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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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://www-jove-com.remotexs.ntu.edu.sg/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 border="0" cellpadding="0" cellspacing="0" width="1148">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine</td>
			<td style="width:141px;">Avanti Polar Lipids</td>
			<td style="width:132px;">850457C</td>
			<td style="width:193px;">POPC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium salt)</td>
			<td>Avanti Polar Lipids</td>
			<td>840457C</td>
			<td>POPG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-an-glycero-3-phosphocholine</td>
			<td>Avanti Polar Lipids</td>
			<td>810130C</td>
			<td>NBD-PC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid</td>
			<td>VWR Scientific</td>
			<td>EM-5330</td>
			<td>HEPES</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma</td>
			<td>S7653-1KG</td>
			<td>NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dodecyl-&#946;-d-maltoside&#160;</td>
			<td>Anatrace</td>
			<td>D310 5 GM</td>
			<td>DDM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorimeter cuvettes</td>
			<td>sigma</td>
			<td>C0918-100EA</td>
			<td>cuvettes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectrofluorometer</td>
			<td colspan="2">Photon Technology International, Inc.</td>
			<td>fluorimeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydrosulfite technical grade, 85%</td>
			<td>Sigma</td>
			<td>157953-5G</td>
			<td>dithionite</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GraphPad Prism 5 software</td>
			<td></td>
			<td></td>
			<td>Prism</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>VWR</td>
			<td>JTX171-3</td>
			<td>Tris</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIPEX 10 mL extruder&#160;</td>
			<td>Northern Lipids, Inc.</td>
			<td></td>
			<td>Extruder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">Whatman, Drain disc, PE, 25 mm</td>
			<td>Sigma</td>
			<td style="width:132px;">28156-243</td>
			<td>Disc support</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">Whatman Nuclepore Track-Etched Membranes, 0.4 &#181;m, 25 mm diameter</td>
			<td>Sigma</td>
			<td style="width:132px;">WHA110607</td>
			<td>400 nm membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">Whatman Nucleopore Track-Etched Membranes, 0.2 &#181;m, 25 mm diameter</td>
			<td>Sigma</td>
			<td style="width:132px;">WHA110606</td>
			<td>200 nm membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium phosphate</td>
			<td>Sigma</td>
			<td>S3264-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VWR Culture Tubes, Disposable, Borosilicate Glass, 13x100 mm</td>
			<td>VWR Scientific</td>
			<td>47729-572</td>
			<td>glass tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perchloric acid</td>
			<td>Sigma</td>
			<td>30755-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Molybdate Tetrahydrate</td>
			<td>Sigma</td>
			<td>A-7302</td>
			<td>ammonium molybdate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(+)-Sodium L-ascorbate</td>
			<td>Sigma</td>
			<td>A7631-25G</td>
			<td>sodium ascorbate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bio-Beads SM2 adsorbents</td>
			<td>Bio Rad</td>
			<td>1523920</td>
			<td>polystyrene beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;2.0 mL Microtubes clear</td>
			<td>VWR Scientific</td>
			<td>10011-742</td>
			<td>Reconstitution tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reconstitution glass tube</td>
			<td>VWR Scientific</td>
			<td style="width:132px;">53283-800</td>
			<td>Reconstitution glass tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zetasizer&#160;</td>
			<td>Malvern&#160;</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...

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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

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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

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

的pH依赖性活性的检测<em&gt;大肠杆菌</em&gt;陪护HdeB<em&gt;体外</em&gt;和<em&gt;在体内</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

科研

生物化学

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

S100A12的表达，纯化和抗菌活性

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

映射代谢: 直接在组织中监测乳酸脱氢酶活性

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released from many cell types and play a pivotal role in regulating cell-cell communication and a diverse range of biological processes. Many different methods for the characterization of EVs have been described. However, most of these methods have the disadvantage that the preparation and characterization of the samples are very time-consuming, or it is extremely difficult to analyze specific markers of interest due to their small size and due to the lack of discrete populations. While methods for analysis of EVs have been considerably improved over the last decade, there is still no standardized method for characterization of single EVs. Here, we demonstrate a semi-automated method for characterization of single EVs by fluorescence-based nanoparticle-tracking analysis. The protocol that is presented addresses the common problem of many researchers in this field and provides the complete workflow for rapid isolation of EVs and characterization with PKH67, a general cell membrane linker, as well as with specific surface markers such as CD63, CD9, vimentin, and lysosomal-associated membrane protein 1 (LAMP-1). The presented results show a high level of reproducibility, as confirmed by other methods, such as Western blotting. In the conducted experiments, we exclusively used EVs isolated from human serum samples, but this method is also suitable for plasma or other body fluids and can be adjusted for characterization of EVs from cell culture supernatants. Irrespective of the future progress of research on EV biology, the protocol that is presented here provides a rapid and reliable method for rapid characterization of single EVs with specific markers.

In this protocol, we describe the complete workflow for rapid isolation of extracellular vesicles from human whole blood and characterization of specific markers by fluorescence-based nanoparticle-tracking analysis. The presented results show a high level of reproducibility and can be adjusted to cell culture supernatants.

基于荧光的人体血液单细胞外囊快速表征

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

一种用于蛋白质溶解活性的荧光肽裂解检测: 冠状病毒穗蛋白活化的应用

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

果蝇梅拉诺加斯特的锡酸盐合成酶活性的色度测定

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

丝状真菌曲霉菌的单步富集蛋白脱乙酰化酶的酶活性测定

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

使用核酸探针进行核酸活性的动力学筛选

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...