title: Determination of nucleoside triphosphatase activities from measurement of true inorganic phosphate in the presence of labile phosphate compounds.
abstract: 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 Piin the presence of labile organic phosphate compounds (OPCs). Since NTPase activity assays typically require a large excess of OPCs, such as nucleotides, selectivity for Piin 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 Pireleased, which achieves selectivity for Piin 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093, United States.
authors.forename: Faith E H
authors.initials: FEH
authors.lastname: Katz
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093, United States.
authors.forename: Xinying
authors.initials: X
authors.lastname: Shi
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093, United States.
authors.forename: Cedric P
authors.initials: CP
authors.lastname: Owens
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093, United States.
authors.forename: Simpson
authors.initials: S
authors.lastname: Joseph
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093, United States. Electronic address: tezcan@ucsd.edu.
authors.forename: F Akif
authors.initials: FA
authors.lastname: Tezcan
mesh_terms: D003124:Colorimetry; D006868:Hydrolysis; D008982:Molybdenum / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D043583:Nucleoside-Triphosphatase / Q000378:metabolism*; D010710:Phosphates / Q000032:analysis / Q000378:metabolism*; D010756:Phosphoric Acids / Q000032:analysis / Q000378:metabolism; D010758:Phosphorus / Q000737:chemistry
chemical_list: D010710:Phosphates; D010756:Phosphoric Acids; D010758:Phosphorus; D008982:Molybdenum; D043583:Nucleoside-Triphosphatase; C581523:molybdic acid; C003125:phosphomolybdic acid
keywords: EF-G; Inorganic phosphate; Nitrogenase; Nucleotide hydrolysis; Phosphomolybdate; Ribosome