Figure 4.

Hydrolysis of cyclic phosphodiester substrates by CthPnkp. (A) Reaction mixtures (10 µl) containing 50 mM Tris–HCl (pH 7.5), 0.5 mM MnCl₂, 10 mM substrate as specified and either 4 µg wild-type CthPnkp, 1.4 µg CthPnkp-H189D, or calf intestine phosphatase (CIP; 1 U) where indicated by + were incubated for 30 min at 45°C. The reactions were quenched by adding EDTA (20 mM final concentration) and then 1 ml of malachite green reagent. Release of phosphate was determined by measuring A₆₂₀ and interpolating the value to a phosphate standard curve. Each datum in the bar graph is the average of two separate experiments. S.E. bars are shown. (B) Kinetics. Reaction mixtures (per 10 µl) containing 50 mM Tris–HCl (pH 7.5), 0.5 mM MnCl₂, 10 mM 2′,3′ cGMP and 1.4 µg of CthPnkp-H189D were incubated at 45°C. Samples (10 µl) were withdrawn at the times specified and quenched immediately with EDTA. Aliquots (1 µl) of each sample were applied to a cellulose-F TLC plate (EMD chemicals). Markers 2′,3′ cGMP and 3′GMP (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.