Figure 7.

Cyclic phosphodiesterase activity of λ-Pase. (A) Reaction mixtures (10 µl) containing 100 mM Tris–HCl (pH 7.5), 0.5 mM MnCl₂, 10 mM DTT, 10 mM substrate as specified and either 32 pmol λ-Pase or 1 U CIP (where indicated by +) were incubated for 30 min at 37°C. Each datum in the bar graph is the average of two separate experiments. S.E. bars are shown. (B) Reaction mixtures (per 10 µl) containing 100 mM Tris–HCl (pH 7.5), 0.5 mM MnCl₂, 10 mM DTT, 10 mM 2′,3′ cAMP and 64 pmol λ-Pase were incubated at 37°C. Aliquots (10 µl) were withdrawn at the times specified and quenched immediately with EDTA. One micro liter of each sample was applied to a cellulose-F TLC plate. Markers 2′-AMP, 3′-AMP and 2′,3′ cAMP (5 nmol each) were spotted in lane M. The TLC plate was developed with buffer containing saturated ammonium sulfate/3 M sodium acetate/isopropanol (80/6/2). The nucleotides were visualized by photography under UV light.