Figure 4.

Dose-response and time-course experiments reveal phosphoribohydrolase activity of BP1253. (A) Schematic representation of phosphoribohydrolase (PRH) catalyzed reaction. (B) Experiments performed at 30 °C in the presence of 20 mM AMP as substrate. On the left, dose-response experiment carried out after 6 hours of incubation. The BP1253 concentrations used were 6, 12, 24 and 48 µM. In the center, time-course assay carried out with 24 µM of enzyme at different incubation time as shown in the figure. On the right, the TLC with the protein inactivated at 99 °C for 10 min. as negative control (H-I) after 8 hours of incubation at 30 °C. After heat inactivation of the assay mixture at 95 °C for 5 min, the products were separated by TLC with 1 M NaCl as mobile phase on Cellulose F plastic sheets. The dots were identified under UV light at 264 nm. Arrows indicate the substrate AMP and the cleaved product adenine. (C) Assays performed at 30 °C in the presence of 20 mM GMP. Dose-response experiment carried out in 20 minutes with 1.2, 2.4 and 4.8 µM enzymatic concentrations of BP1253 and time-course experiment performed with 2.4 µM of BP1253 at different incubation times as shown in the Figure. The reaction mixture was inactivated with 1 M NaOH. The products of the reaction were separated by TLC and dots visualized as described above. Arrows indicate the substrate GMP and the cleaved product guanine. (D) Assays carried out with 2.4 µM of BP1253 at 30 °C in the presence of 20 mM CMP for 20 minutes. The control reaction was performed with BP1253 heat-inactivated at 99 °C for 10 min. (H–I). The products of the enzymatic reaction were separated by TLC on Cellulose F plastic sheets with acetone/water 30/70 (v/v) with the addition of 0.2 mol/l NaCl as mobile phase, while dots were visualized as described above. Arrows indicate the substrate CMP and the cleaved product cytosine.