Ault-Riché D, Fraley C D, Tzeng C M, Kornberg A
Department of Biochemistry, Stanford University School of Medicine, California 94305-5307, USA.
J Bacteriol. 1998 Apr;180(7):1841-7. doi: 10.1128/JB.180.7.1841-1847.1998.
A major impediment to understanding the biological roles of inorganic polyphosphate (polyP) has been the lack of sensitive definitive methods to extract and quantitate cellular polyP. We show that polyP recovered in extracts from cells lysed with guanidinium isothiocynate can be bound to silicate glass and quantitatively measured by a two-enzyme assay: polyP is first converted to ATP by polyP kinase, and the ATP is hydrolyzed by luciferase to generate light. This nonradioactive method can detect picomolar amounts of phosphate residues in polyP per milligram of extracted protein. A simplified procedure for preparing polyP synthesized by polyP kinase is also described. Using the new assay, we found that bacteria subjected to nutritional or osmotic stress in a rich medium or to nitrogen exhaustion had large and dynamic accumulations of polyP. By contrast, carbon exhaustion, changes in pH, temperature upshifts, and oxidative stress had no effect on polyP levels. Analysis of Escherichia coli mutants revealed that polyP accumulation depends on several regulatory genes, glnD (NtrC), rpoS, relA, and phoB.
理解无机多聚磷酸盐（polyP）生物学作用的一个主要障碍是缺乏灵敏可靠的方法来提取和定量细胞中的多聚磷酸盐。我们发现，用异硫氰酸胍裂解细胞后提取物中回收的多聚磷酸盐可与硅酸盐玻璃结合，并通过双酶测定法定量测量：多聚磷酸盐首先被多聚磷酸激酶转化为ATP，然后ATP被荧光素酶水解产生光。这种非放射性方法能够检测每毫克提取蛋白质中皮摩尔量的多聚磷酸盐中的磷酸残基。本文还描述了一种制备由多聚磷酸激酶合成的多聚磷酸盐的简化方法。使用这种新测定法，我们发现，在丰富培养基中遭受营养或渗透胁迫、或氮耗尽的细菌，其多聚磷酸盐会大量动态积累。相比之下，碳耗尽、pH值变化、温度升高和氧化应激对多聚磷酸盐水平没有影响。对大肠杆菌突变体的分析表明，多聚磷酸盐的积累取决于几个调控基因，即谷氨酰胺D（NtrC）、RNA聚合酶S、松弛因子A和磷酸调节蛋白B。
