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

PINTUS, FRANCESCA;Spanò D;Bellelli A;Angelucci F;Floris G;MEDDA, ROSARIA

2009-01-01

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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.