Analysis of 1- and 3-Phosphohistidine (pHis) Protein Modification Using Model Enzymes Expressed in Bacteria.

Analysis of 1- and 3-Phosphohistidine (pHis) Protein Modification Using Model Enzymes Expressed in Bacteria.
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使用细菌中表达的模型酶分析 1- 和 3-磷酸组氨酸 (pHis) 蛋白质修饰。

DOI:
10.1007/978-1-4939-9884-5_5
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发表时间:
2020
期刊:
Methods in molecular biology (Clifton, N.J.)
影响因子:
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通讯作者:
Clubbs Coldron AKM
Clubbs Coldron AKM
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文献类型:
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作者:
Clubbs Coldron AKM

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尽管近六十年前发现了蛋白质组氨酸(His)磷酸化,但测量和定量这种不稳定的翻译后修饰(PTM)的困难限制了其在原核和真核信号转导中的机制分析。在这里,我们描述了可靠的程序,亲和纯化,辅因子结合分析和基于抗体的检测磷酸组氨酸(pHis),对推定的人类组氨酸激酶NME 1(NDPK-A)和NME 2(NDPK-B)和糖酵解磷酸甘油酸酯PGEAM 1。通过利用异构体特异性单克隆N1-pHis和N3-pHis抗体,我们描述了强大的协议,免疫检测和异构体歧视的位点特异性pHis,包括N3-pHis对His 11的PGAM 1。
Despite the discovery of protein histidine (His) phosphorylation nearly six decades ago, difficulties in measuring and quantifying this unstable post-translational modification (PTM) have limited its mechanistic analysis in prokaryotic and eukaryotic signaling. Here, we describe reliable procedures for affinity purification, cofactor-binding analysis and antibody-based detection of phosphohistidine (pHis), on the putative human His kinases NME1 (NDPK-A) and NME2 (NDPK-B) and the glycolytic phosphoglycerate mutase PGAM1. By exploiting isomer-specific monoclonal N1-pHis and N3-pHis antibodies, we describe robust protocols for immunological detection and isomer discrimination of site-specific pHis, including N3-pHis on His 11 of PGAM1.
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