Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2004 Jan 15;377(Pt 2):419–428. doi: 10.1042/BJ20030947

Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures.

Gale G Bozzo 1, Kashchandra G Raghothama 1, William C Plaxton 1
PMCID: PMC1223867  PMID: 14521509

Abstract

An intracellular acid phosphatase (IAP) from P(i)-starved (-P(i)) tomato ( Lycopersicon esculentum ) suspension cells has been purified to homogeneity. IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (lambda(max)=546 nm) and was insensitive to L-tartrate. PAGE, periodic acid-Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (alpha-subunit) and 57 kDa (beta-subunit) polypeptides. However, the nine N-terminal amino acids of the alpha- and beta-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs. Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to P(i)-deprivation was correlated with similar increases in the amount of antigenic IAP alpha- and beta-subunits. IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg(2+), but was potently inhibited by Zn(2+), Cu(2+), Fe(3+), molybdate, vanadate, fluoride and P(i). Although IAP demonstrated broad substrate selectivity, its specificity constant ( V (max)/ K (m)) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate. IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg(2+) or molybdate. This IAP is proposed to scavenge P(i) from intracellular phosphate esters in -P(i) tomato. A possible secondary IAP role in the metabolism of reactive oxygen species is discussed. IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of -P(i) tomato cells [Bozzo, Raghothama and Plaxton (2002) Eur. J. Biochem. 269, 6278-6286].

Full Text

The Full Text of this article is available as a PDF (185.1 KB).

Selected References

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

  1. Baldwin J. C., Karthikeyan A. S., Raghothama K. G. LEPS2, a phosphorus starvation-induced novel acid phosphatase from tomato. Plant Physiol. 2001 Feb;125(2):728–737. doi: 10.1104/pp.125.2.728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bozzo Gale G., Raghothama Kashchandra G., Plaxton William C. Purification and characterization of two secreted purple acid phosphatase isozymes from phosphate-starved tomato (Lycopersicon esculentum) cell cultures. Eur J Biochem. 2002 Dec;269(24):6278–6286. doi: 10.1046/j.1432-1033.2002.03347.x. [DOI] [PubMed] [Google Scholar]
  3. Brooks S. P. A simple computer program with statistical tests for the analysis of enzyme kinetics. Biotechniques. 1992 Dec;13(6):906–911. [PubMed] [Google Scholar]
  4. Carswell M. C., Grant B. R., Plaxton W. C. Disruption of the phosphate-starvation response of oilseed rape suspension cells by the fungicide phosphonate. Planta. 1997 Sep;203(1):67–74. doi: 10.1007/s00050166. [DOI] [PubMed] [Google Scholar]
  5. Chrambach A., Rodbard D. Polyacrylamide gel electrophoresis. Science. 1971 Apr 30;172(3982):440–451. doi: 10.1126/science.172.3982.440. [DOI] [PubMed] [Google Scholar]
  6. Drueckes P., Schinzel R., Palm D. Photometric microtiter assay of inorganic phosphate in the presence of acid-labile organic phosphates. Anal Biochem. 1995 Sep 1;230(1):173–177. doi: 10.1006/abio.1995.1453. [DOI] [PubMed] [Google Scholar]
  7. Duff S. M., Lefebvre D. D., Plaxton W. C. Purification and Characterization of a Phosphoenolpyruvate Phosphatase from Brassica nigra Suspension Cells. Plant Physiol. 1989 Jun;90(2):734–741. doi: 10.1104/pp.90.2.734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Duff S. M., Plaxton W. C., Lefebvre D. D. Phosphate-starvation response in plant cells: de novo synthesis and degradation of acid phosphatases. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9538–9542. doi: 10.1073/pnas.88.21.9538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Durmus A., Eicken C., Sift B. H., Kratel A., Kappl R., Hüttermann J., Krebs B. The active site of purple acid phosphatase from sweet potatoes (Ipomoea batatas) metal content and spectroscopic characterization. Eur J Biochem. 1999 Mar;260(3):709–716. doi: 10.1046/j.1432-1327.1999.00230.x. [DOI] [PubMed] [Google Scholar]
  10. Gellatly K. S., Moorhead GBG., Duff SMG., Lefebvre D. D., Plaxton W. C. Purification and Characterization of a Potato Tuber Acid Phosphatase Having Significant Phosphotyrosine Phosphatase Activity. Plant Physiol. 1994 Sep;106(1):223–232. doi: 10.1104/pp.106.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hegeman C. E., Grabau E. A. A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings. Plant Physiol. 2001 Aug;126(4):1598–1608. doi: 10.1104/pp.126.4.1598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kaija Helena, Alatalo Sari L., Halleen Jussi M., Lindqvist Ylva, Schneider Gunter, Vänänen H. Kalervo, Vihko Pirkko. Phosphatase and oxygen radical-generating activities of mammalian purple acid phosphatase are functionally independent. Biochem Biophys Res Commun. 2002 Mar 22;292(1):128–132. doi: 10.1006/bbrc.2002.6615. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Laine A. C., Faye L. Significant immunological cross-reactivity of plant glycoproteins. Electrophoresis. 1988 Dec;9(12):841–844. doi: 10.1002/elps.1150091210. [DOI] [PubMed] [Google Scholar]
  15. Lebansky B. R., McKnight T. D., Griffing L. R. Purification and characterization of a secreted purple phosphatase from soybean suspension cultures. Plant Physiol. 1992 Jun;99(2):391–395. doi: 10.1104/pp.99.2.391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Li Dongping, Zhu Huifen, Liu Kunfan, Liu Xin, Leggewie Georg, Udvardi Michael, Wang Daowen. Purple acid phosphatases of Arabidopsis thaliana. Comparative analysis and differential regulation by phosphate deprivation. J Biol Chem. 2002 May 20;277(31):27772–27781. doi: 10.1074/jbc.M204183200. [DOI] [PubMed] [Google Scholar]
  17. Lindqvist Y., Schneider G., Vihko P. Crystal structures of rat acid phosphatase complexed with the transition-state analogs vanadate and molybdate. Implications for the reaction mechanism. Eur J Biochem. 1994 Apr 1;221(1):139–142. doi: 10.1111/j.1432-1033.1994.tb18722.x. [DOI] [PubMed] [Google Scholar]
  18. Mildner P., Barbarić S., Golubić Z., Ries B. Purification of protoplast-secreted acid phosphatase from baker's yeast. Effect on adenosine triphosphatase activity. Biochim Biophys Acta. 1976 Mar 11;429(1):274–282. doi: 10.1016/0005-2744(76)90050-4. [DOI] [PubMed] [Google Scholar]
  19. Moorhead G. B., Plaxton W. C. Purification and characterization of cytosolic aldolase from carrot storage root. Biochem J. 1990 Jul 1;269(1):133–139. doi: 10.1042/bj2690133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Moraes T. F., Plaxton W. C. Purification and characterization of phosphoenolpyruvate carboxylase from Brassica napus (rapeseed) suspension cell cultures: implications for phosphoenolpyruvate carboxylase regulation during phosphate starvation, and the integration of glycolysis with nitrogen assimilation. Eur J Biochem. 2000 Jul;267(14):4465–4476. doi: 10.1046/j.1432-1327.2000.01495.x. [DOI] [PubMed] [Google Scholar]
  21. Nakazato H., Okamoto T., Nishikoori M., Washio K., Morita N., Haraguchi K., Thompson G. A., Jr, Okuyama H. The glycosylphosphatidylinositol-anchored phosphatase from Spirodela oligorrhiza is a purple acid phosphatase. Plant Physiol. 1998 Nov;118(3):1015–1020. doi: 10.1104/pp.118.3.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Olczak Mariusz, Olczak Teresa. Diphosphonucleotide phosphatase/phosphodiesterase from yellow lupin (Lupinus luteus L.) belongs to a novel group of specific metallophosphatases. FEBS Lett. 2002 May 22;519(1-3):159–163. doi: 10.1016/s0014-5793(02)02740-0. [DOI] [PubMed] [Google Scholar]
  23. Pinkse M. W., Merkx M., Averill B. A. Fluoride inhibition of bovine spleen purple acid phosphatase: characterization of a ternary enzyme-phosphate-fluoride complex as a model for the active enzyme-substrate-hydroxide complex. Biochemistry. 1999 Aug 3;38(31):9926–9936. doi: 10.1021/bi990446w. [DOI] [PubMed] [Google Scholar]
  24. Plaxton W. C., Moorhead G. B. Peptide mapping by CNBr fragmentation using a sodium dodecyl sulfate-polyacrylamide minigel system. Anal Biochem. 1989 May 1;178(2):391–393. doi: 10.1016/0003-2697(89)90658-1. [DOI] [PubMed] [Google Scholar]
  25. Raghothama K. G. PHOSPHATE ACQUISITION. Annu Rev Plant Physiol Plant Mol Biol. 1999 Jun;50(NaN):665–693. doi: 10.1146/annurev.arplant.50.1.665. [DOI] [PubMed] [Google Scholar]
  26. Schenk G., Ge Y., Carrington L. E., Wynne C. J., Searle I. R., Carroll B. J., Hamilton S., de Jersey J. Binuclear metal centers in plant purple acid phosphatases: Fe-Mn in sweet potato and Fe-Zn in soybean. Arch Biochem Biophys. 1999 Oct 15;370(2):183–189. doi: 10.1006/abbi.1999.1407. [DOI] [PubMed] [Google Scholar]
  27. Schenk G., Guddat L. W., Ge Y., Carrington L. E., Hume D. A., Hamilton S., de Jersey J. Identification of mammalian-like purple acid phosphatases in a wide range of plants. Gene. 2000 May 30;250(1-2):117–125. doi: 10.1016/s0378-1119(00)00186-4. [DOI] [PubMed] [Google Scholar]
  28. Thomas Howard, Ougham Helen J., Wagstaff Carol, Stead Anthony D. Defining senescence and death. J Exp Bot. 2003 Apr;54(385):1127–1132. doi: 10.1093/jxb/erg133. [DOI] [PubMed] [Google Scholar]
  29. Tiwari Budhi Sagar, Belenghi Beatrice, Levine Alex. Oxidative stress increased respiration and generation of reactive oxygen species, resulting in ATP depletion, opening of mitochondrial permeability transition, and programmed cell death. Plant Physiol. 2002 Apr;128(4):1271–1281. doi: 10.1104/pp.010999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Turner W. L., Plaxton W. C. Purification and characterization of banana fruit acid phosphatase. Planta. 2001 Dec;214(2):243–249. doi: 10.1007/s004250100607. [DOI] [PubMed] [Google Scholar]
  31. Vincent J. B., Crowder M. W., Averill B. A. Hydrolysis of phosphate monoesters: a biological problem with multiple chemical solutions. Trends Biochem Sci. 1992 Mar;17(3):105–110. doi: 10.1016/0968-0004(92)90246-6. [DOI] [PubMed] [Google Scholar]
  32. Vincent J. B., Crowder M. W., Averill B. A. Multiple binding sites for tetrahedral oxyanion inhibitors of bovine spleen purple acid phosphatase. Biochemistry. 1992 Mar 31;31(12):3033–3037. doi: 10.1021/bi00127a001. [DOI] [PubMed] [Google Scholar]
  33. WOLD F., BALLOU C. E. Studies on the enzyme enolase. I. Equilibrium studies. J Biol Chem. 1957 Jul;227(1):301–312. [PubMed] [Google Scholar]
  34. Wu F. S., Wang M. Y. Extraction of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis from protease-rich plant tissues. Anal Biochem. 1984 May 15;139(1):100–103. doi: 10.1016/0003-2697(84)90394-4. [DOI] [PubMed] [Google Scholar]
  35. del Pozo J. C., Allona I., Rubio V., Leyva A., de la Peña A., Aragoncillo C., Paz-Ares J. A type 5 acid phosphatase gene from Arabidopsis thaliana is induced by phosphate starvation and by some other types of phosphate mobilising/oxidative stress conditions. Plant J. 1999 Sep;19(5):579–589. doi: 10.1046/j.1365-313x.1999.00562.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES