FIG. 2.
PEP-dependent phosphorylation of His6-IIALacS by His6-HPr(His∼P). The reactions were carried out in 50 mM Tris-acetate (pH 7.5) containing 1 mM DTT, 1 mM MgCl2, 0.8 μM His6-EI, 24 μM His6-HPr, 5.8 μM His6-IIALacS, and 1 mM [32P]PEP (30 μCi/μmol). The reactions were stopped by adding an equal volume of a solution containing 180 mM Tris-HCl (pH 6.8), 200 mM SDS, 30% glycerol, 2 M β-mercaptoethanol, and 0.003% bromophenol blue. Proteins were separated by SDS-PAGE, and phosphoproteins were revealed by autoradiography. (A) Lanes: 1, control experiment conducted without EI and HPr; 2, control experiment conducted without EI and IIALacS; 3, control experiment conducted without HPr and IIALacS; 4, control experiment conducted without IIALacS. (B) PEP-dependent phosphorylation experiment conducted in a medium containing EI, HPr, and IIALacS. Samples were withdrawn at the intervals indicated on the autoradiogram.