Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Nov 15;328(Pt 1):257–262. doi: 10.1042/bj3280257

Adenosine 5'-tetraphosphate phosphohydrolase from yellow lupin seeds: purification to homogeneity and some properties.

A Guranowski 1, E Starzyńska 1, P Brown 1, G M Blackburn 1
PMCID: PMC1218915  PMID: 9359862

Abstract

Adenosine 5'-tetraphosphate phosphohydrolase (EC 3.6.1.14) has been purified to homogeneity from the meal of yellow lupin (Lupinus luteus) seeds. The enzyme is a single polypeptide chain of 25+/-1 kDa. It catalyses the hydrolysis of a nucleoside 5'-tetraphosphate to a nucleoside triphosphate and orthophosphate, and hydrolysis of tripolyphosphate but neither pyrophosphate nor tetraphosphate. A divalent cation, Mg2+, Co2+, Ni2+ or Mn2+, is required for these reactions. The pH optimum for hydrolysis of adenosine 5'-tetraphosphate (p4A) is 8.2, Vmax is 21+/-1.7 micromol/min per mg of protein and the Km for p4A is 3+/-0.6 microM. At saturating p4A concentrations, the rate constant for the reaction is 8.5+/-0.7 s-1 [at 30 degrees C, in 50 mM Hepes/KOH (pH8.2)/5 mM MgCl2/0.1 mM dithiothreitol]. p4A and guanosine 5'-tetraphosphate are hydrolysed at the same rate. Adenosine 5'-pentaphosphate (p5A) is degraded 1/200 as fast and is converted into ATP and two molecules of orthophosphate, which are liberated sequentially. This contrasts with the cleavage of p5A by the lupin diadenosine tetraphosphate hydrolase (EC 3.6.1.17), which gives ATP and pyrophosphate. Zn2+, F- and Ca2+ ions inhibit the hydrolysis of p4A with I50 values of 0.1, 0.12 and 0.2 mM respectively.

Full Text

The Full Text of this article is available as a PDF (279.6 KB).

Selected References

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

  1. Andrews P. Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem J. 1964 May;91(2):222–233. doi: 10.1042/bj0910222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barnes L. D., Garrison P. N., Siprashvili Z., Guranowski A., Robinson A. K., Ingram S. W., Croce C. M., Ohta M., Huebner K. Fhit, a putative tumor suppressor in humans, is a dinucleoside 5',5"'-P1,P3-triphosphate hydrolase. Biochemistry. 1996 Sep 10;35(36):11529–11535. doi: 10.1021/bi961415t. [DOI] [PubMed] [Google Scholar]
  3. Costas M. J., Pinto R. M., Fernández A., Canales J., García-Agúndez J. A., Cameselle J. C. Purification to homogeneity of rat liver dinucleoside tetraphosphatase by affinity elution with adenosine 5'-tetraphosphate. J Biochem Biophys Methods. 1990 Jun;21(1):25–33. doi: 10.1016/0165-022x(90)90042-b. [DOI] [PubMed] [Google Scholar]
  4. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  5. Guranowski A. B., Chiang P. K., Cantoni G. L. 5'-Methylthioadenosine nucleosidase. Purification and characterization of the enzyme from Lupinus luteus seeds. Eur J Biochem. 1981 Feb;114(2):293–299. doi: 10.1111/j.1432-1033.1981.tb05148.x. [DOI] [PubMed] [Google Scholar]
  6. Guranowski A., Blanquet S. Phosphorolytic cleavage of diadenosine 5',5'''-P1,P4-tetraphosphate. Properties of homogeneous diadenosine 5',5'''-P1,P4-tetraphosphate alpha, beta-phosphorylase from Saccharomyces cerevisiae. J Biol Chem. 1985 Mar 25;260(6):3542–3547. [PubMed] [Google Scholar]
  7. Guranowski A., Brown P., Ashton P. A., Blackburn G. M. Regiospecificity of the hydrolysis of diadenosine polyphosphates catalyzed by three specific pyrophosphohydrolases. Biochemistry. 1994 Jan 11;33(1):235–240. doi: 10.1021/bi00167a031. [DOI] [PubMed] [Google Scholar]
  8. Guranowski A. Fluoride is a strong and specific inhibitor of (asymmetrical) Ap4A hydrolases. FEBS Lett. 1990 Mar 26;262(2):205–208. doi: 10.1016/0014-5793(90)80190-t. [DOI] [PubMed] [Google Scholar]
  9. Guranowski A., Günther Sillero M. A., Sillero A. Adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate are synthesized by yeast acetyl coenzyme A synthetase. J Bacteriol. 1994 May;176(10):2986–2990. doi: 10.1128/jb.176.10.2986-2990.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Guranowski A. Plant adenosine kinase: purification and some properties of the enzyme from Lupinus luteus seeds. Arch Biochem Biophys. 1979 Aug;196(1):220–226. doi: 10.1016/0003-9861(79)90569-1. [DOI] [PubMed] [Google Scholar]
  11. Guranowski A., Starzyńska E., Bojarska E., Stepiński J., Darzynkiewicz E. Dinucleoside 5', 5"'-P1, P3-triphosphate hydrolase from yellow lupin (Lupinus luteus) seeds: purification to homogeneity and hydrolysis of mRNA 5'-cap analogs. Protein Expr Purif. 1996 Dec;8(4):416–422. doi: 10.1006/prep.1996.0119. [DOI] [PubMed] [Google Scholar]
  12. Guranowski A., Starzyńska E., Rataj-Guranowska M., Günther Sillero M. A. Purification of apyrase from yellow lupin cotyledons after extraction with perchloric acid. Protein Expr Purif. 1991 Aug;2(4):235–239. doi: 10.1016/1046-5928(91)90078-w. [DOI] [PubMed] [Google Scholar]
  13. Jakubowski H., Guranowski A. Enzymes hydrolyzing ApppA and/or AppppA in higher plants. Purification and some properties of diadenosine triphosphatase, diadenosine tetraphosphatase, and phosphodiesterase from yellow lupin (Lupinus luteus) seeds. J Biol Chem. 1983 Aug 25;258(16):9982–9989. [PubMed] [Google Scholar]
  14. Jakubowski H. Sporulation of the yeast Saccharomyces cerevisiae is accompanied by synthesis of adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2378–2382. doi: 10.1073/pnas.83.8.2378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lazewska D., Starzyńska E., Guranowski A. Human placental (Asymmetrical) diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase: purification to homogeneity and some properties. Protein Expr Purif. 1993 Feb;4(1):45–51. doi: 10.1006/prep.1993.1007. [DOI] [PubMed] [Google Scholar]
  16. MEJBAUM-KATZENELLENBOGEN W. Turbidymetryczna mikrometoda oznaczania białek tanina. Acta Biochim Pol. 1955;2(3):279–296. [PubMed] [Google Scholar]
  17. McLennan A. G., Mayers E., Hankin S., Thorne N. M., Prescott M., Powls R. The green alga Scenedesmus obliquus contains both diadenosine 5',5'''-P1,P4-tetraphosphate (asymmetrical) pyrophosphohydrolase and phosphorylase activities. Biochem J. 1994 May 15;300(Pt 1):183–189. doi: 10.1042/bj3000183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pintor J., Díaz-Rey M. A., Torres M., Miras-Portugal M. T. Presence of diadenosine polyphosphates--Ap4A and Ap5A--in rat brain synaptic terminals. Ca2+ dependent release evoked by 4-aminopyridine and veratridine. Neurosci Lett. 1992 Mar 2;136(2):141–144. doi: 10.1016/0304-3940(92)90034-5. [DOI] [PubMed] [Google Scholar]
  19. Pintor J., Rotllán P., Torres M., Miras-Portugal M. T. Characterization and quantification of diadenosine hexaphosphate in chromaffin cells: granular storage and secretagogue-induced release. Anal Biochem. 1992 Feb 1;200(2):296–300. doi: 10.1016/0003-2697(92)90469-n. [DOI] [PubMed] [Google Scholar]
  20. Schlüter H., Offers E., Brüggemann G., van der Giet M., Tepel M., Nordhoff E., Karas M., Spieker C., Witzel H., Zidek W. Diadenosine phosphates and the physiological control of blood pressure. Nature. 1994 Jan 13;367(6459):186–188. doi: 10.1038/367186a0. [DOI] [PubMed] [Google Scholar]
  21. Sillero M. A., Del Valle M., Zaera E., Michelena P., García A. G., Sillero A. Diadenosine 5',5"-P1,P4-tetraphosphate (Ap4A), ATP and catecholamine content in bovine adrenal medulla, chromaffin granules and chromaffin cells. Biochimie. 1994;76(5):404–409. doi: 10.1016/0300-9084(94)90116-3. [DOI] [PubMed] [Google Scholar]
  22. Small G. D., Cooper C. Purification and properties of nucleoside tetraphosphate hydrolase from rabbit muscle. Biochemistry. 1966 Jan;5(1):14–26. doi: 10.1021/bi00865a003. [DOI] [PubMed] [Google Scholar]
  23. Weber K., Pringle J. R., Osborn M. Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 1972;26:3–27. doi: 10.1016/s0076-6879(72)26003-7. [DOI] [PubMed] [Google Scholar]

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

RESOURCES