Bru Samuel, Jiménez Javier, Canadell David, Ariño Joaquín, Clotet Josep
Department of Basic Sciences, Faculty of Medicine and Health Sciences. Universitat Internacional de Catalunya. Barcelona, Spain.
Departament de Bioquímica i Biologia Molecular and Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona. Cerdanyola del Vallès, Spain.
Microb Cell. 2016 Dec 29;4(1):6-15. doi: 10.15698/mic2017.01.551.
Polyphosphate (polyP) is an abundant and physiologically important biomolecule for virtually any living cell. Therefore, determination of changes in cellular content of polyP is crucial for its functional characterization. Determination of cellular polyP has been performed by many different methods, and the lack of a standardized procedure is possibly responsible for the large dispersion of results found in the relevant literature. For a relatively simple organism, such as the yeast , this variation can be up to 12-fold. polyP extraction and determination of free phosphate released by enzymatic degradation of the polymer is a method quite common and relatively straightforward for polyP determination. By using the yeast as model, we have experimentally evaluated the different steps in this procedure in order to identify critical issues that might explain the disparate reported results. As the main output of this evaluation we propose a straightforward and robust procedure that can be used as gold standard protocol for cellular polyP purification and determination from unicellular organisms, thus providing consistency to measurements and facilitating inter-laboratory comparisons and biological interpretation of the results.
多聚磷酸盐（polyP）对于几乎任何活细胞来说都是一种丰富且具有重要生理意义的生物分子。因此，确定细胞内多聚磷酸盐含量的变化对于其功能表征至关重要。细胞内多聚磷酸盐的测定已通过许多不同方法进行，而缺乏标准化程序可能是相关文献中结果差异较大的原因。对于像酵母这样相对简单的生物体，这种差异可能高达12倍。多聚磷酸盐的提取以及通过聚合物酶促降解释放的游离磷酸盐的测定是一种相当常见且相对直接的多聚磷酸盐测定方法。以酵母为模型，我们通过实验评估了该程序中的不同步骤，以确定可能解释所报道的不同结果的关键问题。作为该评估的主要成果，我们提出了一种直接且可靠的程序，可作为从单细胞生物中纯化和测定细胞内多聚磷酸盐的金标准方案，从而使测量结果具有一致性，并便于实验室间的比较以及对结果进行生物学解释。
