CN1348503A - 用于检测磷酸基－n－乙酰胞壁酰五肽移位酶活性的测定方法 - Google Patents
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Abstract
本发明提供一种检测磷酸基－N－酰胞壁酰五肽移位酶的酶活性的测定方法,其包含下列步骤:(1)培养反应混合物,其包含,在含水介质中,N－琥珀酰亚胺基[2,3－3H]丙酸酯取代的UDP－MurNAc－五肽,N－琥珀酰亚胺基丙酸酯取代的UDP－MurNAc－五肽(非－放射性的),二价金属离子源,十一异戊二烯基磷酸酯源,去污剂和移位酶源,在适于酶活性发生的条件下;(2)在PH～4.2,用含季铵盐的适当缓冲液酸化反应混合物,以终止步骤(1)的酶反应;(3)提取所形成的任一十一异戊烯醇－焦磷酸酯－[2,3－3H]丙酸酯－N－乙酰胞壁酰五肽产物,并使用闪烁计数器测量放射性,且提供在其中使用的试剂盒。
Description
本发明涉及一种检测磷酸基-N-乙酰胞壁酰五肽移位酶(以下称为“移位酶”)酶活性的新的测定方法。
肽聚糖是细菌细胞壁的主要组成部分，其提供细胞壁的形状和强度。它是细菌所独有的，且能够在所有的细菌，包括革兰氏阳性菌和革兰氏阴性菌中找到。肽聚糖是聚糖链的聚合物，其是通过短肽桥交联的。它是由N-乙酰胞壁酸(MurNAc)和N-乙酰葡糖胺的交替β1-4连接的残基组成的。肽聚糖链附着在MurNAc(MurNAc-五肽)上，且肽聚糖聚合物是通过这些肽链交联的。
肽聚糖的生物合成可以分成三个阶段：第一，细胞质中前体的合成，第二，前体至脂类载体分子的转移和，第三，前体插入到细胞壁中，并偶联到存在的肽聚糖上。
负责细菌细胞壁的肽聚糖组分生物合成的酶是研制新抗菌素的新靶。由于对目前抗菌素耐受的细菌菌株的广泛出现，必须研制新的抗微生物剂。移位酶催化肽聚糖生物合成的膜循环中的第一步，即磷酸基-N-乙酰胞壁酰-L-丙氨酸-γ-D-谷氨酸-m-二氨基庚二酸-D-丙氨酸-D-丙氨酸自尿苷5’-二磷酸酯磷酸基-N-乙酰胞壁酰-L-丙氨酸-γ-D-谷氨酸-m-二氨基庚二酸-D-丙氨酸-D-丙氨酸(以下称为“UDP-MurNAc-五肽”或“UDP-MPP”)至膜-束缚脂类载体，十一异戊二烯基磷酸酯(undecaprenyl phosphate)的转移。移位酶是在大肠杆菌中由mraY基因编码的。
移位酶对于细菌的生存很重要(见D.Mengin-Lecreulx，L.Texier，M.Rousseaue和J.Van Heijernoot，J.Bacteriol.，(1991)，173，4625-4636)。
目前商业上使用的抗菌素还没有针对移位酶的。因此，提供一种新的抗细菌剂的靶，其至今还没有被开发。
移位酶通常是通过放射性标记UDP-MurNAc-五肽，并监测磷酸基-N-乙酰胞壁酰五肽自UDP-MurNAc-五肽至十一异戊二烯基磷酸酯的转移，导致脂类中间体，Lipid I的形成而测定的。放射性标记通常是通过使用酶连接酶以标记D-丙氨酸-D-丙氨酸末端或通过在体内掺入到膜上而完成的。这些方法的产率均较低，因而不能有效的研制高-通量-筛选(HTS)的测定方法。
移位酶的活性可以两者择一地使用Brandish et al.，J.Biol.Chem.，(1996)，271，7609-7614中描述的荧光底物，如丹磺酰氯来测定。然而，某些化合物可以猝灭荧光，从而导致检出酶反应的假抑制剂。
人们期望研制出一种移位酶的测定方法，其适于高-通量的筛选。
根据本发明，提供一种检测磷酸基-N-乙酰胞壁酰五肽移位酶的酶活性的测定方法，其包含下列步骤：
(1)培养反应混合物，其包含，在含水介质中，N-琥珀酰亚胺基[2，3-3H]丙酸酯取代的UDP-MurNAc-五肽，N-琥珀酰亚胺基丙酸酯取代的UDP-MurNAc-五肽(非-放射性的)，二价金属离子源，十一异戊二烯基磷酸酯源，去污剂和移位酶源，在适于酶活性发生的条件下；
(2)在PH～4.2，用含季铵盐的适当缓冲液酸化反应混合物，以终止步骤(1)的酶反应；
(3)提取所形成的任一十一异戊烯醇(undecapreol)-焦磷酸酯-[2，3-3H]丙酸酯-N-乙酰胞壁酰五肽产物，并使用闪烁计数器测量放射性。
在步骤(1)中，所用的UDP-MPP可以是任一天然存在的肽聚糖。它们通常是自细菌中提纯的，或用前体自细菌酶促制成的，例如使用与Blaauwen et al.，J.Bacteriol.(1990)，172，63-70所描述的类似方法。二者择一地，可以通过Lugtenberg et al.，J.Bacteriol.(1972)，109，326-335所描述的方法自枯草杆菌W23分离它。所使用的优选UDP-MPP是来源于蜡样芽胞杆菌的UDP-MurNAc-L-丙氨酸-γ-D-谷氨酸-m-二氨基庚二酸-D-丙氨酸-D-丙氨酸。
这样得到的UDP-MPP与N-琥珀酰亚胺基[2，3-3H]丙酸酯(商业上应用来源于Amersham Ltd.的)反应，得到N-琥珀酰亚胺基[2，3-3H]丙酸酯取代的UDP-MurNAc-五肽(以下称为“3H-丙酸酯化的UDP-MPP”)。
测定中使用的3H-丙酸酯化的UDP-MPP的浓度在2-50μM，优选2-40μM，更优选2-25μM之间。
反应中所用未标记的，非-放射性的N-琥珀酰亚胺基丙酸酯取代的UDP-MPP(以下称为“丙酸酯化的非-放射性UDP-MPP”)的浓度为5-70μM，优选5-50μM，特别是8-30μM。
反应中所用的二价金属离子优选是镁离子。适当的镁离子源是氯化镁。所用二价金属离子的浓度为20mM-100mM，优选20mM-80mM，更优选20mM-50mM，例如25mM。
此外，可以将50mM-100mM浓度的氯化钾加到反应混合物中。
可以便利地使用大肠杆菌的膜，实际上它是优选的移位酶和十一异戊二烯基磷酸酯源。每50μl反应混合物所用膜的量为5-200μg，优选50μg。该膜可以通过本领域已知的方法制备。
步骤(1)中所用的含水介质优选是缓冲液，例如三[羟甲基]氨基甲烷氢氯化物(“Tris-HCl”)，其具有大约7.5的PH。商业上应用来源于Sigma Aldrich Co.Ltd的Tris-HCl。
反应混合物还可以另外包含0.01个单位的碱性磷酸酶。
所用去污剂可以是，例如，0.1％w/v浓度的Triton X-100.如果使用这些，去污剂可以有效地溶解细菌膜。
如果将该测定方法用作鉴定抗-细菌化合物，其为移位酶的拮抗剂，的筛选，则步骤(1)中的反应混合物还可以进一步包含一种或多种不同浓度的试验化合物。因为移位酶是肽聚糖合成第一步中所需要的酶，因而它提供研制抗-细菌药物的适宜的靶。
将步骤(1)中的反应混合物保持在20℃-37℃，优选25℃的温度下，较短的时间，例如，最多可达10分钟，特别是6-8分钟。
酶反应是通过，例如，6M吡啶嗡醋酸酯和正-丁醇(PH-4.2)的2∶1混合物的加入而终止的。这构成了步骤(2)。
在步骤(3)中，使用，例如，正-丁醇提取产物。然后用闪烁计数器量化。
参考下列实施例进一步举例说明本发明。
实施例1
首先将40μl的反应混合物分别填装到微量离心管中。反应混合物是由100mM的Tris-HCl(三[羟甲基]氨基甲烷氢氯化物)含水缓冲液，25mM氯化镁，50mM的氯化钾，0.1％w/v的Triton X-100，8μM的丙酸酯化的非-放射性UDP-MPP，室温下的2μM的3H-丙酸酯化的UDP-MPP，10μl的酶，其浓度为5μg/ml，和17.5μl的水组成的。向其中加入各种浓度的试验化合物(例如，衣霉素)溶液。衣霉素是已知的移位酶拮抗剂。反应是以酶的加入开始的。
大肠杆菌膜，其作为酶源，是用下列方法制备的。
该膜是从商业上应用的原生质球粒制备的。每个粒均包含一定量的大肠杆菌Hfr H。4℃过夜融化上述粒。称重该粒，并将数字乘以7.5。这为它提供了所加入缓冲液的体积。所用缓冲液是20μM PH8.0的Tris-HCl，含20％的蔗糖。使用磁搅拌器轻轻地冷却搅拌混合物10分钟。向其中加入蛋清溶菌酶的溶液，直到得到0.2mg/ml的最终浓度。在冰上又搅拌10分钟。在超过1小时的时段内，向其中慢慢地加入PH8.0，0.2M浓度的乙二胺四乙酸(EDTA)，商购于Sigma AldrichCo.Ltd.，的20mM Tris-HCl溶液，直到得到0.02M的最终浓度。此加入是在4-8℃的冷室中进行的。然后在12,000g离心混合物20分钟。然后又在PH7.5的，按照前段计算的相同体积的50mM Tris-HCl缓冲液中混悬，含，20μg/ml的DNAse，20μg/ml的RNAse，1mM的氯化镁和1mM的β-巯基乙醇。在室温搅拌1小时，直到样品变得均质。通过在1,00,000g下超速离心旋转1小时回收膜部分。
用下列方法制备用作底物的3H-丙酸酯化的UDP-MPP。
在262nm的波长下，将4 O.D.固定容积，即，大约450-500μg未标记的UDP-MPP装入空的微量离心管中。在另一个微量离心管中，装入0.5mCi的含氚的N-琥珀酰亚胺基丙酸酯(N-succinidimyl[2，3-3H]丙酸酯)，向其中加入大约180μl的1％NaHCO3。将第二个管混合均匀，并转移到第一个管中。将第二个管冲洗干净，加入180μl的1％NaHCO3，再转移到第一个管中。该管，含混合物，在振荡器中室温过夜以利于标记。用类似的方法，制备丙酸酯化的非放射性UDP-MPP，使用N-琥珀酰亚胺基丙酸酯代替放射性化合物。
如下提纯它，
(1)用1ml商购的sephadex A’-25装填玻璃柱。
(2)用10柱床体积的水洗柱子。
(3)然后用10柱床体积的1％NaHCO3平衡。
(4)将反应混合物装填在柱子上，收集穿过的液流。
(5)使液流通过柱子两次，以利于结合。
(6)然后用0.5ml的1％NaHCO3洗柱子。
(7)在同一管中收集洗液和液流，并标记。
(8)用6ml的1％NaHCO3洗柱子两次。
(9)在同一管中收集两次的洗液，并标记。
(10)用1ml的1％NaHCO3，其含0.4M氯化锂，洗脱柱子。
(11)收集组分并标记。
(12)计算组分，在反应中使用最纯的。
将含反应混合物的微量离心管在37℃培养大约4-60分钟，然后，每两分钟，加入50μl吡啶嗡醋酸酯和正-丁醇的2∶1混合物，以终止反应。
用饱和的正-丁醇提取产物，并用50μl的水洗。然后在闪烁计数器上，每次数25-50μl。
已知用移位酶催化的反应是可逆的。为了显示可逆的反应，继续酶反应10分钟，以形成放射性产物，十一异戊烯醇-焦磷酸酯-[2，3-3H]丙酸酯-N-乙酰胞壁酰五肽，Lipid I。这是从它的有机相分离的。一旦形成了放射性产物，则使用上述吡啶嗡醋酸酯和正-丁醇的2∶1混合物终止一系列反应。在一平行的系列反应中，加入1μM的UMP。然后在大约3-4分钟之后终止反应。脂类部分可以从两组反应中提取。发现有机相中的放射性数降低至具有UMP的基础水平。这表明Lipid I至水溶性前体的逆转。
此实施例表明本发明的测定系统仅仅对于移位酶反应是特殊的，甚至是将特殊的膜用作酶源时。
图1是表示基于从100％对照所得到的读数，相对于时间的每分次数(cpm)的图。
图2是表示与对照组(用■表示)相比，0.3μg/ml浓度的衣霉素(用▲表示)对移位酶的抑制速率。这证实了衣霉素是移位酶的拮抗剂。
Claims (10)
1.一种检测磷酸基-N-乙酰胞壁酰五肽移位酶的酶活性的测定方法，其包含下列步骤：
(1)培养反应混合物，其包含，在含水介质中，N-琥珀酰亚胺基[2，3-3H]丙酸酯取代的UDP-MurNAc-五肽，N-琥珀酰亚胺基丙酸酯取代的UDP-MurNAc-五肽(非-放射性的)，二价金属离子源，十一异戊二烯基磷酸酯源，去污剂和移位酶源，在适于酶活性发生的条件下；
(2)在PH～4.2，用含季铵盐的适当缓冲液酸化反应混合物，以终止步骤(1)的酶反应；和
(3)提取所形成的任一十一异戊烯醇-焦磷酸酯-[2，3-3H]丙酸酯-N-乙酰胞壁酰五肽产物，并使用闪烁计数器测量放射性。
2.根据权利要求1的测定方法，其中被取代的UDP-N-乙酰胞壁酰五肽是UDP-MurNAc-L-丙氨酸-γ-D-谷氨酸-m-二氨基庚二酸-D-丙氨酸-D-丙氨酸。
3.根据权利要求1或权利要求2的测定方法，其中将氯化镁用作二价金属离子源。
4.根据权利要求1-3中任何一项的测定方法，其中细菌细胞膜被用作十一异戊二烯基磷酸酯和移位酶中的一个或两个的源。
5.根据权利要求4的测定方法，其中的细菌细胞膜来源于大肠杆菌。
6.根据权利要求1-5中任何一项的测定方法，其中步骤(1)的反应混合物进一步包含试验化合物。
7.根据权利要求6的测定方法，其中的试验化合物是移位酶的拮抗剂。
8.根据权利要求1-7中任何一项的方法，其中使用吡啶嗡醋酸酯和正-丁醇的2∶1混合物终止步骤(2)中的反应。
9.根据权利要求1-7中任何一项的测定方法，其中步骤(3)中的产物是使用正-丁醇提取的。
10.用于进行前述任何一项权利要求的测定方法的试剂盒，其包含：
(1)N-琥珀酰亚胺基[2，3-3H]丙酸酯取代的UDP-MurNAc-五肽，
(2)N-琥珀酰亚胺基丙酸酯取代的UDP-MurNAc-五肽(非-放射性的)，
(3)二价金属离子源，
(4)十一异戊二烯基磷酸酯源，
(5)磷酸基-N-乙酰胞壁酰五肽移位酶源，和
(6)去污剂。
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| US7439043B2 (en) * | 2001-10-10 | 2008-10-21 | Neose Technologies, Inc. | Galactosyl nucleotide sugars |
| US7173003B2 (en) | 2001-10-10 | 2007-02-06 | Neose Technologies, Inc. | Granulocyte colony stimulating factor: remodeling and glycoconjugation of G-CSF |
| US7157277B2 (en) | 2001-11-28 | 2007-01-02 | Neose Technologies, Inc. | Factor VIII remodeling and glycoconjugation of Factor VIII |
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| CA2519092C (en) | 2003-03-14 | 2014-08-05 | Neose Technologies, Inc. | Branched water-soluble polymers and their conjugates |
| PL1615945T3 (pl) | 2003-04-09 | 2012-03-30 | Ratiopharm Gmbh | Sposoby glikopegylacji i białka/peptydy wytwarzane tymi sposobami |
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| US9005625B2 (en) | 2003-07-25 | 2015-04-14 | Novo Nordisk A/S | Antibody toxin conjugates |
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| KR101439880B1 (ko) | 2004-01-08 | 2014-09-12 | 라티오팜 게엠베하 | 펩티드의 오-결합형 글리코실화 |
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| NZ556436A (en) | 2005-01-10 | 2010-11-26 | Biogenerix Ag | Glycopegylated granulocyte colony stimulating factor |
| EP2386571B1 (en) | 2005-04-08 | 2016-06-01 | ratiopharm GmbH | Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants |
| JP5216580B2 (ja) | 2005-05-25 | 2013-06-19 | ノヴォ ノルディスク アー／エス | グリコペグ化第ｉｘ因子 |
| US20070105755A1 (en) | 2005-10-26 | 2007-05-10 | Neose Technologies, Inc. | One pot desialylation and glycopegylation of therapeutic peptides |
| US20090048440A1 (en) | 2005-11-03 | 2009-02-19 | Neose Technologies, Inc. | Nucleotide Sugar Purification Using Membranes |
| ES2516694T3 (es) | 2006-07-21 | 2014-10-31 | Ratiopharm Gmbh | Glicosilación de péptidos a través de secuencias de glicosilación con unión en O |
| WO2008057683A2 (en) | 2006-10-03 | 2008-05-15 | Novo Nordisk A/S | Methods for the purification of polypeptide conjugates |
| BRPI0809670A8 (pt) | 2007-04-03 | 2018-12-18 | Biogenerix Ag | métodos para aumentar a produção de célula tronco, para aumentar o número de granulócitos em um indivíduo, para prevenir, tratar e aliviar a mielossupressão que resulta de uma terapia contra o câncer, para tratar uma condição em um indivíduo, para tratar neutropenia e trombocitopenia em um mamífero, para expandir células tronco hematopoiéticas em cultura, para aumentar a hematopoiese em um indivíduo, para aumentar o número de célulars progenitoras hematopoiéticas em um indivíduo, e para fornecer enxerto estável da medula óssea, e, forma de dosagem oral. |
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