Biboy Jacob, Bui Nhat Khai, Vollmer Waldemar
Institute for Cell and Molecular Biosciences, The Centre for Bacterial Cell Biology, Newcastle University, Newcastle upon Tyne, UK.
Methods Mol Biol. 2013;966:273-88. doi: 10.1007/978-1-62703-245-2_17.
Bacterial cell wall peptidoglycan is synthesized from lipid II precursor by two reactions. Glycosyltransferases polymerize the glycan chains and transpeptidases form the peptide cross-links. The bifunctional class A penicillin-binding proteins catalyze both of these reactions. Here, we describe an in vitro peptidoglycan synthesis assay utilizing radiolabeled lipid II substrate to monitor simultaneously peptidoglycan glycosyltransferase and transpeptidase activities. The products of the reaction are separated by high-pressure liquid chromatography and quantified by flow-through scintillation counting.
细菌细胞壁肽聚糖由脂质II前体通过两个反应合成。糖基转移酶使聚糖链聚合，转肽酶形成肽交联。双功能A类青霉素结合蛋白催化这两个反应。在此，我们描述了一种体外肽聚糖合成测定法，利用放射性标记的脂质II底物同时监测肽聚糖糖基转移酶和转肽酶活性。反应产物通过高压液相色谱分离，并通过流通闪烁计数进行定量。
