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. 1985 Mar 15;226(3):645–651. doi: 10.1042/bj2260645

5'-Nucleotidase from rat heart membranes. Inhibition by adenine nucleotides and related compounds.

Y Naito, J M Lowenstein
PMCID: PMC1144761  PMID: 2985044

Abstract

ADP and ATP and their analogues were evaluated as inhibitors of 5'-nucleotidase purified from heart plasma membrane. ADP analogues are more powerful inhibitors than the corresponding ATP analogues. The most powerful inhibitor found is adenosine 5'-[alpha beta-methylene]diphosphate (AOPCP) for which the enzyme shows a Ki of 5 nM at pH 7.2. Measurements of pKi values for ADP and AOPCP as a function of pH indicate that the major inhibitory species of both nucleotides is the dianion. In the physiological range of pH values, AOPCP is a more powerful inhibitor than ADP principally because a higher percentage of AOPCP exists in the dianion form. The methylenephosphonate analogue of AMP (ACP), though not a substrate, is a moderately effective inhibitor. The corresponding analogues of ADP (ACPOP) and ATP (ACPOPOP) are as good inhibitors as ADP and ATP respectively. The thiophosphate analogues of ADP all inhibit 5'-nucleotidase, although not as powerfully as ADP, the most effective of these analogues being adenosine 5'-O-(1-thiodiphosphate) diastereoisomer B (ADP[alpha S](B)]. Other nucleotides inhibit the enzyme, but none is as effective as AOPCP. Inorganic tripolyphosphate and methylenediphosphonate are better inhibitors of the enzyme than is inorganic pyrophosphate. Inorganic thiophosphate is a better inhibitor than is orthophosphate. Hill plots of the ADP and AOPCP inhibition yield slopes close to 1; Hill plots of the ATP inhibition yield slopes of about 0.6. MgADP- is not an inhibitor, and MgATP2- is at best a very weak inhibitor of the enzyme.

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Selected References

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  1. 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]
  2. Burger R. M., Lowenstein J. M. Preparation and properties of 5'-nucleotidase from smooth muscle of small intestine. J Biol Chem. 1970 Dec 10;245(23):6274–6280. [PubMed] [Google Scholar]
  3. Cathala G., Brunel C. Bovine kidney alkaline phosphatase. Purification, subunit structure, and metalloenzyme properties. J Biol Chem. 1975 Aug 10;250(15):6040–6045. [PubMed] [Google Scholar]
  4. Cleland W. W. Statistical analysis of enzyme kinetic data. Methods Enzymol. 1979;63:103–138. doi: 10.1016/0076-6879(79)63008-2. [DOI] [PubMed] [Google Scholar]
  5. Cusack N. J., Pearson J. D., Gordon J. L. Stereoselectivity of ectonucleotidases on vascular endothelial cells. Biochem J. 1983 Sep 15;214(3):975–981. doi: 10.1042/bj2140975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Dornand J., Bonnafous J. C., Mani J. C. Purification and properties of 5'-nucleotidase from lymphocyte plasma membranes. Eur J Biochem. 1978 Jul 3;87(3):459–465. doi: 10.1111/j.1432-1033.1978.tb12396.x. [DOI] [PubMed] [Google Scholar]
  8. Eckstein F., Bär H. P. Enzymatic hydrolysis of adenosine 3'-5'-cyclic phosphorothioate. Biochim Biophys Acta. 1969 Nov 4;191(2):316–321. doi: 10.1016/0005-2744(69)90250-2. [DOI] [PubMed] [Google Scholar]
  9. Eckstein F. Nucleoside phosphorothioates. J Am Chem Soc. 1970 Jul 29;92(15):4718–4723. doi: 10.1021/ja00718a039. [DOI] [PubMed] [Google Scholar]
  10. Edwards M. J., Maguire M. H. Purification and properties of rat heart 5'-nucleotidase. Mol Pharmacol. 1970 Nov;6(6):641–648. [PubMed] [Google Scholar]
  11. Edwards N. L., Recker D., Manfredi J., Rembecki R., Fox I. H. Regulation of purine metabolism by plasma membrane and cytoplasmic 5'-nucleotidases. Am J Physiol. 1982 Nov;243(5):C270–C277. doi: 10.1152/ajpcell.1982.243.5.C270. [DOI] [PubMed] [Google Scholar]
  12. Evans W. H., Gurd J. W. Properties of a 5'-nucleotidase purified from mouse liver plasma membranes. Biochem J. 1973 May;133(1):189–199. doi: 10.1042/bj1330189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hill A. V. The Combinations of Haemoglobin with Oxygen and with Carbon Monoxide. I. Biochem J. 1913 Oct;7(5):471–480. doi: 10.1042/bj0070471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jones G. H., Moffatt J. G. The synthesis of 6'-deoxyhomonucleoside-6'-phosphonic acids. J Am Chem Soc. 1968 Sep 11;90(19):5336–5338. doi: 10.1021/ja01021a086. [DOI] [PubMed] [Google Scholar]
  15. KREBS H. A., HEMS R. Some reactions of adenosine and inosine phosphates in animal tissues. Biochim Biophys Acta. 1953 Sep-Oct;12(1-2):172–180. doi: 10.1016/0006-3002(53)90136-x. [DOI] [PubMed] [Google Scholar]
  16. Krishnaswamy M., Kenkare U. W. The effect of pH, temperature, and organic solvents on the kinetic parameters of Escherichia coli alkaline phosphatase. J Biol Chem. 1970 Aug 10;245(15):3956–3963. [PubMed] [Google Scholar]
  17. MYERS T. C., NAKAMURA K., DANIELZADEH A. B. PHOSPHONIC ACID ANALOGS OF NUCLEOSIDE PHOSPHATES. 3. THE SYNTHESIS OF ADENOSINE-5'-METHYLENEDIPHOSPHONATE, A PHOSPHONIC ACID ANALOG OF ADENOSINE-5'-DIPHOSPHATE. J Org Chem. 1965 May;30:1517–1520. doi: 10.1021/jo01016a043. [DOI] [PubMed] [Google Scholar]
  18. Murray A. W., Atkinson M. R. Adenosine 5'-phosphorothioate. A nucleotide analog that is a substrate, competitive inhibitor, or regulator of some enzymes that interact with adenosine 5'-phosphate. Biochemistry. 1968 Nov;7(11):4023–4029. doi: 10.1021/bi00851a032. [DOI] [PubMed] [Google Scholar]
  19. Murray A. W., Friedrichs B. Inhibition of 5'-nucleotidase from Ehrlich ascites-tumour cells by nucleoside triphosphates. Biochem J. 1969 Jan;111(1):83–89. doi: 10.1042/bj1110083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Naito Y., Lowenstein J. M. 5'-Nucleotidase from rat heart. Biochemistry. 1981 Sep 1;20(18):5188–5194. doi: 10.1021/bi00521a014. [DOI] [PubMed] [Google Scholar]
  21. PLOCKE D. J., LEVINTHAL C., VALLEE B. L. Alkaline phosphatase of Escherichia coli: a zinc metalloenzyme. Biochemistry. 1962 May 25;1:373–378. doi: 10.1021/bi00909a001. [DOI] [PubMed] [Google Scholar]
  22. Phillips R. C., George P., Rutman R. J. Thermodynamic studies of the formation and ionization of the magnesium(II) complexes of ADP and ATP over the pH range 5 to 9. J Am Chem Soc. 1966 Jun 20;88(12):2631–2640. doi: 10.1021/ja00964a002. [DOI] [PubMed] [Google Scholar]
  23. Schliselfeld L. H., Burt C. T., Labotka R. J. 31P nuclear magnetic resonance of phosphonic acid analogues of adenosine nucleotides as functions of pH and magnesium ion concentration. Biochemistry. 1982 Jan 19;21(2):317–320. doi: 10.1021/bi00531a018. [DOI] [PubMed] [Google Scholar]
  24. Stefanovic V., Mandel P., Rosenberg A. Ecto-5'-nucleotidase of intact cultured C6 rat glioma cells. J Biol Chem. 1976 Jul 10;251(13):3900–3905. [PubMed] [Google Scholar]
  25. Sullivan J. M., Alpers J. B. In vitro regulation of rat heart 5'-nucleotidase by adenine nucleotides and magnesium. J Biol Chem. 1971 May 10;246(9):3057–3063. [PubMed] [Google Scholar]
  26. Tanaka R., Teruya A., Morita H. Effects of divalent cations on 5'-mononucleotidase of bovine cerebral cortex. Can J Biochem. 1973 Jun;51(6):841–848. doi: 10.1139/o73-104. [DOI] [PubMed] [Google Scholar]
  27. Vogel H. J., Bridger W. A. Phosphorus-31 nuclear magnetic resonance studies of the methylene and fluoro analogues of adenine nucleotides. Effects of pH and magnesium ion binding. Biochemistry. 1982 Jan 19;21(2):394–401. doi: 10.1021/bi00531a029. [DOI] [PubMed] [Google Scholar]
  28. WEITZEL G., SPEHR T. Zink-Komplexe von Purinen, Nucleosiden und Nucleotiden. Hoppe Seylers Z Physiol Chem. 1958;313:212–226. doi: 10.1515/bchm2.1958.313.1.212. [DOI] [PubMed] [Google Scholar]
  29. Yount R. G. ATP analogs. Adv Enzymol Relat Areas Mol Biol. 1975;43:1–56. doi: 10.1002/9780470122884.ch1. [DOI] [PubMed] [Google Scholar]

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