Adenosine phosphorylase, a purine nucleoside phosphorylase endowed with high specificity for adenine nucleosides, was purified 117-fold from vegetative forms of Bacillus cereus. The purification procedure included ammonium sulphate fractionation, pH 4 treatment, ion exchange chromatography on DEAE-Sephacel, gel filtration on Sephacryl S-300 HR and affinity chromatography on N(6)-adenosyl agarose. The enzyme shows a good stability to both temperature and pH. It appears to be a homohexamer of 164+/-5 kDa. Kinetic characterization confirmed the specificity of this phosphorylase for 6-aminopurine nucleosides. Adenosine was the preferred substrate for nucleoside phosphorolysis (k(cat)/K(m) 2.1x10(6) s(-1) M(-1)), followed by 2'-deoxyadenosine (k(cat)/K(m) 4.2x10(5) s(-1) M(-1)). Apparently, the low specificity of adenosine phosphorylase towards 6-oxopurine nucleosides is due to a slow catalytic rate rather than to poor substrate binding.

Characterization of the adenine nucleoside specific phosphorylase of Bacillus cereus

ALLEGRINI S;CAMICI, MARCELLA;TOZZI, MARIA GRAZIA;
2007-01-01

Abstract

Adenosine phosphorylase, a purine nucleoside phosphorylase endowed with high specificity for adenine nucleosides, was purified 117-fold from vegetative forms of Bacillus cereus. The purification procedure included ammonium sulphate fractionation, pH 4 treatment, ion exchange chromatography on DEAE-Sephacel, gel filtration on Sephacryl S-300 HR and affinity chromatography on N(6)-adenosyl agarose. The enzyme shows a good stability to both temperature and pH. It appears to be a homohexamer of 164+/-5 kDa. Kinetic characterization confirmed the specificity of this phosphorylase for 6-aminopurine nucleosides. Adenosine was the preferred substrate for nucleoside phosphorolysis (k(cat)/K(m) 2.1x10(6) s(-1) M(-1)), followed by 2'-deoxyadenosine (k(cat)/K(m) 4.2x10(5) s(-1) M(-1)). Apparently, the low specificity of adenosine phosphorylase towards 6-oxopurine nucleosides is due to a slow catalytic rate rather than to poor substrate binding.
Sgarrella, F; Frassetto, L; Allegrini, S; Camici, Marcella; Carta, Mc; Fadda, P; Tozzi, MARIA GRAZIA; Ipata, Pl
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11568/173579
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