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Recombinant Human and Mouse Purple Acid Phosphatases: Expression and Characterization

https://doi.org/10.1006/abbi.1997.0250Get rights and content

Abstract

The mammalian purple acid phosphatases (also called tartrate-resistant acid phosphatases) are expressed primarily in actively resorbing osteoclasts and activated macrophages. The enzymes are characterized by the presence of a binuclear iron center at the active site. Recent studies on transgenic mice lacking purple acid phosphatase implicate the osteoclast enzyme in both bone resorption and bone mineralization. To characterize the mammalian enzymes in more detail, particularly with respect to their substrate specificity at the low pH of the osteoclastic resorptive space (2.5–3), we have purified the recombinant human and mouse enzymes from baculovirus-infected insect cells. The properties of the recombinant mouse enzyme are compared with those of the nonrecombinant enzyme isolated from mouse spleen. The kinetics of hydrolysis of the substratesp-nitrophenyl phosphate, phosphotyrosine, and pyrophosphate and a phosphotyrosyl peptide by the recombinant human and mouse enzymes and the nonrecombinant mouse and pig enzymes were analyzed. For all the enzymes the ratiokcat/Kmwas typically ∼106m−1s−1and was higher at pH 2.5 than at 4.9. The increase was attributable to a large decrease inKmat the lower pH value. The results indicate that the enzyme exhibits high catalytic efficiency toward substrates such as pyrophosphate and acidic phosphotyrosine-containing peptides, particularly at low pH values typical of the bone resorptive space. The implications of the results for the physiological function of the enzyme are discussed.

References (39)

  • C.J. Wynne et al.

    Arch. Biochem. Biophys.

    (1995)
  • K. Nash et al.

    Anal. Biochem.

    (1993)
  • J. Sibille et al.

    J. Biol. Chem.

    (1987)
  • A.R. Hayman et al.

    J. Biol. Chem.

    (1994)
  • B. Ek-Rylander et al.

    J. Biol. Chem.

    (1994)
  • H.D. Campbell et al.

    Biochem. Biophys. Res. Commun.

    (1978)
  • H.D. Campbell et al.

    Biochem. Biophys. Res. Commun.

    (1973)
  • C.M. Ketcham et al.

    J. Biol. Chem.

    (1985)
  • A.J. Janckila et al.

    Clin. Biochem.

    (1992)
  • J.J. Stepán et al.

    Biochem. Biophys. Res. Commun.

    (1990)
  • A.I. Cassady et al.

    Gene

    (1993)
  • J.V. Frangioni et al.

    Anal. Biochem.

    (1993)
  • J.B. Shatton et al.

    Anal. Biochem.

    (1983)
  • J.L. Beck et al.

    Biochim. Biophys. Acta

    (1984)
  • T. Klabunde et al.

    FEBS Lett.

    (1995)
  • M.E. Flores et al.

    Exp. Cell Res.

    (1992)
  • A.R. Hayman et al.

    Biochem. J.

    (1989)
  • H.J. Radzun et al.

    Hematol. Oncol.

    (1983)
  • J. Schindelmeiser et al.

    Histochemistry

    (1987)
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    T. S. WorkE. Work, Eds.

    1

    To whom correspondence should be addressed at Department of Biochemistry, University of Queensland, St. Lucia, Queensland 4072, Australia. Fax: 61-7-33654699.

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