Department of Biomolecular Science, Faculty of Science, Toho University, Funabashi, Chiba, Japan.
Department of Chemistry, Wakayama Medical University, Wakayama, Wakayama, Japan.
Biosci Biotechnol Biochem. 2024 Mar 22;88(4):429-436. doi: 10.1093/bbb/zbae001.
Dephosphorylation of undecaprenyl diphosphate is a crucial step in the synthesis of undecaprenyl phosphate, which is essential for cell wall synthesis. We have developed a method for the quantification of intracellular polyprenyl diphosphates, which have never before been measured directly. Polyprenyl phosphates and diphosphates prepared by chemical phosphorylation of polyprenols from Staphylococcus aureus were used to establish the conditions for fractionation by ion-exchange chromatography and high-performance liquid chromatography (HPLC). By using an elution solvent containing tetraethylammonium phosphate as an ion-pair reagent for HPLC, polyprenyl phosphate and polyprenyl diphosphate with carbon numbers from 40 to 55 could be detected as separate peaks from the reversed-phase column. This analytical method was applied to lipids extracted from Escherichia coli to determine the intracellular levels of octaprenyl phosphate, undecaprenyl phosphate, octaprenyl diphosphate, and undecaprenyl diphosphate. This is the first report of separate measurement of cellular levels of polyprenyl phosphates and polyprenyl diphosphates.
去磷酸化的十一碳烯焦磷酸是十一磷酸胞苷合成的关键步骤，十一磷酸胞苷对于细胞壁的合成是必不可少的。我们已经开发出一种用于定量分析细胞内多萜二磷酸的方法，这些物质以前从未被直接测量过。使用来自金黄色葡萄球菌的多萜醇化学磷酸化制备的多萜磷酸酯和二磷酸酯，建立了离子交换色谱和高效液相色谱（HPLC）分离的条件。通过使用含有四乙基铵磷酸盐的洗脱溶剂作为 HPLC 的离子对试剂，可以从反相柱上检测到碳数从 40 到 55 的多萜磷酸酯和多萜二磷酸酯作为单独的峰。该分析方法应用于从大肠杆菌中提取的脂质，以确定八磷酸胞苷、十一磷酸胞苷、八磷酸二磷酸酯和十一磷酸二磷酸酯的细胞内水平。这是首次分别测量细胞内多萜磷酸酯和多萜二磷酸酯水平的报道。
