Ukr.Biochem.J. 2013; Volume 85, Issue 3, May-Jun, pp. 31-37

doi: http://dx.doi.org/10.15407/ubj87.03.031

Purification and properties of a catalytically active fragment of soluble nucleoside triphosphatase from bovine kidney

26.10.2015   Uncategorized   No comments

А. F. Makarchikov

Grodno State Agricultural University, Belarus;
e-mail: a_makarchikov@yahoo.com

A catalytic fragment of soluble NTPase has been isolated from bovine kidneys.The 236-fold purification was carried out to obtain the preparation with a specific activity of 37.7 U/mg of protein. The purification scheme included the enzyme extraction followed by four column chromatography steps. The catalytic fragment was activated with divalent metal ions, had a pH optimum of 7.0, and possessed specificity for ITP, GTP, UTP and XTP. The apparent Km for Mg–ITP, Mg–GTP and Mg–UTP complexes were calculated from Hanes plots to be 1.70 mM, 0.93 mM and 0.48 mM, respectively. As estimated by gel filtration and SDS-PAAGE­, the catalytic fragment has Mw 54.7 kDa being composed of two identical poly­peptide chains. Our results suppose soluble NTPase from bovine kidney to consist of regulatory and catalytic structural units.

Keywords: , , ,

References:

  1. BRENDA. The Comprehensive Enzyme Information System [Electronic resource].  Regime of access: http:// www.brenda-enzymes.info.
  2. Makarchikov AF, Chernikevich IP. Thiamine triphosphatase activity in bovine kidney. Biochem Mol Biol Int. 1998 Sep;46(1):115-23. PubMed,  CrossRef
  3. Makarchikov AF. Partial purification and characterization of a soluble nucleoside triphosphatase from bovine kidney. J Biochem Mol Biol Biophys. 2001;5:525-531.
  4. Rusina I, Makarchikov A, Lukashenko Y. Study on regulatory properties of a soluble nucleoside triphosphatase from bovine kidney. Vesnik of Yanka Kupala State University of Grodno. 2011; (Ser. 5 (1)):128-133.
  5. Sapru MK, Geetha H, Shetty KT. A single reagent method of phosphate estimation in phosphatase(s) assay. Indian J Biochem Biophys. 1987 Dec;24(6):340-3. PubMed
  6. Weber K, Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406-12. PubMed
  7. Davis BJ. Disc electrophoresis. ii. method and application to human serum proteins. Ann N Y Acad Sci. 1964 Dec 28;121:404-27. PubMed, CrossRef
  8. Bradford MM. 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;72:248-54. PubMed, CrossRef
  9. Darbre A. Practical protein chemistry / Darbre A. M .: Mir, 1989. 623 p.
  10. Agutter PS, Cockrill JB, Lavine JE, McCaldin B, Sim RB. Properties of mammalian nuclear-envelope nucleoside triphosphatase. Biochem J. 1979 Sep 1;181(3):647-58.
    PubMed, PubMedCentral, CrossRef
  11. Lewis M, Weissman S. Properties of a soluble nucleoside triphosphatase activity in mammalian liver. Arch Biochem Biophys. 1965 Mar;109:490-8. PubMed, CrossRef
  12. Naryzhny SN, Krutiakov VM. Non-specific acid nucleoside triphosphatase from cytosol and chromatin of rat liver: partial purification and general properties. Biokhimiia. 1982 Apr;47(4):569-74.  Russian. PubMed
  13. Dahlmann N, Kirchgesser M. Acid nucleoside triphosphatase: partial purification and characterization of a new enzyme from human serum. Biochem Int. 1990;20(2):317-27. PubMed
  14. Williams MV. Purification and properties of a nucleoside triphosphatase from Sinclair swine. Enzyme. 1984;32(4):201-7. PubMed
  15. McCarty DR, Selman BR. Partial purification of a nucleoside triphosphatase from the inner membrane of the chloroplast envelope of pea. Arch Biochem Biophys. 1986 Aug 1;248(2):523-31. PubMed, CrossRef
  16. Mukherjee J, Biswas S. Purification & characterization of a nucleoside triphosphatase from Mimosa pudica. Indian J Biochem Biophys. 1980 Dec;17(6):452-6. PubMed
  17. Nishimune T, Ito S, Abe M, Kimoto M, Hayashi R. Nucleoside-triphosphatase and hydrolysis of thiamin triphosphate in Escherichia coli. Biochim Biophys Acta. 1987 Jan 20;923(1):74-82. PubMed, CrossRef
  18. Romani AM. Cellular magnesium homeostasis. Arch Biochem Biophys. 2011 Aug 1;512(1):1-23. Review. PubMed, PubMedCentral, CrossRef
  19. Ash DE, Schramm VL. Determination of free and bound manganese(II) in hepatocytes from fed and fasted rats. J Biol Chem. 1982 Aug 25;257(16):9261-4.  PubMed
  20. Metzler DE. Biochemistry. The chemical reactions of living cells. Harcourt. Academic Press, 2001;1:937 p.
  21. Dixon M, Webb E. Enzymes. M: Mir, 1982;2:515 p.
  22. Smith RM, Martell AE, Chen Y. Critical evaluation of stability constants for nucleotide complexes with protons and metal ions and the accompanying enthalpy changes. Pure Appl Chem. 1991;63(7):1015-1080. CrossRef
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