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. 1985 Aug 15;230(1):255–260. doi: 10.1042/bj2300255

Enzymic determination of inorganic phosphates, organic phosphates and phosphate-liberating enzymes by use of nucleoside phosphorylase-xanthine oxidase (dehydrogenase)-coupled reactions.

H de Groot, H de Groot, T Noll
PMCID: PMC1152609  PMID: 2996493

Abstract

Coupled enzyme assays are described for measuring inorganic phosphates, organic phosphates and phosphate-liberating enzymes in biological material. The assays all determine Pi by its reaction with inosine, catalysed by nucleoside phosphorylase; this yields ribose 1-phosphate and hypoxanthine. The hypoxanthine is oxidized to uric acid by xanthine oxidase, and may be measured either by the absorbance of the uric acid, or by the formazan formed when a tetrazolium salt is used as the oxidant. The coupled enzyme assays are characterized by high sensitivity, quantitative utilization of phosphates and stoichiometric formation of the measurable products, measurement at pH 6.0-8.5, determination of phosphates within a single analytical step, and continuous measurement of phosphohydrolase activity in a corresponding rate assay. Examples include determinations of substrates such as Pi, PPi and AMP, and of enzymes such as 5'-nucleotidase, inorganic pyrophosphatase and glucose-6-phosphatase. Directions for further examples are given.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arion W. J., Wallin B. K., Lange A. J., Ballas L. M. On the involvement of a glucose 6-phosphate transport system in the function of microsomal glucose 6-phosphatase. Mol Cell Biochem. 1975 Feb 28;6(2):75–83. doi: 10.1007/BF01732001. [DOI] [PubMed] [Google Scholar]
  2. Baginski E. S., Foà P. P., Zak B. Microdetermination of inorganic phosphate, phospholipids, and total phosphate in biologic materials. Clin Chem. 1967 Apr;13(4):326–332. [PubMed] [Google Scholar]
  3. Berenblum I., Chain E. An improved method for the colorimetric determination of phosphate. Biochem J. 1938 Feb;32(2):295–298. doi: 10.1042/bj0320295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Black M. J., Jones M. E. Inorganic phosphate determination in the presence of a labile organic phosphate: assay for carbamyl phosphate phosphatase activity. Anal Biochem. 1983 Nov;135(1):233–238. doi: 10.1016/0003-2697(83)90756-x. [DOI] [PubMed] [Google Scholar]
  5. DRYER R. L., TAMMES A. R., ROUTH J. I. The determination of phosphorus and phosphatase with N-phenyl-p-phenylenediamine. J Biol Chem. 1957 Mar;225(1):177–183. [PubMed] [Google Scholar]
  6. Hwang W. I., Cha S. A new enzymatic method for the determination of inorganic phosphate and its application to the nucleoside diphosphatase assay. Anal Biochem. 1973 Oct;55(2):379–387. doi: 10.1016/0003-2697(73)90126-7. [DOI] [PubMed] [Google Scholar]
  7. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  8. Lane R. J., Watmough N. J. Measurement of phosphate concentration in the presence of very labile phosphate esters. Anal Biochem. 1984 Aug 1;140(2):424–427. doi: 10.1016/0003-2697(84)90188-x. [DOI] [PubMed] [Google Scholar]
  9. MARSH B. B. The estimation of inorganic phosphate in the presence of adenosine triphosphate. Biochim Biophys Acta. 1959 Apr;32:357–361. doi: 10.1016/0006-3002(59)90607-9. [DOI] [PubMed] [Google Scholar]
  10. NACHLAS M. M., MARGULIES S. I., SELIGMAN A. M. A colorimetric method for the estimation of succinic dehydrogenase activity. J Biol Chem. 1960 Feb;235:499–503. [PubMed] [Google Scholar]
  11. SCHLESINGER M. J., COON M. J. Hydrolysis of nucleoside diand triphosphates by crystalline preparations of yeast inorganic pyrophosphatase. Biochim Biophys Acta. 1960 Jun 17;41:30–36. doi: 10.1016/0006-3002(60)90365-6. [DOI] [PubMed] [Google Scholar]
  12. TAUSSKY H. H., SHORR E. A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem. 1953 Jun;202(2):675–685. [PubMed] [Google Scholar]
  13. Takagahara I., Yamauti J., Fujii K., Yamashita J., Horio T. Theoretical and experimental analyses of coupled enzyme reactions. J Biochem. 1983 Apr;93(4):1145–1157. doi: 10.1093/oxfordjournals.jbchem.a134240. [DOI] [PubMed] [Google Scholar]
  14. de Groot H., Haas W. O2-independent damage of cytochrome P450 by CCl4-metabolites in hepatic microsomes. FEBS Lett. 1980 Jun 30;115(2):253–256. doi: 10.1016/0014-5793(80)81180-x. [DOI] [PubMed] [Google Scholar]
  15. de Groot H., Noll T. Halothane-induced lipid peroxidation and glucose-6-phosphatase inactivation in microsomes under hypoxic conditions. Anesthesiology. 1985 Jan;62(1):44–48. doi: 10.1097/00000542-198501000-00009. [DOI] [PubMed] [Google Scholar]

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