FIG. 4.

Autophosphorylation and phosphotransfer activities of KdpD wild type (A) and derivatives (B) under different iso-osmotic conditions. Time-dependent autophosphorylation of membrane vesicles containing KdpD, KdpD(G492R), KdpD(E509K), or KdpD(Y516C) was carried out as described in Materials and Methods. The 0.5 M NaCl of the phosphorylation buffer was partially replaced with KCl, K⁺-glutamate, or NH₄Cl as indicated (represented by X). After 5 min of autophosphorylation, purified KdpE was added, and the phosphotransfer capacity was followed. At different time points aliquots were removed and subjected to SDS-polyacrylamide gel electrophoresis. The amounts of KdpD∼P and KdpE∼P were quantified with a phosphorimager, using [γ-³²P]ATP as a standard. Please note the different scale for the enzymatic activities.