title: Colorimetric inorganic pyrophosphate assay using a double cycling enzymatic method.
abstract: Pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) from the hyperthermophile Thermotoga maritima was biochemically characterized with the aim of establishing a colorimetric assay for inorganic pyrophosphate (PPi). When heterologously expressed in Escherichia coli, T. maritima PPDK (TmPPDK) was far more stable any other PPDK reported so far: it retained >90% of its activity after incubation for 1 h at 80°C, and >80% of its activity after incubation for 20 min at pHs ranging from 6.5 to 10.5 (50°C). In contrast to PPDKs from protozoa and plants, this TmPPDK showed very long-term stability at low temperature: full activity was retained even after storage for at least 2 years at 4°C. TmPPDK was successfully applied to a novel colorimetric PPi assay, which employed (i) a PPi cycling reaction using TmPPDK and nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) from Saccharomyces cerevisiae and (ii) a NAD cycling reaction to accumulate reduced nitroblue tetrazolium (diformazan). This enabled detection of 0.2 μM PPi, making this method applicable for preliminary measurement of PPi levels in PCR products in an automatic clinical analyzer.
authors.affiliation: Asahi Kasei Pharma Corporation, Shizuoka 410-4321, Japan. sakasegawa.sb@om.asahi-kasei.co.jp
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Sakasegawa
authors.forename: Junji
authors.initials: J
authors.lastname: Hayashi
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Ikura
authors.forename: Shigeru
authors.initials: S
authors.lastname: Ueda
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Imamura
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Kumazawa
authors.forename: Atsuhisa
authors.initials: A
authors.lastname: Nishimura
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Ohshima
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Sakuraba
mesh_terms: D003124:Colorimetry / Q000379:methods*; D011756:Diphosphates / Q000032:analysis*; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000378:metabolism*; D011772:Pyruvate, Orthophosphate Dikinase / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D020124:Thermotoga maritima / Q000201:enzymology
chemical_list: D011756:Diphosphates; D009612:Nicotinamide-Nucleotide Adenylyltransferase; D011772:Pyruvate, Orthophosphate Dikinase