Figure 6.

Phosphodiesterase activity of λ-Pase. (A) Reaction mixtures (25 µl) containing 0.2 mM MnCl₂, 10 mM DTT and either 100 mM Tris–HCl pH 8.0 and 10 mM p-nitrophenyl phosphate (filled square) or 100 mM Tris–HCl pH 7.0 and 10 mM bis-p-nitrophenyl phosphate (open circle) or p-nitrophenyl phenylphosphonate (filled circle), and λ-Pase as specified were incubated for 15 min at 37°C. (B) Reaction mixtures (25 µl) containing 100 mM Tris–acetate (filled circle) or Tris–HCl (open circle) at the pH specified, 0.5 mM MnCl₂, 10 mM DTT, 10 mM bis-p-nitrophenyl phosphate and 2 pmol λ-Pase were incubated for 10 min at 37°C. (C) Kinetics. Reaction mixtures (per 25 µl) containing 100 mM Tris–HCl (pH 7.0), 0.2 mM MnCl₂, 10 mM DTT, 10 mM bis-p-nitrophenyl phosphate and 2 pmol λ-Pase were incubated at 37°C. Two aliquots (25 µl) were withdrawn at the times specified, quenched immediately with EDTA and assayed for p-nitrophenol (filled circle) and inorganic phosphate (open circle).