This work received support through grant HA 6136/1-1 from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) to MH. JAT is supported by Program and Project Grants from the National Health and Medical Research Council of Australia.

注:脾脏衍生自小鼠（6-10周龄），所有动物实验均根据彼得MacCallum肿瘤中心（E486）的动物伦理准则进行。 1.准备样品。 <ol><li>准备激活小鼠NK细胞。 <ol><li> / - - （GZMB基因为null）小鼠通过阴性选择使用市售的试剂盒，从C57BL / 6或B6.GrzmB的脾脏的单细胞悬液分离主幼稚NK细胞。 </li><li>按照制造商的协议。简要地说，磁标签"非NK"细胞如T和B细胞，树突状细胞，粒细胞，巨噬细胞，并使用生物素缀合的抗体针对其特定的表面标记的红血细胞。使用磁分离与抗生物素珠粒耗尽"非NK"细胞。 </li><li>培养在24孔板中分离NK细胞5-7天以含IL-2（1000单位/毫升）的介质以700000个细胞/ ml的在37 &#160; ℃。 </li><li>可替代地，使用活化的T细胞19，初级抗原限制性CTL从OT1 T细胞受体转基因小鼠20，CTL在混合的淋巴细胞培养物21，或从上清液活化NK细胞产生的，以确定分泌GZMB 19的活性。 </li></ol></li><li>维护人类NK细胞和初级NK细胞的隔离。 <ol><li>维持人NK细胞白血病（YT细胞）在补充有10％胎牛血清罗斯威尔公园纪念研究所（RPMI）培养基。可替代地，净化从健康受试者的外周血主NK细胞和刺激2-3天在培养基含IL-2，在100单位/毫升，如前所述22。 </li></ol></li><li>代细胞裂解物。 <ol><li>在PBS（pH7.4）中洗涤细胞三次，然后悬浮于的Nonidet P-40的裂解缓冲液（0.1％NP-40，250 mM氯化钠，25毫摩尔的Hepes，2.5mM的EDTA）中。理想的是，溶解至少5×10 6个</s向上&gt; NK细胞，或1×10 7个T细胞在100μl缓冲液中。不添加蛋白酶抑制剂，因为许多丝氨酸蛋白酶的不可逆抑制剂，特别tryptases如GzmA。在冰上孵育20分钟，然后沉淀核在微量离心以15000×g离心10分钟。弃去核沉淀，上清液转移到新的管中。 </li></ol></li><li>确定的裂解物的蛋白质浓度。 <ol><li>使用市售的选择，例如，布拉德福德或二喹啉酸（BCA）的协议，并确定蛋白浓度。确保该浓度是〜2毫克/毫升最小。商店裂解物在-20℃下直到需要（粒酶的活性是稳定许多个月，在-20℃）。 </li></ol></li></ol> 2.颗粒酶B活性分析<ol><li>试剂的制备<ol><li>在DMSO准备基板（BOC-AAD-S-BZL或AC-IEPD-PNA）的10毫米股票（〜5毫克/毫升），并存储在分装在-20＆＃176℃。准备工作稀释新鲜和丢弃的每一天后，中银-AAD-S-BZL部分水解的存储DMSO。 </li><li>准备的比色试剂（5,5&#39;-二硫 - 双（2-硝基苯甲酸），DTNB）在DMSO中的250mM的股票（0.09微克/毫升），并存储在-20℃。每天准备一个新的工作稀释。该试剂只需要用于与SBzl指示符组，不pNA的衬底。 </li><li>弥补了新鲜的缓冲液（0.1M HEPES，0.05M的氯化镁2，pH值7.3），每2-3周。 </li></ol></li><li>带来DTNB和合成底物至室温。稀基板（1:16 如 1.6 ml缓冲液100微升）和DTNB（1:250， 如 10微升于2.5毫升缓冲）拌匀。 </li><li>稀至少在缓冲各样品的100微克蛋白质裂解物，以160微升的最终体积。使用多通道移液器以一式三份加入50微升每孔（〜33.3微克/孔）在一个""U""底部乙烯基96孔板。有一个"缓冲 - 只有"控制（也一式三份）。 </li><li>加入100微升稀释DTNB到样品/缓冲控制AC-IEPD-PNA工作时（省略这一步。 注:AC-IEPD-PNA由GZMB的裂解直接释放荧光PNA（ 图4B）。 </li><li>使用多道移液器快速添加50微升稀释基材每组一式三份，先从缓冲区那么任何负面的控制，预计阳性标本整理。 </li><li>将板在板读数器。测量在酶标仪在OD 405nm处为Boc - 丙氨酸 - 丙氨酸 - 天冬氨酸 - S-BZL / AC-IEPD-pNA的水解天冬氨酸酶的活性。使用动力学分析方案，以25的读数，每隔15秒（约6分钟的总阅读时间）。 </li><li>使用在每个时间点所产生的吸光率读数手动计算底物裂解的速率，如果板读取器的软件不能生成的Vmax值。 </li></ol>大3.分析TA 注意:产生被表示为最大速度的数据，定义为OD的变化随时间（MOD /分钟）。 <ol><li>分析数据，确定衬底解理，它是从吸光度变化率计算出的最大速度。自动使用平板阅读器软件，它通常有这个选项做到这一点。 </li><li>观看吸光度变化，这是在酶标仪动力学实验过程中产生的动能地块，手动后或者选择数据点，给出最陡峭的直线。对于大多数试验的最大速率是该测定的前2分钟内实现。 </li><li>减去初始OD读数从最高的OD读数在此直线和除以时间间隔。传输原始数据到Microsoft Excel /图板进行统计分析和图形显示。 </li></ol>

<ol>
	<li>Trapani, J. A., Browne, K. A., Dawson, M., Smyth, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunopurification+of+functional+Asp-ase+(natural+killer+cell+granzyme+B)+using+a+monoclonal+antibody).">Immunopurification of functional Asp-ase (natural killer cell granzyme B) using a monoclonal antibody).</a> Biochem Biophys Res Commun. 195 (2), 910-920 (1993).</li><li>Ewen, C. L., Kane, K. P., Bleackley, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quarter+century+of+granzymes.">A quarter century of granzymes.</a> Cell Death Differ. 19 (1), 28-35 (2012).</li><li>Hoves, S., Trapani, J. A., Voskoboinik, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+battlefield+of+perforin/granzyme+cell+death+pathways.">The battlefield of perforin/granzyme cell death pathways.</a> J Leukoc Biol. , (2009).</li><li>Peters, P. J., 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=Cytotoxic+T+lymphocyte+granules+are+secretory+lysosomes,+containing+both+perforin+and+granzymes.">Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes.</a> J Exp Med. 173 (5), 1099-1109 (1991).</li><li>Berthou, C., 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=Acquisition+of+granzyme+B+and+Fas+ligand+proteins+by+human+keratinocytes+contributes+to+epidermal+cell+defense.">Acquisition of granzyme B and Fas ligand proteins by human keratinocytes contributes to epidermal cell defense.</a> J Immunol. 159 (11), 5293-5300 (1997).</li><li>Tschopp, C. 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=Granzyme+B,+a+novel+mediator+of+allergic+inflammation:+its+induction+and+release+in+blood+basophils+and+human+asthma.">Granzyme B, a novel mediator of allergic inflammation: its induction and release in blood basophils and human asthma.</a> Blood. 108 (7), 2290-2299 (2006).</li><li>Strik, M. C., 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=Human+mast+cells+produce+and+release+the+cytotoxic+lymphocyte+associated+protease+granzyme+B+upon+activation.">Human mast cells produce and release the cytotoxic lymphocyte associated protease granzyme B upon activation.</a> Mol Immunol. 44 (14), 3462-3472 (2007).</li><li>Jahrsdorfer, B., 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=Granzyme+B+produced+by+human+plasmacytoid+dendritic+cells+suppresses+T+cell+expansion.">Granzyme B produced by human plasmacytoid dendritic cells suppresses T cell expansion.</a> Blood. , (2009).</li><li>Hagn, 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=Human+B+cells+secrete+granzyme+B+when+recognizing+viral+antigens+in+the+context+of+the+acute+phase+cytokine+IL-21.">Human B cells secrete granzyme B when recognizing viral antigens in the context of the acute phase cytokine IL-21.</a> J Immunol. 183 (3), 1838-1845 (2009).</li><li>Hagn, 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=Human+B+cells+differentiate+into+granzyme+B-secreting+cytotoxic+B+lymphocytes+upon+incomplete+T-cell+help.">Human B cells differentiate into granzyme B-secreting cytotoxic B lymphocytes upon incomplete T-cell help.</a> Immunol Cell Biol. 90 (4), 457-467 (2012).</li><li>Froelich, C. J., Pardo, J., Simon, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granule-associated+serine+proteases:+granzymes+might+not+just+be+killer+proteases.">Granule-associated serine proteases: granzymes might not just be killer proteases.</a> Trends Immunol. 30 (3), 117-123 (2009).</li><li>Hagn, M., Jahrsdorfer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+human+B+cells+secrete+granzyme+B?+Insights+into+a+novel+B-cell+differentiation+pathway.">Why do human B cells secrete granzyme B? Insights into a novel B-cell differentiation pathway.</a> Oncoimmunology. 1 (8), 1368-1375 (2012).</li><li>Susanto, O., Trapani, J. A., Brasacchio, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controversies+in+granzyme+biology.">Controversies in granzyme biology.</a> Tissue Antigens. 80 (6), 477-487 (2012).</li><li>Walch, 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=Cytotoxic+cells+kill+intracellular+bacteria+through+granulysin-mediated+delivery+of+granzymes.">Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes.</a> Cell. 157 (6), 1309-1323 (2014).</li><li>Odake, S., 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=Human+and+murine+cytotoxic+T+lymphocyte+serine+proteases:+subsite+mapping+with+peptide+thioester+substrates+and+inhibition+of+enzyme+activity+and+cytolysis+by+isocoumarins.">Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins.</a> Biochemistry. 30 (8), 2217-2227 (1991).</li><li>Casciola-Rosen, L., 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=Mouse+and+human+granzyme+B+have+distinct+tetrapeptide+specificities+and+abilities+to+recruit+the+bid+pathway.">Mouse and human granzyme B have distinct tetrapeptide specificities and abilities to recruit the bid pathway.</a> J Biol Chem. 282 (7), 4545-4552 (2007).</li><li>Kaiserman, D., 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=The+major+human+and+mouse+granzymes+are+structurally+and+functionally+divergent.">The major human and mouse granzymes are structurally and functionally divergent.</a> J Cell Biol. 175 (4), 619-630 (2006).</li><li>Powers, J. C., Kam, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+thioester+substrates+for+serine+peptidases+and+metalloendopeptidases.">Peptide thioester substrates for serine peptidases and metalloendopeptidases.</a> Methods Enzymol. 248, 3-18 (1995).</li><li>Hagn, 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=Activated+mouse+B+cells+lack+expression+of+granzyme.">Activated mouse B cells lack expression of granzyme.</a> B. J Immunol. 188 (2), 3886-3892 (2012).</li><li>Konjar, S., 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=Human+and+mouse+perforin+are+processed+in+part+through+cleavage+by+the+lysosomal+cysteine+proteinase+cathepsin+L.">Human and mouse perforin are processed in part through cleavage by the lysosomal cysteine proteinase cathepsin L.</a> Immunology. 131 (2), 257-267 (2010).</li><li>Sutton, V. R., 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=Residual+active+granzyme+B+in+cathepsin+C-null+lymphocytes+is+sufficient+for+perforin-dependent+target+cell+apoptosis.">Residual active granzyme B in cathepsin C-null lymphocytes is sufficient for perforin-dependent target cell apoptosis.</a> J Cell Biol. 176 (4), 425-433 (2007).</li><li>Trapani, J. A., Smyth, M. J., Apostolidis, V. A., Dawson, M., Browne, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granule+serine+proteases+are+normal+nuclear+constituents+of+natural+killer+cells.">Granule serine proteases are normal nuclear constituents of natural killer cells.</a> J Biol Chem. 269 (28), 18359-18365 (1994).</li><li>Cullen, S. P., Adrain, C., Luthi, A. U., Duriez, P. J., Martin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+murine+granzyme+B+exhibit+divergent+substrate+preferences.">Human and murine granzyme B exhibit divergent substrate preferences.</a> J Cell Biol. 176 (4), 435-444 (2007).</li><li>Thornberry, N. A., 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=A+combinatorial+approach+defines+specificities+of+members+of+the+caspase+family+and+granzyme+B.+Functional+relationships+established+for+key+mediators+of+apoptosis.">A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.</a> J Biol Chem. 272 (29), 17907-17911 (1997).</li><li>Bird, C. H., 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=Selective+regulation+of+apoptosis:+the+cytotoxic+lymphocyte+serpin+proteinase+inhibitor+9+protects+against+granzyme+B-mediated+apoptosis+without+perturbing+the+Fas+cell+death+pathway.">Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway.</a> Mol Cell Biol. 18 (11), 6387-6398 (1998).</li><li>Bots, M., Medema, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serpins+in+T+cell+immunity.">Serpins in T cell immunity.</a> J Leukoc Biol. 84 (5), 1238-1247 (2008).</li><li>Sun, J., 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=A+new+family+of+10+murine+ovalbumin+serpins+includes+two+homologs+of+proteinase+inhibitor+8+and+two+homologs+of+the+granzyme+B+inhibitor+(proteinase+inhibitor+9).">A new family of 10 murine ovalbumin serpins includes two homologs of proteinase inhibitor 8 and two homologs of the granzyme B inhibitor (proteinase inhibitor 9).</a> J Biol Chem. 272 (24), 15434-15441 (1997).</li><li>Bird, C. H., Hitchen, C., Prescott, M., Harper, I., Bird, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunodetection+of+granzyme+B+tissue+distribution+and+cellular+localisation.">Immunodetection of granzyme B tissue distribution and cellular localisation.</a> Methods Mol Biol. 844, 237-250 (2012).</li><li>Jenkins, M. R., 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=Visualizing+CTL+activity+for+different+CD8++effector+T+cells+supports+the+idea+that+lower+TCR/epitope+avidity+may+be+advantageous+for+target+cell+killing.">Visualizing CTL activity for different CD8+ effector T cells supports the idea that lower TCR/epitope avidity may be advantageous for target cell killing.</a> Cell Death Differ. 16 (4), 537-542 (2009).</li><li>Edwards, K. M., Kam, C. M., Powers, J. C., Trapani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+cytotoxic+T+cell+granule+serine+protease+granzyme+H+has+chymotrypsin-like+(chymase)+activity+and+is+taken+up+into+cytoplasmic+vesicles+reminiscent+of+granzyme+B-containing+endosomes.">The human cytotoxic T cell granule serine protease granzyme H has chymotrypsin-like (chymase) activity and is taken up into cytoplasmic vesicles reminiscent of granzyme B-containing endosomes.</a> J Biol Chem. 274 (43), 30468-30473 (1999).</li><li>Sutton, V. R., 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=Initiation+of+apoptosis+by+granzyme+B+requires+direct+cleavage+of+bid,+but+not+direct+granzyme+B-mediated+caspase+activation.">Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation.</a> J Exp Med. 192 (10), 1403-1414 (2000).</li></ol>

The authors have nothing to disclose.

在历史上，粒酶被鉴定为细胞毒性淋巴细胞（CTL和NK细胞）能够诱导快速的凋亡性死亡的靶细胞的关键效应分子。这主要是由于GZMB的动作，这裂解靶底物分子在天门冬氨酸（D）残基，因而能由这两种裂解亲胱天蛋白酶激活的胱天蛋白酶级联，以及若干的其下游目标。然而，现在认识到，GZMB表达并不局限于淋巴细胞毒性细胞，其功能可被扩展远远超出靶细胞识别和细胞死亡。 ...

粒酶是一类在自然杀伤（NK）细胞和细胞毒性T淋巴细胞（CTL）的1的分泌溶酶体发现丝氨酸蛋白酶。在人类中（A，B，H，K和M）存在五种不同的颗粒酶和10小鼠（A - G，K，M和N）2,3。颗粒酶A和颗粒酶B（GzmA，GZMB）是最丰富的，并已被广泛研究，在人类和啮齿类动物的设置。 GZMB经典功能是细胞凋亡的诱导在与孔形成蛋白穿孔，其允许粒酶访问目标细胞胞质溶胶4一起执行靶细胞。虽然GZMB表达在细胞毒性淋巴细胞明确发现，最近的研究已经解决了各种其他GZMB表达的细胞类型，包括但不限于角质细胞5，嗜碱性粒细胞6，肥大细胞7，浆细胞样树突细胞8，和B细胞9， 10。在这种情况下，非凋亡GZMB功能进行了揭示，从参与炎症过程，组织重塑和其他免疫调节性质11-14。 鉴于一个更广泛的生物作用已被提议用于GZMB比先前怀疑，研究人员需要针对其检测可靠和专用工具。的优点是GZMB的具体要求来切割上的天冬氨酸残基，一个属性之间的真核丝氨酸蛋白酶15独特的羧基侧。小鼠，人和大鼠GZMB在结构上非常相似，但是小鼠GZMB的扩展的底物特异性不同巧妙地从人类和鼠16，这意味着某些通用基板用Asp在终端（P1）可以通过GZMB有效切割的所有三个品种，而其他底材具有更复杂的序列上游P1可能会给广泛不同的结果。在过去和最近的立terature，这一事实造成了相当大的混乱和一些实验结果的生物学意义的曲解，即使严格控制的，动力学研究试图纠正这种情况17。 在本文中，我们已经设法示出了这些点使用两个市售底物，即的Boc - 丙氨酸 - 丙氨酸 - 天冬氨酸 - SBzl和N-乙酰基异亮氨酸 - 谷氨酸 - 亲天冬氨酸 - 对硝基苯胺。的两种试剂做产生不同的反应基团以下裂解（游离巯与荧光游离对硝基酰苯胺），但这种不具有任何对蛋白水解裂解的影响。所描述的协议是一个非常古老的协议，18现代适应，但应该帮助调查人员适当地使用不同的GZMB基板，同时也提供了一个方法框架，用于检测其他颗粒酶，如GzmA和GzmH的活动。

<table border="0" cellpadding="0" cellspacing="0" width="1133">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Product</td>
			<td style="width:241px;">Company</td>
			<td style="width:181px;">Catalogue number</td>
			<td style="width:427px;">Comment/description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boc-Ala-Ala-Asp-S-Bzl</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SMSB05</td>
			<td>Granzyme B substrate (mouse and human)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ac-IEPD-pNA&#160;</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SMPNA009</td>
			<td>Granzyme B substrate (only human)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-a-CBZ-L-lysine-S-Bzl</td>
			<td>Sigma-Aldrich</td>
			<td>C3647</td>
			<td>Granzyme A substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suc-Phe-Leu-Phe-S-Bzl</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SB025</td>
			<td>Granzyme H substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5,5&#8217;-dithio-bis(2-nitrobenzoic acid)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D8130</td>
			<td>&#160;DTNB, Ellman&#8217;s Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NK cell isolation kit II mouse</td>
			<td>Miltenyi Biotec GmbH</td>
			<td style="width:181px;">130-096-892</td>
			<td style="width:427px;">negative selection kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NK cell isolation kit human</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-092-657</td>
			<td style="width:427px;">negative selection kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate reader</td>
			<td>Biorad iMark</td>
			<td></td>
			<td>Biorad Microplate Manager Software Version MPM6.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serocluster U-bottom vinyl 96-well plate</td>
			<td>Corning, MA, USA</td>
			<td>2797</td>
			<td></td>
		</tr>
	</tbody>
</table>

a colorimetric assay that specifically measures granzyme b proteolytic activity: hydrolysis of boc-ala-ala-asp-s-bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB&#8217;s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

将Boc-AAD-S- BZL基板特异于GZMB 丝氨酸蛋白酶GZMB是细胞毒性淋巴细胞（CTL和NK细胞）的主要成分，是主要负责诱导快速凋亡性死亡的靶细胞，如病毒感染或转化的细胞。这主要是由于它的底物优先用于某些特定的天冬氨酸残基中选择的蛋白质后裂解，属性与胱天，这也裂解天冬氨酸残基后，但是从一个不同的类蛋白酶，半胱氨酸蛋白酶家族15共享。在蛋白水?...

Watch this Scientific Journal Video about A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl at JoVE.com

A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

比色测定的具体措施颗粒酶B蛋白水解活动:水解的Boc-丙氨酸 - 丙氨酸-ASP-S-BZL的

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is ...

a-colorimetric-assay-that-specifically-measures-granzyme-b

Research

JoVE Journal

Chemistry

12.9K 次观看.  Peter MacCallum Cancer Centre. We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

视频: A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios &lt; 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios &gt; 1.53.
However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at &asymp;280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 &lt; ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).

A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

快速比色测定RNA和DNA生物分子样品进行定性区分

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in ...

科研

化学

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

稳态，稳定前的状态，单营业额动力学测量DNA糖基化酶活性

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.

PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

PAD4活动通过亲荧光底物类似物基于荧光监测

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein ...

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

氯离子的动态电化学测试

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

A Method to Manipulate Surface Tension of a Liquid Metal via Surface Oxidation and Reduction

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where interfacial tension is a dominant force. A variety of methods exist for controlling the interfacial tension of aqueous and organic liquids on this scale; however, these techniques have limited utility for liquid metals due to their large interfacial tension.
Liquid metals can form soft, stretchable, and shape-reconfigurable components in electronic and electromagnetic devices. Although it is possible to manipulate these fluids via mechanical methods (e.g., pumping), electrical methods are easier to miniaturize, control, and implement. However, most electrical techniques have their own constraints: electrowetting-on-dielectric requires large (kV) potentials for modest actuation, electrocapillarity can affect relatively small changes in the interfacial tension, and continuous electrowetting is limited to plugs of the liquid metal in capillaries.
Here, we present a method for actuating gallium and gallium-based liquid metal alloys via an electrochemical surface reaction. Controlling the electrochemical potential on the surface of the liquid metal in electrolyte rapidly and reversibly changes the interfacial tension by over two orders of magnitude ( &#820;500 mN/m to near zero). Furthermore, this method requires only a very modest potential (&#60; 1 V) applied relative to a counter electrode. The resulting change in tension is due primarily to the electrochemical deposition of a surface oxide layer, which acts as a surfactant; removal of the oxide increases the interfacial tension, and vice versa. This technique can be applied in a wide variety of electrolytes and is independent of the substrate on which it rests.

We present a method to control the interfacial energy of a liquid metal in an electrolyte via electrochemical deposition (or removal) of a surface oxide layer. This simple method can control the capillary behavior of gallium-based liquid metals by tuning the interfacial energy rapidly, significantly, and reversibly using modest voltages.

方法通过表面氧化还原操纵液态金属的表面张力

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where ...

Preparation and Reactivity of a Triphosphenium Bromide Salt: A Convenient and Stable Source of Phosphorus(I)

We present herein the optimized synthesis of a triphosphenium bromide salt. Apart from being a versatile metathesis reagent, this unusually stable low-valent-phosphorus-containing compound acts as a useful P+ transfer agent. Unlike traditional methods employed to access low-coordinate phosphorus species which usually require pyrophoric phosphorus-containing precursors (white phosphorus, Tris(trimethylsilyl)phosphine, etc.), or harsh reducing agents (alkali metals, potassium graphite, etc.), the current approach does not involve pyrophoric or explosive reagents and can be done on large scales (&#62;20 g) in excellent yields by undergraduates with basic air-free synthetic training. The bromide counter ion is readily exchanged with other anions such as tetraphenyl borate (described herein) using typical salt metathesis reagents to obtain materials with desired properties and reactivities. The versatility of this P+ transfer approach is exemplified by the reactions of these triphosphenium precursors with an N-heterocyclic carbene and an anionic bisphosphine, each of which readily displace the neutral bisphosphine to give an NHC-stabilized phosphorus(I) cation and a phosphorus(I) containing zwitterion, respectively.

Preparation and Reactivity of a Triphosphenium Bromide Salt: A Convenient and Stable Source of PhosphorusI

The synthesis of a triphosphenium bromide salt is described and its use as a P+ transfer agent is outlined by reactions with an N-heterocyclic carbene and an anionic bisphosphine, yielding an NHC-stabilized P(I) cation and a P(I) containing zwitterion, respectively.

制备及反应性的Triphosphenium溴化物盐:磷方便稳定来源（I）

We present herein the optimized synthesis of a triphosphenium bromide salt. Apart from being a versatile metathesis reagent, this unusually stable ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

同时测量 HDAC1 和 HDAC6 活动在 HeLa 细胞使用 UHPLC 女士

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation usually consists in the formation of droplets containing low molar mass liquid crystals at elevated temperatures. Subsequently, these particle precursors are oriented in the flow field of the capillary and solidified by a crosslinking polymerization, which produces the final actuating particles. The optimization of the process is necessary to obtain the actuating particles and the proper variation of the process parameters (temperature and flow rate) and allows variations of size and shape (from oblate to strongly prolate morphologies) as well as the magnitude of actuation. In addition, it is possible to vary the type of actuation from elongation to contraction depending on the director profile induced to the droplets during the flow in the capillary, which again depends on the microfluidic process and its parameters. Furthermore, particles of more complex shapes, like core-shell structures or Janus particles, can be prepared by adjusting the setup. By the variation of the chemical structure and the mode of crosslinking (solidification) of the liquid crystalline elastomer, it is also possible to prepare actuating particles triggered by heat or UV-vis irradiation.

This article describes the microfluidic process and parameters to prepare actuating particles from liquid crystalline elastomers. This process allows the preparation of actuating particles and the variation of their size and shape (from oblate to strongly prolate, core-shell, and Janus morphologies) as well as the magnitude of actuation.

液晶弹性体致动器的微流控制备

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation ...

A Two-Step Protocol for Umpolung Functionalization of Ketones Via Enolonium Species

&#945;-Functionalization of ketones via umpolung of enolates by hypervalent iodine reagents is an important concept in synthetic organic chemistry. Recently, we have developed a two-step strategy for ketone enolate umpolung that has enabled the development of methods for chlorination, azidation, and amination using azoles. In addition, we have developed C-C bond&#8211;forming arylation and allylation reactions. At the heart of these methods is the preparation of the intermediate and highly reactive enolonium species prior to addition of a reactive nucleophile. This strategy is thus reminiscent of the preparation and use of metal enolates in classical synthetic chemistry. This strategy allows the use of nucleophiles that would otherwise be incompatible with the strongly oxidizing hypervalent iodine reagents. In this paper we present a detailed protocol for chlorination, azidation, N-heteroarylation, arylation, and allylation. The products include motifs prevalent in medicinally active products. This article will greatly assist others in using these methods.

A two step one-pot protocol for the umpolung of ketone enolates to enolonium species and addition of a nucleophile to the &#945;-position is described. Nucleophiles include chloride, azide, azoles, allyl-silanes, and aromatic compounds.

Enolonium 种极性转换酮功能化的两步协议

&#945;-Functionalization of ketones via umpolung of enolates by hypervalent iodine reagents is an important concept in synthetic organic chemistry. ...

A Rhodopsin Transport Assay by High-Content Imaging Analysis

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. We previously performed a luminescence-based high-throughput screen (HTS) and identified a group of pharmacological chaperones that rescued the transport of the P23H rhodopsin from ER to the plasma membrane. Here, using an immunostaining method followed by a high-content imaging analysis, we quantified the mutant rhodopsin protein amount in the whole cell and on the plasma membrane. This method is informative and effective to identify true hits from false positives following HTS. Additionally, the high-content image analysis enabled us to quantify multiple parameters from a single experiment to evaluate the pharmacological properties of each compound. Using this assay, we analyzed the effect of 11 different compounds towards six RP associated rhodopsin mutants, obtaining a 2-D pharmacological profile for a quantitative and qualitative understanding about the structural stability of these rhodopsin mutants and efficacy of different compounds towards these mutants.

Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell.

高含量成像分析研究。

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding ...