Determination of nucleoside triphosphatase activities from measurement of true inorganic phosphate in the presence of labile phosphate compounds

详细信息    查看全文

• 作者：Faith E.H. Katz ; Xinying Shi ; Cedric P. Owens¹ ; Simpson Joseph ; F. Akif Tezcan ; ^{tezcan@ucsd.edu}
• 关键词：Nitrogenase ; EF-G ; Ribosome ; Inorganic phosphate ; Nucleotide hydrolysis ; Phosphomolybdate
• 刊名：Analytical Biochemistry
• 出版年：2017
• 出版时间：1 March 2017
• 年：2017
• 卷：520
• 期：Complete
• 页码：62-67
• 全文大小：713 K
• 卷排序：520

文摘

One of the most common assays for nucleoside triphosphatase (NTPase) activity entails the quantification of inorganic phosphate (Pi) as a colored phosphomolybdate complex at low pH. While this assay is very sensitive, it is not selective for Pi in the presence of labile organic phosphate compounds (OPCs). Since NTPase activity assays typically require a large excess of OPCs, such as nucleotides, selectivity for Pi in the presence of OPCs is often critical in evaluating enzyme activity. Here we present an improved method for the measurement of enzymatic nucleotide hydrolysis as Pi released, which achieves selectivity for Pi in the presence of OPCs while also avoiding the costs and hazards inherent in other methods for measuring nucleotide hydrolysis. We apply this method to the measurement of ATP hydrolysis by nitrogenase and GTP hydrolysis by elongation factor G (EF-G) in order to demonstrate the broad applicability of our method for the determination of nucleotide hydrolysis in the presence of interfering OPCs.