FIG 4.

Analysis of the phosphoribohydrolase activity of SiRe_0427 with increasing concentrations of protein and substrate. (A) Phosphoribohydrolase activity of SiRe_0427 as a function of concentrations. The experiment was performed using increasing concentrations of wild-type SiRe_0427 protein (columns 1 to 9: 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, and 51.2 μM, respectively) and 20 mM AMP substrate. The 20-μl reaction mixtures (pH 7.4) were incubated at 65°C for 3 h. (B) Quantification of the results in panel A using ImagQuant. (C) Phosphoribohydrolase activity of SiRe_0427 protein with increased concentrations of AMP. The reaction was performed at 65°C using 12 μM SiRe_0427 protein and increasing concentrations of AMP (5, 10, 20, 30, 40, and 50 mM) in 20-μl reaction mixtures (see Materials and Methods). One to two microliters of samples was dotted onto each TLC sheet. After separation, the plate was dried and visualized by UV illumination at 254 nm. The AMP and adenine standards are on the left side of the figure. (D) Quantification of the results in panel C. The reaction time (hours) is indicated at the bottom of the figure. All experiments were repeated at least three times.