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. 1975 Apr;147(1):111–118. doi: 10.1042/bj1470111

Properties of inorganic pyrophosphatase of pig scapula cartilage.

R Felix, H Fleisch
PMCID: PMC1165380  PMID: 239696

Abstract

The properties of a highly purified inorganic pyrophosphatase (pyrophosphate phosphohydrolase; EC 3.6.1.1) from pig scapula cartilage were studied. The enzyme had a molecular weight of 66 000 and a pH optimum of 7-8. It was markedly activated by magnesium, but not, or only to a much smaller degree, by other metal ions. PP1 was the only substrate found and had a Km value of 11 muM. The enzyme was not inhibited by phosphate and other inhibitors of alkaline phosphatase such as CN- minus, amino acids and theophylline; it was slightly inhibited by tartrate, formaldehyde and ammonium molybdate and strongly inhibited by F- minus, Ca2+ and other metal ions. The properties of the enzyme in the presence of concentrations of PP1 present in plasma (3.5 muM) were similar to those found at higher (2 mM) concentrations of PP1. The diphosphonates ethane-1-hydroxy-1,1-diphosphonate and dichloromethylenediphosphonate inhibited the enzyme in the presence of low PP1 concentrations. The characteristics of this enzyme are therefore similar to pyrophosphatases from other sources, such as from yeast and erythrocytes, and do not support a specific role of this enzyme in the calcification process.

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Selected References

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  1. Alcock N. W. Calcification of cartilage. Clin Orthop Relat Res. 1972 Jul-Aug;86:287–311. doi: 10.1097/00003086-197207000-00040. [DOI] [PubMed] [Google Scholar]
  2. Alcock N. W., Shils M. E. Association of inorganic pyrophosphatase activity with normal calcification of rat costal cartilage in vivo. Biochem J. 1969 May;112(4):505–510. doi: 10.1042/bj1120505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson H. C. Vesicles associated with calcification in the matrix of epiphyseal cartilage. J Cell Biol. 1969 Apr;41(1):59–72. doi: 10.1083/jcb.41.1.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Andrews P. The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem J. 1965 Sep;96(3):595–606. doi: 10.1042/bj0960595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brunel C., Cathala G. Imidazole: an inhibitor of L-phenylalanine-insensitive alkaline phosphatases of tissues other than intestine and placenta. Biochim Biophys Acta. 1972 May 12;268(2):415–421. doi: 10.1016/0005-2744(72)90337-3. [DOI] [PubMed] [Google Scholar]
  6. Burton P. M., Josse J. Constitutive inorganic pyrophosphatase of Escherichia coli. VI. Inactivation by chemical modification of lysine residues. J Biol Chem. 1970 Sep 10;245(17):4358–4364. [PubMed] [Google Scholar]
  7. Chersi A., Bernardi A., Bernardi G. Studies on acid hydrolases. II. Isolation and properties of spleen acid phosphomonoesterase. Biochim Biophys Acta. 1966 Oct 24;129(1):12–22. [PubMed] [Google Scholar]
  8. Cooperman B. S., Chiu N. Y. Yeast inorganic pyrophosphatase. 3. Active-site mapping by electrophilic reagents and binding measurements. Biochemistry. 1973 Apr 24;12(9):1676–1682. doi: 10.1021/bi00733a003. [DOI] [PubMed] [Google Scholar]
  9. FISHMAN W. H., GREEN S., INGLIS N. I. Organ-specific behavior exhibited by rat intestine and liver alkaline phosphatase. Biochim Biophys Acta. 1962 Aug 13;62:363–375. doi: 10.1016/0006-3002(62)90266-4. [DOI] [PubMed] [Google Scholar]
  10. FLEISH H., NEUMAN W. F. Mechanisms of calcification: role of collagen, polyphosphates, and phosphatase. Am J Physiol. 1961 Jun;200:1296–1300. doi: 10.1152/ajplegacy.1961.200.6.1296. [DOI] [PubMed] [Google Scholar]
  11. Fawaz E. N., Tejirian A. Inhibition of alkaline phosphatase by theophylline in vitro. Hoppe Seylers Z Physiol Chem. 1972 Nov;353(11):1779–1783. doi: 10.1515/bchm2.1972.353.2.1779. [DOI] [PubMed] [Google Scholar]
  12. Felix R., Fleisch H. Purification and heterogeneity of inorganic pyrophosphatase of pig scapula cartilage. Biochem J. 1975 Apr;147(1):103–109. doi: 10.1042/bj1470103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Felix R., Fleisch H. The pyrophosphatase and (Ca2+-Mg2+)-ATPase activity of purified calf bone alkaline phosphatase. Biochim Biophys Acta. 1974 May 20;350(1):84–94. doi: 10.1016/0005-2744(74)90205-8. [DOI] [PubMed] [Google Scholar]
  14. Fernley H. N., Walker P. G. Studies on alkaline phosphatase. Inhibition by phosphate derivatives and the substrate specificity. Biochem J. 1967 Sep;104(3):1011–1018. doi: 10.1042/bj1041011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fishman W. H., Sie H. G. Organ-specific inhibition of human alkaline phosphatase isoenzymes of liver, bone, intestine and placenta; L-phenylalanine, L-tryptophan and L homoarginine. Enzymologia. 1971 Sep 30;41(3):141–167. [PubMed] [Google Scholar]
  16. Fleisch H. A., Russell R. G., Bisaz S., Mühlbauer R. C., Williams D. A. The inhibitory effect of phosphonates on the formation of calcium phosphate crystals in vitro and on aortic and kidney calcification in vivo. Eur J Clin Invest. 1970 Mar;1(1):12–18. doi: 10.1111/j.1365-2362.1970.tb00591.x. [DOI] [PubMed] [Google Scholar]
  17. HALL R. J. An improved method for the micro-determination of inorganic phosphate in small volumes of biological fluids. J Med Lab Technol. 1963 Apr;20:97–103. [PubMed] [Google Scholar]
  18. Hansen G., Eifler R., Heitmann P. The subunit structure of inorganic pyrophosphatase from baker's yeast. Acta Biol Med Ger. 1972;28(6):977–987. [PubMed] [Google Scholar]
  19. Howell D. S., Pita J. C., Marquez J. F., Madruga J. E. Partition of calcium, phosphate, and protein in the fluid phase aspirated at calcifying sites in epiphyseal cartilage. J Clin Invest. 1968 May;47(5):1121–1132. doi: 10.1172/JCI105801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Höhne W. E., Rapoport T. A. Slow conformational changes of the inorganic pyrophosphatase from bakers' yeast induced by divalent metal ions. Eur J Biochem. 1973 Mar 1;33(2):323–331. doi: 10.1111/j.1432-1033.1973.tb02686.x. [DOI] [PubMed] [Google Scholar]
  21. Irie M., Yabuta A., Kimura K., Shindo Y., Tomita K. Distribution and properties of alkaline pyrophosphatases of rat liver. J Biochem. 1970 Jan;67(1):47–58. doi: 10.1093/oxfordjournals.jbchem.a129233. [DOI] [PubMed] [Google Scholar]
  22. Josse J. Constitutive inorganic pyrophosphatase of Escherichia coli. 1. Purification and catalytic properties. J Biol Chem. 1966 May 10;241(9):1938–1947. [PubMed] [Google Scholar]
  23. Josse J. Constitutive inorganic pyrophosphatase of Escherichia coli. II. Nature and binding of active substrate and the role of magnesium. J Biol Chem. 1966 May 10;241(9):1948–1955. [PubMed] [Google Scholar]
  24. KUHLMAN R. E. PHOSPHATASES IN EPIPHYSEAL CARTILAGE; THEIR POSSIBLE ROLE IN TISSUE SYNTHESIS. J Bone Joint Surg Am. 1965 Apr;47:545–550. [PubMed] [Google Scholar]
  25. KUNITZ M. Crystalline inorganic pyrophosphatase isolated from baker's yeast. J Gen Physiol. 1952 Jan;35(3):423–450. doi: 10.1085/jgp.35.3.423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Moe O. A., Butler L. G. Yeast inorganic pyrophosphatase. 3. Kinetics of Ca 2+ inhibition. J Biol Chem. 1972 Nov 25;247(22):7315–7319. [PubMed] [Google Scholar]
  27. Moe O. A., Butler L. G. Yeast inorganic pyrophosphatase. II. Kinetics of Mg 2+ activation. J Biol Chem. 1972 Nov 25;247(22):7308–7314. [PubMed] [Google Scholar]
  28. NORDLIE R. C., ARION W. J. EVIDENCE FOR THE COMMON IDENTITY OF GLUCOSE 6-PHOSPHATASE, INORGANIC PYROPHOSPHATASE, AND PYROPHOSPHATE-GLUCOSE PHOSPHOTRANSFERASE. J Biol Chem. 1964 Jun;239:1680–1685. [PubMed] [Google Scholar]
  29. Negi T., Samejima T., Irie M. Studies on the tryptophan residues of yeast inorganic pyrophosphatase in relation to the enzymatic activity. J Biochem. 1972 Jan;71(1):29–37. doi: 10.1093/oxfordjournals.jbchem.a129743. [DOI] [PubMed] [Google Scholar]
  30. Pynes G. D., Younathan E. S. Purification and some properties of inorganic pyrophosphatase from human erythrocytes. J Biol Chem. 1967 May 10;242(9):2119–2123. [PubMed] [Google Scholar]
  31. REINER J. M., TSUBOI K. K., HUDSON P. B. Acid phosphatase. IV. Fluoride inhibition of prostatic acid phosphatase. Arch Biochem Biophys. 1955 May;56(1):165–183. doi: 10.1016/0003-9861(55)90346-5. [DOI] [PubMed] [Google Scholar]
  32. Rapoport T. A., Höhne W. E., Heitmann P., Rapoport S. Binding of ligands to the inorganic pyrophosphatase of bakers' yeast. Eur J Biochem. 1973 Mar 1;33(2):341–347. doi: 10.1111/j.1432-1033.1973.tb02688.x. [DOI] [PubMed] [Google Scholar]
  33. Rapoport T. A., Höhne W. E., Reich J. G., Heitmann P., Rapoport S. M. A kinetic model for the action of the inorganic pyrophosphatase from bakers' yeast. The activating influence of magnesium ions. Eur J Biochem. 1972 Mar 27;26(2):237–246. doi: 10.1111/j.1432-1033.1972.tb01761.x. [DOI] [PubMed] [Google Scholar]
  34. Ridlington J. W., Butler L. G. Yeast inorganic pyrophosphatase. I. Binding of pyrophosphate, metal ion, and metal ion-pyrophosphate complexes. J Biol Chem. 1972 Nov 25;247(22):7303–7307. [PubMed] [Google Scholar]
  35. Russell R. G., Bisaz S., Donath A., Morgan D. B., Fleisch H. Inorganic pyrophosphate in plasma in normal persons and in patients with hypophosphatasia, osteogenesis imperfecta, and other disorders of bone. J Clin Invest. 1971 May;50(5):961–969. doi: 10.1172/JCI106589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Russell R. G., Mühlbauer R. C., Bisaz S., Williams D. A., Fleisch H. The influence of pyrophosphate, condensed phosphates, phosphonates and other phosphate compounds on the dissolution of hydroxyapatite in vitro and on bone resorption induced by parathyroid hormone in tissue culture and in thyroparathyroidectomised rats. Calcif Tissue Res. 1970;6(3):183–196. doi: 10.1007/BF02196199. [DOI] [PubMed] [Google Scholar]
  37. SCHACHMAN H. K. Ultracentrifuge, electrophoresis, and viscosity studies on crystalline pyrophosphatase. J Gen Physiol. 1952 Jan;35(3):451–454. doi: 10.1085/jgp.35.3.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sperow J. W., Moe O. A., Ridlington J. W., Butler L. G. Yeast inorganic pyrophosphatase. VI. Studies on specificity and mechanism. J Biol Chem. 1973 Mar 25;248(6):2062–2065. [PubMed] [Google Scholar]
  39. Tono H., Kornberg A. Biochemical studies of bacterial sporulation. 3. Inorganic pyrophosphatase of vegetative cells and spores of Bacillus subtilis. J Biol Chem. 1967 May 25;242(10):2375–2382. [PubMed] [Google Scholar]
  40. Wong S. C., Hall D. C., Josse J. Constitutive inorganic pyrophosphatase of Escherichia coli. 3. Molecular weight and physical properties of the enzyme and its subunits. J Biol Chem. 1970 Sep 10;245(17):4335–4345. [PubMed] [Google Scholar]
  41. Wöltgens J., Ahsmann W. Determination of orthophosphate in the presence of inorganic pyrophosphate in the assay of inorganic pyrophosphatase activity. Anal Biochem. 1970 Jun;35(2):526–529. doi: 10.1016/0003-2697(70)90217-4. [DOI] [PubMed] [Google Scholar]

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