with regard to their substrate specificities and physiological functions. Here we provide a protocol to optimize enzyme activities, search for natural substrates, and propose physiological functions for these enzymes.

定义底物特异性的脂肪酶和磷脂酶考生

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. ...

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.

We present a new technique to measure the lateral diffusion of a surface active species at the fluid-fluid interface by merging a droplet monolayer onto a flat monolayer.

合并一个液滴后荧光恢复来衡量磷脂单层的二维扩散

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after ...

生物工程

Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main common features required for this activity resulted from the interactions of CPPs with the plasma membrane (lipids) and more particularly with components of the extracellular matrix of the membrane itself (heparan sulphate). Indeed, independent of the direct translocation or the endocytosis-dependent internalization, lipid bilayers are involved in the internalization process both at the level of the plasma membrane and at the level of intracellular traffic (endosomal vesicles). In this article, we present a detailed protocol describing the different steps of a large unilamellar vesicles (LUVs) formulation, purification, characterization, and application in fluorescence leakage assay in order to detect possible CPP-membrane destabilization/interaction and to address their role in the internalization mechanism. LUVs with a lipid composition reflecting the plasma membrane content are generated in order to encapsulate both a fluorescent dye and a quencher. The addition of peptides in the extravesicular medium and the induction of peptide-membrane interactions on the LUVs might thus induce in a dose-dependent manner a significant increase in fluorescence revealing a leakage. Examples are provided here with the recently developed tryptophan (W)- and arginine (R)-rich Amphipathic Peptides (WRAPs), which showed a rapid and efficient siRNA delivery in various cell lines. Finally, the nature of these interactions and the affinity for lipids are discussed to understand and to improve the membrane translocation and/or the endosomal escape.

The fluorescence leakage assay is a simple method that enables the investigation of peptide/membrane interactions in order to understand their involvement in several biological processes and especially the ability of cell-penetrating peptides to disturb phospholipids bilayers during a direct cellular translocation process.

荧光泄漏测定研究细胞穿透肽对膜的破坏稳定性

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main ...

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

Here, we describe protocols using fluorescent lipid sensors and liposomes to determine whether a protein extracts and transports phosphatidylserine or phosphatidylinositol 4-phosphate in vitro.

基于荧光的测量磷酸酯/磷酸二醇 4-磷酸盐膜之间的交换

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

短杆菌肽为基础的荧光分析法，确定小分子的修改脂质双层属性的潜在

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

生物学

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

脂质囊泡介导的亲和层析法，使用磁性细胞分选（LIMACS）：一种新的方法来分析蛋白质 - 脂质相互作用

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

单脂蛋白的SNARE介导融合在一个微流控流细胞拴支持双分子层通过偏光显微镜TIRF监控

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

神经科学

pH Modulation Assay on Supported Lipid Bilayers to Detect Protein-Phosphoinositide Interactions

Source: Shengjuler, D., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/55869">PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions.</a> J. Vis. Exp. (2017)
In this video, we demonstrate protein-phosphoinositide interactions in a microfluidic pattern using the pH modulation assay, using pH-sensitive fluorescent dye tagged to phosphatidylethanolamine lipid of a supported lipid bilayer, called a microfluidic platform. The modulations in pH after protein binding to phospholipid cause fluorescence quenching.