Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 1990 May 15;268(1):117–122. doi: 10.1042/bj2680117

Partial purification and properties of an AMP-specific soluble 5'-nucleotidase from pigeon heart.

A C Skladanowski 1, A C Newby 1
PMCID: PMC1131399  PMID: 2344353

Abstract

A soluble 5'-nucleotidase was purified 200-fold from pigeon heart. The enzyme (1) had an apparent molecular mass close to 150 kDa, (2) had a neutral pH optimum and hydrolysed a wide range of nucleoside 5'-monophosphates with a 15-fold preference for AMP over IMP, (3) at near-physiological concentrations of AMP was activated by ADP but not by ATP, (4) was inhibited by high Mg2+ concentration and high ionic strength, (5) was weakly inhibited by p-nitrophenol phosphate and Pi, and (6) was non-competitively inhibited more potently by 5'-deoxy-5'-isobutylthioinosine than by 5'-deoxy-5'-isobutylthioadenosine, but not by [alpha,beta-methylene]ADP. The data show that the enzyme is distinct from the ecto-5'-nucleotidase and from the previously purified IMP-specific 5'-nucleotidase. They also predict that the enzyme is activated during ATP catabolism and hence will generate a more-than-linear increase in the adenosine-formation rate in response to an increase in cytosolic AMP concentration.

Full text

PDF
118

Selected References

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

  1. Bontemps F., Van den Berghe G., Hers H. G. 5'-Nucleotidase activities in human erythrocytes. Identification of a purine 5'-nucleotidase stimulated by ATP and glycerate 2,3-bisphosphate. Biochem J. 1988 Mar 15;250(3):687–696. doi: 10.1042/bj2500687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Burger R. M., Lowenstein J. M. 5'-Nucleotidase from smooth muscle of small intestine and from brain. Inhibition of nucleotides. Biochemistry. 1975 Jun 3;14(11):2362–2366. doi: 10.1021/bi00682a014. [DOI] [PubMed] [Google Scholar]
  4. Bünger R., Soboll S. Cytosolic adenylates and adenosine release in perfused working heart. Comparison of whole tissue with cytosolic non-aqueous fractionation analyses. Eur J Biochem. 1986 Aug 15;159(1):203–213. doi: 10.1111/j.1432-1033.1986.tb09854.x. [DOI] [PubMed] [Google Scholar]
  5. DIXON M. The determination of enzyme inhibitor constants. Biochem J. 1953 Aug;55(1):170–171. doi: 10.1042/bj0550170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Frick G. P., Lowenstein J. M. Studies of 5'-nucleotidase in the perfused rat heart. Including measurements of the enzyme in perfused skeletal muscle and liver. J Biol Chem. 1976 Oct 25;251(20):6372–6378. [PubMed] [Google Scholar]
  7. Fritzson P., Haugen T. B., Tjernshaugen H. The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5'-nucleotidase. Biochem J. 1986 Oct 1;239(1):185–190. doi: 10.1042/bj2390185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gibson W. B., Drummond G. I. Properties of 5'-nucleotidase from avian heart. Biochemistry. 1972 Jan 18;11(2):223–229. doi: 10.1021/bi00752a013. [DOI] [PubMed] [Google Scholar]
  9. Headrick J. P., Willis R. J. 5'-Nucleotidase activity and adenosine formation in stimulated, hypoxic and underperfused rat heart. Biochem J. 1989 Jul 15;261(2):541–550. doi: 10.1042/bj2610541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Imai S., Chin W. P., Jin H., Nakazawa M. Production of AMP and adenosine in the interstitial fluid compartment of the isolated perfused normoxic guinea pig heart. Pflugers Arch. 1989 Aug;414(4):443–449. doi: 10.1007/BF00585055. [DOI] [PubMed] [Google Scholar]
  11. Itaya K., Ui M. A new micromethod for the colorimetric determination of inorganic phosphate. Clin Chim Acta. 1966 Sep;14(3):361–366. doi: 10.1016/0009-8981(66)90114-8. [DOI] [PubMed] [Google Scholar]
  12. Itoh R., Oka J. Evidence for existence of a cytosol 5'-nucleotidase in chicken heart: comparison of some properties of heart and liver enzymes. Comp Biochem Physiol B. 1985;81(1):159–163. doi: 10.1016/0305-0491(85)90177-4. [DOI] [PubMed] [Google Scholar]
  13. Itoh R., Oka J., Ozasa H. Regulation of rat heart cytosol 5'-nucleotidase by adenylate energy charge. Biochem J. 1986 May 1;235(3):847–851. doi: 10.1042/bj2350847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Itoh R. Purification and some properties of cytosol 5'-nucleotidase from rat liver. Biochim Biophys Acta. 1981 Feb 13;657(2):402–410. doi: 10.1016/0005-2744(81)90326-0. [DOI] [PubMed] [Google Scholar]
  15. Itoh R. Regulation of cytosol 5'-nucleotidase by adenylate energy charge. Biochim Biophys Acta. 1981 May 14;659(1):31–37. doi: 10.1016/0005-2744(81)90268-0. [DOI] [PubMed] [Google Scholar]
  16. Itoh R. Studies on some molecular properties of cytosol 5'-nucleotidase from rat liver. Biochim Biophys Acta. 1982 May 5;716(1):110–113. doi: 10.1016/0304-4165(82)90208-2. [DOI] [PubMed] [Google Scholar]
  17. Itoh R., Usami C., Nishino T., Tsushima K. Kinetic properties of cytosol 5'-nucleotidase from chicken liver. Biochim Biophys Acta. 1978 Sep 11;526(1):154–162. doi: 10.1016/0005-2744(78)90300-5. [DOI] [PubMed] [Google Scholar]
  18. Le Hir M., Dubach U. C. An ATP-inhibited soluble 5'-nucleotidase of rat kidney. Am J Physiol. 1988 Feb;254(2 Pt 2):F191–F195. doi: 10.1152/ajprenal.1988.254.2.F191. [DOI] [PubMed] [Google Scholar]
  19. Madrid-Marina V., Fox I. H. Human placental cytoplasmic 5'-nucleotidase. Kinetic properties and inhibition. J Biol Chem. 1986 Jan 5;261(1):444–452. [PubMed] [Google Scholar]
  20. Marszalek J., Kostrowicki J., Spychala J. LEHM: a convenient non-linear regression microcomputer program for fitting Michaelis-Menten and Hill models to enzyme kinetic data. Comput Appl Biosci. 1989 Jul;5(3):239–240. doi: 10.1093/bioinformatics/5.3.239. [DOI] [PubMed] [Google Scholar]
  21. Meghji P., Holmquist C. A., Newby A. C. Adenosine formation and release from neonatal-rat heart cells in culture. Biochem J. 1985 Aug 1;229(3):799–805. doi: 10.1042/bj2290799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Meghji P., Middleton K. M., Newby A. C. Absolute rates of adenosine formation during ischaemia in rat and pigeon hearts. Biochem J. 1988 Feb 1;249(3):695–703. doi: 10.1042/bj2490695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Meghji P., Rubio R., Berne R. M. Intracellular adenosine formation and its carrier-mediated release in cultured embryonic chick heart cells. Life Sci. 1988;43(23):1851–1859. doi: 10.1016/s0024-3205(88)80002-x. [DOI] [PubMed] [Google Scholar]
  24. Naito Y., Tsushima K. Cytosol 5'-nucleotidase from chicken liver. Purification and some properties. Biochim Biophys Acta. 1976 Jun 7;438(1):159–168. doi: 10.1016/0005-2744(76)90232-1. [DOI] [PubMed] [Google Scholar]
  25. Newby A. C. The pigeon heart 5'-nucleotidase responsible for ischaemia-induced adenosine formation. Biochem J. 1988 Jul 1;253(1):123–130. doi: 10.1042/bj2530123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schütz W., Schrader J., Gerlach E. Different sites of adenosine formation in the heart. Am J Physiol. 1981 Jun;240(6):H963–H970. doi: 10.1152/ajpheart.1981.240.6.H963. [DOI] [PubMed] [Google Scholar]
  27. Skladanowski A. C., Sala G. B., Newby A. C. Inhibition of IMP-specific cytosolic 5'-nucleotidase and adenosine formation in rat polymorphonuclear leucocytes by 5'-deoxy-5'-isobutylthio derivatives of adenosine and inosine. Biochem J. 1989 Aug 15;262(1):203–208. doi: 10.1042/bj2620203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Składanowski A., Kaletha K., Zydowo M. Potassium-dependent regulation by ATP and ADP of AMP-deaminase from beef heart. Int J Biochem. 1979;10(2):177–181. doi: 10.1016/0020-711x(79)90114-9. [DOI] [PubMed] [Google Scholar]
  29. Spychała J., Madrid-Marina V., Fox I. H. High Km soluble 5'-nucleotidase from human placenta. Properties and allosteric regulation by IMP and ATP. J Biol Chem. 1988 Dec 15;263(35):18759–18765. [PubMed] [Google Scholar]
  30. Stochaj U., Flocke K., Mathes W., Mannherz H. G. 5'-Nucleotidases of chicken gizzard and human pancreatic adenocarcinoma cells are anchored to the plasma membrane via a phosphatidylinositol-glycan. Biochem J. 1989 Aug 15;262(1):33–40. doi: 10.1042/bj2620033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Truong V. L., Collinson A. R., Lowenstein J. M. 5'-Nucleotidases in rat heart. Evidence for the occurrence of two soluble enzymes with different substrate specificities. Biochem J. 1988 Jul 1;253(1):117–121. doi: 10.1042/bj2530117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Worku Y., Newby A. C. Nucleoside exchange catalysed by the cytoplasmic 5'-nucleotidase. Biochem J. 1982 Sep 1;205(3):503–510. doi: 10.1042/bj2050503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Worku Y., Newby A. C. The mechanism of adenosine production in rat polymorphonuclear leucocytes. Biochem J. 1983 Aug 15;214(2):325–330. doi: 10.1042/bj2140325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. van den Berghe G., van Pottelsberghe C., Hers H. G. A kinetic study of the soluble 5'-nucleotidase of rat liver. Biochem J. 1977 Mar 15;162(3):611–616. doi: 10.1042/bj1620611. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES