FIG.4.

Dephosphorylation of His₆-IIA^LacS(His∼³²P) by HPr(Ser-P) and HPr. The synthesis and purification of His₆-IIA^LacS(His∼³²P) is described in Materials and Methods. His₆-IIA^LacS(His∼³²P) was dephosphorylated by HPr and HPr(Ser-P) in 50 mM Tris-acetate (pH 7.5) containing 1 mM DTT, 2 mM MgCl₂, and 20 μM HPr or HPr(Ser-P) in a total volume of 15 μl. After the mixture was incubated for 10 min at 37°C, His₆-IIA^LacS(His∼³²P) was added to a final concentration of 2 μM, and the incubation was extended for 5 min. The proteins were separated by SDS-PAGE and revealed by autoradiography. Lanes: 1, purified His₆-IIA^LacS(His∼³²P); 2, phosphorylated products resulting from the incubation of His₆-IIA^LacS(His∼³²P) with HPr(Ser-P); 3, phosphorylated products resulting from the incubation of His₆-IIA^LacS(His∼³²P) with HPr.