An authentic soluble metallo-protein nucleotide pyrophosphatase/ phosphodiesterase (ELNPP) was purified to homogeneity from Euphorbia characias latex. The native protein had a molecular mass of 80 5 kDa and was shown to be formed by two apparently identical subunits, each containing 1 Ca2+ and 1 Mg2+ ion. Whereas Mg2+ was shown to be strongly bound to the enzyme, Ca2+ was easily removed by treatment with EDTA. Ca2+-demetalated enzyme was shown to be almost totally inactive and the activity was fully restored incubating the demetalated ELNPP with Ca2+ ions. ELNPP exhibited hydrolytic activities toward pyrophosphate/phosphodiester bonds of a broad range of substrates and very efficiently hydrolyzed the artificial substrate thymidine 50 -monophosphate 4-nitrophenyl ester generating 4-nitrophenolate as a final product, and it has been used for enzyme kinetic experiments. ELNPP represents the first example of a nucleotide pyrophosphatase/ phosphodiesterase enzyme purified from the latex of a plant belonging to the large genus Euphorbia.

Nucleotide pyrophosphatase/phosphodiesterase from Euphorbia characias latex: Purification and characterization

ANGELUCCI, Francesco;
2009

Abstract

An authentic soluble metallo-protein nucleotide pyrophosphatase/ phosphodiesterase (ELNPP) was purified to homogeneity from Euphorbia characias latex. The native protein had a molecular mass of 80 5 kDa and was shown to be formed by two apparently identical subunits, each containing 1 Ca2+ and 1 Mg2+ ion. Whereas Mg2+ was shown to be strongly bound to the enzyme, Ca2+ was easily removed by treatment with EDTA. Ca2+-demetalated enzyme was shown to be almost totally inactive and the activity was fully restored incubating the demetalated ELNPP with Ca2+ ions. ELNPP exhibited hydrolytic activities toward pyrophosphate/phosphodiester bonds of a broad range of substrates and very efficiently hydrolyzed the artificial substrate thymidine 50 -monophosphate 4-nitrophenyl ester generating 4-nitrophenolate as a final product, and it has been used for enzyme kinetic experiments. ELNPP represents the first example of a nucleotide pyrophosphatase/ phosphodiesterase enzyme purified from the latex of a plant belonging to the large genus Euphorbia.
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11697/3006
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 12
  • ???jsp.display-item.citation.isi??? 11
social impact