Dynamics of HipA and HipA7 in vitro (auto)phosphorylation measured by autoradiography

For qualitative comparison of HipA and HipA7 autophosphorylation activity and the phosphorylation activity toward GltX, we performed a time-depended kinase assay. Kinase (1 μM) (His6-HipA or His6-HipA7) was incubated with 54 μM ATP and 12 μM [γ-32P]ATP and with or without 6 μM His6-GltX and 19 μM of total E. coli tRNA in the kinase buffer [50 mM tris-HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, and 16 μM ZnSO4]. Reactions with HipA and HipA7 were performed simultaneously using the same ATP stock solution. The reactions were incubated at 37°C for 45 min and stopped by the addition of Laemmli buffer at indicated time points. Reaction mixtures were separated by SDS-PAGE, revealed by phosphorimaging (GE Healthcare), and analyzed using ImageQuant software (GE Healthcare). The intensity of each time point was normalized to the sum of intensities of all time points and presented as a fraction of total intensity. Phosphorylation activity was determined by the linear regression of the linear part of the intensity over time curve.

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