Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Jan 15;321(Pt 2):305–311. doi: 10.1042/bj3210305

Comparative studies of rat recombinant purple acid phosphatase and bone tartrate-resistant acid phosphatase.

B Ek-Rylander 1, T Barkhem 1, J Ljusberg 1, L Ohman 1, K K Andersson 1, G Andersson 1
PMCID: PMC1218069  PMID: 9020859

Abstract

The tartrate-resistant acid phosphatase (TRAP) of rat osteoclasts has been shown to exhibit high (85-94%) identity at the amino acid sequence level with the purple acid phosphatase (PAP) from bovine spleen and with pig uteroferrin. These iron-containing purple enzymes contain a binuclear iron centre, with a tyrosinate-to-Fe(III) charge-transfer transition responsible for the purple colour. In the present study, production of rat osteoclast TRAP could be achieved at a level of 4.3 mg/litre of medium using a baculovirus expression system. The enzyme was purified to apparent homogeneity using a combination of cation-exchange, hydrophobic-interaction, lectin-affinity and gel-permeation chromatography steps. The protein as isolated had a purple colour, a specific activity of 428 units/mg of protein and consisted of the single-chain form of molecular mass 34 kDa, with only trace amounts of proteolytically derived subunits. The recombinant enzyme had the ability to dephosphorylate bone matrix phosphoproteins, as previously shown for bone TRAP. Light absorption spectroscopy of the isolated purple enzyme showed a lambda max at 544 nm, which upon reduction with ascorbic acid changed to 515 nm, concomitant with the transition to a pink colour. EPR spectroscopic analysis of the reduced enzyme at 3.6 K revealed a typical mu-hydr(oxo)-bridged mixed-valent Fe(II)Fe(III) signal with g-values at 1.96, 1.74 and 1.60, proving that recombinant rat TRAP belongs to the family of PAPs. To validate the use of recombinant PAP in substituting for the rat bone counterpart in functional studies, various comparative studies were carried out. The enzyme isolated from bone exhibited a lower K(m) for p-nitrophenyl phosphate and was slightly more sensitive to PAP inhibitors such as molybdate, tungstate, arsenate and phosphate. In contrast with the recombinant enzyme, TRAP from bone was isolated predominantly as the proteolytically cleaved, two-subunit, form. Both the recombinant enzyme and rat bone TRAP were shown to be substituted with N-linked oligosaccharides. A slightly higher apparent molecular mass of the monomeric form and N-terminal chain of bone TRAP compared with the recombinant enzyme could not be accounted for by differential N-glycosylation. Despite differences in specific post-translational modifications, the recombinant PAP should be useful in future studies on the properties and regulation of the mammalian PAP enzyme.

Full Text

The Full Text of this article is available as a PDF (374.7 KB).

Selected References

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

  1. Andersson G., Ek-Rylander B., Hammarström L. Purification and characterization of a vanadate-sensitive nucleotide tri- and diphosphatase with acid pH optimum from rat bone. Arch Biochem Biophys. 1984 Feb 1;228(2):431–438. doi: 10.1016/0003-9861(84)90007-9. [DOI] [PubMed] [Google Scholar]
  2. Antanaitis B. C., Aisen P. Detection of a g' = 1.74 EPR signal in bovine spleen purple acid phosphatase. J Biol Chem. 1982 May 25;257(10):5330–5332. [PubMed] [Google Scholar]
  3. Antanaitis B. C., Aisen P. Effects of perturbants on the pink (reduced) active form of uteroferrin. Phosphate-induced anaerobic oxidation. J Biol Chem. 1985 Jan 25;260(2):751–756. [PubMed] [Google Scholar]
  4. Antanaitis B. C., Aisen P., Lilienthal H. R. Physical characterization of two-iron uteroferrin. Evidence for a spin-coupled binuclear iron cluster. J Biol Chem. 1983 Mar 10;258(5):3166–3172. [PubMed] [Google Scholar]
  5. Baumbach G. A., Ketcham C. M., Richardson D. E., Bazer F. W., Roberts R. M. Isolation and characterization of a high molecular weight stable pink form of uteroferrin from uterine secretions and allantoic fluid of pigs. J Biol Chem. 1986 Sep 25;261(27):12869–12878. [PubMed] [Google Scholar]
  6. Bazer F. W., Chen T. T., Knight J. W., Schlosnagle D., Baldwin N. J., Roberts R. M. Presence of a progesterone-induced, uterine specific, acid phosphatase in allantoic fluid of gilts. J Anim Sci. 1975 Oct;41(4):1112–1119. doi: 10.2527/jas1975.4141112x. [DOI] [PubMed] [Google Scholar]
  7. Bensadoun A., Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem. 1976 Jan;70(1):241–250. doi: 10.1016/s0003-2697(76)80064-4. [DOI] [PubMed] [Google Scholar]
  8. Buhi W. C., Gray W. J., Mansfield E. A., Chun P. W., Ducsay C. A., Bazer F. W., Roberts R. M. Iron content and molecular weight of uteroferrin and a comparison of its iron and copper forms. Biochim Biophys Acta. 1982 Feb 4;701(1):32–38. doi: 10.1016/0167-4838(82)90308-9. [DOI] [PubMed] [Google Scholar]
  9. Burman S., Davis J. C., Weber M. J., Averill B. A. The interaction of phosphate with the purple acid phosphatase from beef spleen: evidence that phosphate binding is accompanied by oxidation of the iron chromophore. Biochem Biophys Res Commun. 1986 Apr 29;136(2):490–497. doi: 10.1016/0006-291x(86)90467-5. [DOI] [PubMed] [Google Scholar]
  10. Campbell H. D., Dionysius D. A., Keough D. T., Wilson B. E., de Jersey J., Zerner B. Iron-containing acid phosphatases: comparison of the enzymes from beef spleen and pig allantoic fluid. Biochem Biophys Res Commun. 1978 May 30;82(2):615–620. doi: 10.1016/0006-291x(78)90919-1. [DOI] [PubMed] [Google Scholar]
  11. Davis J. C., Lin S. S., Averill B. A. Kinetics and optical spectroscopic studies on the purple acid phosphatase from beef spleen. Biochemistry. 1981 Jul 7;20(14):4062–4067. doi: 10.1021/bi00517a018. [DOI] [PubMed] [Google Scholar]
  12. Dietrich M., Münstermann D., Suerbaum H., Witzel H. Purple acid phosphatase from bovine spleen. Interactions at the active site in relation to the reaction mechanism. Eur J Biochem. 1991 Jul 1;199(1):105–113. doi: 10.1111/j.1432-1033.1991.tb16097.x. [DOI] [PubMed] [Google Scholar]
  13. Drexler H. G., Gignac S. M. Characterization and expression of tartrate-resistant acid phosphatase (TRAP) in hematopoietic cells. Leukemia. 1994 Mar;8(3):359–368. [PubMed] [Google Scholar]
  14. Ek-Rylander B., Bergman T., Andersson G. Characterization of a tartrate-resistant acid phosphatase (ATPase) from rat bone: hydrodynamic properties and N-terminal amino acid sequence. J Bone Miner Res. 1991 Apr;6(4):365–373. doi: 10.1002/jbmr.5650060408. [DOI] [PubMed] [Google Scholar]
  15. Ek-Rylander B., Bill P., Norgård M., Nilsson S., Andersson G. Cloning, sequence, and developmental expression of a type 5, tartrate-resistant, acid phosphatase of rat bone. J Biol Chem. 1991 Dec 25;266(36):24684–24689. [PubMed] [Google Scholar]
  16. Ek-Rylander B., Flores M., Wendel M., Heinegård D., Andersson G. Dephosphorylation of osteopontin and bone sialoprotein by osteoclastic tartrate-resistant acid phosphatase. Modulation of osteoclast adhesion in vitro. J Biol Chem. 1994 May 27;269(21):14853–14856. [PubMed] [Google Scholar]
  17. Flores M. E., Norgård M., Heinegård D., Reinholt F. P., Andersson G. RGD-directed attachment of isolated rat osteoclasts to osteopontin, bone sialoprotein, and fibronectin. Exp Cell Res. 1992 Aug;201(2):526–530. doi: 10.1016/0014-4827(92)90305-r. [DOI] [PubMed] [Google Scholar]
  18. Goldberg J., Huang H. B., Kwon Y. G., Greengard P., Nairn A. C., Kuriyan J. Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-1. Nature. 1995 Aug 31;376(6543):745–753. doi: 10.1038/376745a0. [DOI] [PubMed] [Google Scholar]
  19. Griffith J. P., Kim J. L., Kim E. E., Sintchak M. D., Thomson J. A., Fitzgibbon M. J., Fleming M. A., Caron P. R., Hsiao K., Navia M. A. X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex. Cell. 1995 Aug 11;82(3):507–522. doi: 10.1016/0092-8674(95)90439-5. [DOI] [PubMed] [Google Scholar]
  20. Grimes R., Reddy S. V., Leach R. J., Scarcez T., Roodman G. D., Sakaguchi A. Y., Lalley P. A., Windle J. J. Assignment of the mouse tartrate-resistant acid phosphatase gene (Acp5) to chromosome 9. Genomics. 1993 Feb;15(2):421–422. doi: 10.1006/geno.1993.1079. [DOI] [PubMed] [Google Scholar]
  21. Hayman A. R., Cox T. M. Purple acid phosphatase of the human macrophage and osteoclast. Characterization, molecular properties, and crystallization of the recombinant di-iron-oxo protein secreted by baculovirus-infected insect cells. J Biol Chem. 1994 Jan 14;269(2):1294–1300. [PubMed] [Google Scholar]
  22. Keough D. T., Beck J. L., de Jersey J., Zerner B. Iron-containing acid phosphatases: interaction of phosphate with the enzyme from pig allantoic fluid. Biochem Biophys Res Commun. 1982 Oct 29;108(4):1643–1648. doi: 10.1016/s0006-291x(82)80098-3. [DOI] [PubMed] [Google Scholar]
  23. Klabunde T., Sträter N., Krebs B., Witzel H. Structural relationship between the mammalian Fe(III)-Fe(II) and the Fe(III)-Zn(II) plant purple acid phosphatases. FEBS Lett. 1995 Jun 19;367(1):56–60. doi: 10.1016/0014-5793(95)00536-i. [DOI] [PubMed] [Google Scholar]
  24. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  25. Leach R. J., Reus B. E., Hundley J. E., Johnson-Pais T. L., Windle J. J. Confirmation of the assignment of the human tartrate-resistant acid phosphatase gene (ACP5) to chromosome 19. Genomics. 1994 Jan 1;19(1):180–181. doi: 10.1006/geno.1994.1037. [DOI] [PubMed] [Google Scholar]
  26. Ling P., Roberts R. M. Uteroferrin and intracellular tartrate-resistant acid phosphatases are the products of the same gene. J Biol Chem. 1993 Apr 5;268(10):6896–6902. [PubMed] [Google Scholar]
  27. Miyauchi A., Alvarez J., Greenfield E. M., Teti A., Grano M., Colucci S., Zambonin-Zallone A., Ross F. P., Teitelbaum S. L., Cheresh D. Recognition of osteopontin and related peptides by an alpha v beta 3 integrin stimulates immediate cell signals in osteoclasts. J Biol Chem. 1991 Oct 25;266(30):20369–20374. [PubMed] [Google Scholar]
  28. Orlando J. L., Zirino T., Quirk B. J., Averill B. A. Purification and properties of the native form of the purple acid phosphatase from bovine spleen. Biochemistry. 1993 Aug 17;32(32):8120–8129. doi: 10.1021/bi00083a010. [DOI] [PubMed] [Google Scholar]
  29. Pyrz J. W., Sage J. T., Debrunner P. G., Que L., Jr The interaction of phosphate with uteroferrin. Characterization of a reduced uteroferrin-phosphate complex. J Biol Chem. 1986 Aug 25;261(24):11015–11020. [PubMed] [Google Scholar]
  30. Roberts R. M., Bazer F. W. Phosphoprotein phosphatase activity of the progesterone-induced purple glycoprotein of the porcine uterus. Biochem Biophys Res Commun. 1976 Jan 26;68(2):450–455. doi: 10.1016/0006-291x(76)91166-9. [DOI] [PubMed] [Google Scholar]
  31. Schindelmeiser J., Münstermann D., Witzel H. Histochemical investigations on the localization of the purple acid phosphatase in the bovine spleen. Histochemistry. 1987;87(1):13–19. doi: 10.1007/BF00518719. [DOI] [PubMed] [Google Scholar]
  32. Schlosnagle D. C., Bazer F. W., Tsibris J. C., Roberts R. M. An iron-containing phosphatase induced by progesterone in the uterine fluids of pigs. J Biol Chem. 1974 Dec 10;249(23):7574–7579. [PubMed] [Google Scholar]
  33. Sträter N., Klabunde T., Tucker P., Witzel H., Krebs B. Crystal structure of a purple acid phosphatase containing a dinuclear Fe(III)-Zn(II) active site. Science. 1995 Jun 9;268(5216):1489–1492. doi: 10.1126/science.7770774. [DOI] [PubMed] [Google Scholar]
  34. Sørensen E. S., Højrup P., Petersen T. E. Posttranslational modifications of bovine osteopontin: identification of twenty-eight phosphorylation and three O-glycosylation sites. Protein Sci. 1995 Oct;4(10):2040–2049. doi: 10.1002/pro.5560041009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sørensen E. S., Petersen T. E. Identification of two phosphorylation motifs in bovine osteopontin. Biochem Biophys Res Commun. 1994 Jan 14;198(1):200–205. doi: 10.1006/bbrc.1994.1028. [DOI] [PubMed] [Google Scholar]
  36. Tarentino A. L., Plummer T. H., Jr, Maley F. The release of intact oligosaccharides from specific glycoproteins by endo-beta-N-acetylglucosaminidase H. J Biol Chem. 1974 Feb 10;249(3):818–824. [PubMed] [Google Scholar]
  37. Vincent J. B., Crowder M. W., Averill B. A. Spectroscopic and kinetics studies of a high-salt-stabilized form of the purple acid phosphatase from bovine spleen. Biochemistry. 1991 Mar 26;30(12):3025–3034. doi: 10.1021/bi00226a007. [DOI] [PubMed] [Google Scholar]
  38. Wang Z., Ming L. J., Que L., Jr, Vincent J. B., Crowder M. W., Averill B. A. 1H NMR and NOE studies of the purple acid phosphatases from porcine uterus and bovine spleen. Biochemistry. 1992 Jun 16;31(23):5263–5268. doi: 10.1021/bi00138a004. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES