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2011 | 58 | 1 | 131-136
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Plant nucleoside 5'-phosphoramidate hydrolase; simple purification from yellow lupin (Lupinus luteus) seeds and properties of homogeneous enzyme

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Adenosine 5'-phosphoramidate (NH2-pA) is an uncommon natural nucleotide of poorly understood biochemistry and function. We studied a plant enzyme potentially involved in the catabolism of NH2-pA. A fast and simple method comprising extraction of yellow lupin (Lupinus luteus) seed-meal with a low ionic strength buffer, ammonium sulfate and acetone fractionations, removal of contaminating proteins by heat denaturation, and affinity chromatography on AMP-agarose, yielded homogenous nucleoside 5'-phosphoramidase. Mass spectrometric analysis showed that the lupin hydrolase exhibits closest similarity to Arabidopsis thaliana Hint1 protein. The substrate specificity of the lupin enzyme, in particular its ability to split the P-S bond in adenosine 5'-phosphorothioate, is typical of known Hint1 proteins. Adenosine 5'-phosphofluoride and various derivatives of guanosine 5'-phosphoramidate were also substrates. Neither common divalent metal cations nor 10 mM EDTA or EGTA affected the hydrolysis of NH2-pA. The enzyme functions as a homodimer (2 × 15 800 Da). At the optimum pH of 7.0, the Km for NH2-pA was 0.5 µM and kcat 0.8 s-1 (per monomer active site). The properties of the lupin nucleoside 5'-phosphoramidase are compared with those of its counterparts from other organisms.

Physical description
  • Department of Biochemistry and Biotechnology, Poznań, University of Life Sciences, Poznań, Poland
  • Department of Biochemistry and Biotechnology, Poznań, University of Life Sciences, Poznań, Poland
  • Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warszawa, Poland
  • Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warszawa, Poland
  • Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warszawa, Poland
  • Barbier E, Wang JB (2009) Anti-depressant anxiolytic like behaviors in PKCI/HINT 1 knockout mice associated with elevated plasma corticosterone level. BMC Neurosci 10: 132.
  • Bieganowski P, Garrison PN, Hodawadekar SC, Faye G, Barnes LD, Brenner C (2002) Adenosine monophosphoramidase activity of Hint and Hnt1 supports function of Kin28, Cc11, and Tfb3. J Biol Chem 277: 10852-10860.
  • Frankhauser H, Berkowitz GA, Schiff JA (1981a) A nucleotide with the properties of adenosine 5'-phosphoramidate from Chlorella cells. Biochem Biophys Res Commun 101: 524-532.
  • Frankhauser H, Schiff JA, Graber LJ (1981b) Purification and properties of adenylyl sulphate:ammonia adenylyltransferase from Chlorella catalyzing the formation of adenosine phosphoramidate from adenosine 5'-phosphosulphate and ammonia. Biochem J 195: 545-560.
  • Guranowski A (1979) Plant adenosine kinase: purification and some properties of the enzyme from Lupinus luteus seeds. Arch Biochem Biophys 196: 220-226.
  • Guranowski A, Schneider Z (1977) Purification and characterization of adenosine nucleosidase from barley leaves. Biochim Biophys Acta 482: 145-158.
  • Guranowski A, Starzynska E, Bojarska E, Stepinski J, Darzynkiewicz E (1996) 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 8: 416-422.
  • Guranowski A, Wojdyła AM, Pietrowska-Borek M, Bieganowski P, Khurs EN, Cliff MJ, Blackburn GM, Błaziak D, Stec, W J (2008) Fhit proteins can also recognize substrates other than dinucleoside polyphosphates. FEBS Lett 582: 3152-3158.
  • Guranowski A, Bieganowski P, Baraniak J, Rydzik A, Stępiński J, Jemielity J (2006) Catabolism of nucleoside phosphoramidates in higher plants can be controlled by nucleoside phosphoramidase (Hint protein) and dinucleoside triphosphatase (Fhit protein). Acta Biochim Pol 53: (Suppl 1): 183-184.
  • Guranowski A, Wojdyła AM, Zimny J, Wypijewska A, Kowalska J, Jemielity J, Davis RE, Bieganowski P (2010a) Dual activity of certain HIT-proteins: A. thaliana Hint4 and C. elegans DcpS act on adenosine 5'-phosphosulfate as hydrolases (forming AMP) and as phosphorylases (forming ADP). FEBS Lett 584: 93-98.
  • Guranowski A, Wojdyła AM, Zimny J, Wypijewska A, Kowalska J, Łukaszewicz M, Jemielity J, Darżynkiewicz E, Jagiełło A, Bieganowski P (2010b) Recognition of different nucleotidyl-derivatives as substrates of reactions catalyzed by various HIT-proteins. New J Chem 34: 888-893.
  • Jakubowski H, Pawelkiewicz J (1974) Valyl-tRNA synthetase of yellow lupin seeds. Purification and some properties. Acta Biochim Pol 21: 271-282.
  • Jakubowski H, Guranowski A (1983) Enzymes hydrolyzing ApppA and/or AppppA in higher plants. Purification and some properties of diadenosine triphosphatase, diadenosine tetraphosphatase, and phosphodiesterase from yellow lupin (Lupinus lutesu) seeds. J Biol Chem 258: 9982-9989.
  • Kowalska J, Lewdorowicz M, Zuberek J, Grudzien-Nogalska E, Bojarska E, Stepinski J, Rhoads RE, Darzynkiewicz E, Davis RE, Jemielity J (2008) Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS. RNA 14: 1119-1131.
  • Kuba M, Okazaki T, Ohmori H, Kumon A (1994) Nucleoside monophosphoramidate hydrolase from rat liver: purification and characterization. Int J Biochem 26: 235-245.
  • Mejbaum-Katzenelenbogen W (1955) Turbidymetryczna mikrometoda oznaczania białek taniną. Acta Biochim Pol 2: 279-296 (in Polish).
  • Mukaiyama T, Hashimoto M (1971) Phosphorylation by oxidation-reduction condensation. Preparation of active phosphorylating reagents. Bul Chem Soc Japan 44: 2284.
  • Ozga M, Dolot R, Janicka M, Kaczmarek R, Krakowiak A (2010) Histidine triad nucleotide-binding protein 1 (Hint-1) phosphoramidase transforms nucleoside 5'-O-phosphorothioates to nucleoside 5'-phosphates. J Biol Chem 285: 40809-40818.
  • Rossomando EF, Hadjimichael J (1986) Characterization and cAMP inhibition of a lysyl-(N-ε-5'-phospho) adenosyl phosphoamidase in Dictyostelium discoideum. Int J Biochem 18: 481-484.
  • Tasheva B, Dessev G (1983) Artifacts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to 2-mercaptoethanol. Anal Biochem 129: 98-102.
  • Tomasz J, Vaghefi MM, Ratsep PC, Willis RC, Robins RK (1988) Nucleoside imidodiphosphates synthesis and biological activities. Nucleic Acids Res 16: 8645-8664.
  • Van Veldhoven PP, Mannaerts GP (1987) Inorganic and organic phosphate measurements in the nanomolar range. Anal Biochem 161: 45-48.
  • Wittman R (1963) Die Reaction der Phosphorsäuren mit 2,4-Dinitro-fluorobenzol. I: Eine neue Synthese von Monofluorophosphorsäurenmonoestern. Chem Ber 96: 771-779.
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