Purification and Properties of Purine Nucleoside Phosphorylase from Salmonella typhimurium

Purine nucleoside phosphorylase (inosine + Pi ⇌ hypoxanthine + α-d-ribose 1-phosphate, EC 2.4.2.1) from Salmonella typhimurium LT-2 has been purified 230-fold. High speed sedimentation equilibrium studies showed that the molecular weight of the native enzyme was 141,000. A molecular weight of 130,000 ± 10% was determined by Sephadex G-150 filtration. Disc gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate revealed a subunit molecular weight of 23,500, indicating that the enzyme is composed of six subunits. The purified enzyme was found to be specific for the purine ribonucleosides and deoxyribonucleosides, inosine, deoxyinosine, guanosine, deoxyguanosine, adenosine, and deoxyadenosine. The Michaelis constants for inosine, deoxyinosine, and phosphate ion were 50 µm, 47 µm, and 0.37 mm, respectively.