FIG. 4.

Effect on uridylylation of Mg²⁺ or Mn²⁺. The three P_II paralogs (0.5 μM) were incubated with either 250 μM α-ketoglutarate and 25 mM Mg²⁺ (lanes 1, 3, and 5) or 60 μM α-ketoglutarate and 3 mM Mn²⁺ (lanes 2, 4, and 6). For both divalent cations used, 2 mM ATP, 0.13 μM GlnD, and 0.5 mM UTP, supplemented with [α-³²P]UTP, were included in the reaction mixture. Samples were withdrawn from the reaction mixtures after 20 min of incubation and stopped by addition of SDS cocktail. (A) Autoradiogram showing incorporation of [α-³²P]UMP into GlnB (lanes 1 and 2), GlnJ (lanes 3 and 4), or GlnK (lanes 5 and 6). (B) Coomassie-stained SDS-PAGE showing the levels of P_II proteins loaded in panel A. (C) Histogram showing the difference in incorporation of [α-³²P]UMP between the R. rubrum P_II proteins with Mg²⁺ (dark) or Mn²⁺ (gray) in the uridylylation reaction. The amount of labeled, uridylylated GlnB, GlnJ, or GlnK was quantified using the Image Quant program. The data shown are from at least three independent experiments. UTase, uridylyltransferase.