Monitoring the expression of purinoceptors and nucleotide-metabolizing ecto-enzymes with antibodies directed against proteins in native conformation.
Monitoring the expression of purinoceptors and nucleotide-metabolizing ecto-enzymes with antibodies directed against proteins in native conformation.
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DOI：
10.1007/s11302-007-9084-9
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发表时间：
2007-09
影响因子：
3.5
通讯作者：
Koch-Nolte, Friedrich
中科院分区：
文献类型：
--
作者：
Moeller, Sina;Jung, Caroline;Adriouch, Sahil;Dubberke, Gudrun;Seyfried, Fabienne;Seman, Michel;Haag, Friedrich;Koch-Nolte, Friedrich
能量代谢的通用货币ATP和NAD从细胞释放后，作为细胞外信号分子发挥作用。哺乳动物细胞表达许多嘌呤受体，即，核苷酸门控P2 X离子通道和G蛋白偶联P2 Y受体。通过嘌呤受体的信号传导由核苷酸代谢胞外酶控制，其调节胞外核苷酸的可用性。这些酶包括外三磷酸核苷二磷酸水解酶（ENTPD，CD 39家族）和外核苷酸焦磷酸酶/磷酸二酯酶（ENPP，CD 203家族）。这些受体和酶的研究一直受到缺乏可用抗体的阻碍，特别是那些识别这些蛋白质在其天然构象。本研究报告了使用遗传免疫来产生针对P2 X1、P2 X4、P2 X7、ENTPD 1、ENPTD 2、ENPTD 5、ENPTD 6、ENPP 2、ENPP 3、ENPP 4、ENPP 5和ENPP 6的此类抗体。遗传免疫确保了免疫动物的细胞表达天然蛋白质，并产生针对天然构象蛋白质（ADAPINC）的抗体。这种抗体特别适用于免疫荧光和免疫沉淀分析，而针对合成肽的抗体通常仅在Western印迹分析中发挥良好作用。在这里，我们说明了新的抗体的效用，以监测细胞表面的表达和纯化嘌呤能信号的一些关键球员。
Following their release from cells, ATP and NAD, the universal currencies of energy metabolism, function as extracellular signalling molecules. Mammalian cells express numerous purinoceptors, i.e., the nucleotide-gated P2X ion channels and the G-protein-coupled P2Y receptors. Signalling through purinoceptors is controlled by nucleotide-metabolizing ecto-enzymes, which regulate the availability of extracellular nucleotides. These enzymes include ecto-nucleoside triphosphate diphosphohydrolases (ENTPD, CD39 family) and ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPP, CD203 family). Investigation of these receptors and enzymes has been hampered by the lack of available antibodies, especially ones that recognize these proteins in their native conformation. This study reports the use of genetic immunization to generate such antibodies against P2X1, P2X4, P2X7, ENTPD1, ENPTD2, ENPTD5, ENPTD6, ENPP2, ENPP3, ENPP4, ENPP5, and ENPP6. Genetic immunization ensures expression of the native protein by the cells of the immunized animal and yields antibodies directed against proteins in native conformation (ADAPINCs). Such antibodies are especially useful for immunofluorescence and immunoprecipitation analyses, whereas antibodies against synthetic peptides usually function well only in Western-blot analyses. Here we illustrate the utility of the new antibodies to monitor the cell surface expression of and to purify some key players of purinergic signalling.
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DOI：
10.1196/annals.1299.101
发表时间：
2003-01-01
期刊：
APOPTOSIS: FROM SIGNALING PATHWAYS TO THERAPEUTIC TOOLS
影响因子：
--
作者：
Diessenbacher, P;Bartels, K;Haag, F
通讯作者：
Haag, F
影响因子：
2.9
作者：
Zalatan, Jesse G.;Fenn, Timothy D.;Herschlag, Daniel
通讯作者：
Herschlag, Daniel
影响因子：
4.3
作者：
Adriouch, S;Dubberke, G;Koch-Nolte, F
通讯作者：
Koch-Nolte, F
影响因子：
4.3
作者：
Koch-Nolte, F;Glowacki, G;Haag, F
通讯作者：
Haag, F
影响因子：
64.8
作者：
TANG, DC;DEVIT, M;JOHNSTON, SA
通讯作者：
JOHNSTON, SA