Figure 2
Interaction between PorX and PorY.
(A) Affinity assays between rPorY and rPorX by surface plasmon resonance (SPR) analysis. rPorX (0.31–4.92 μM) were injected into the sensor chip immobilized by rPorY. BSA exhibited a negative interaction with rPorY. (B) In vitro phosphorylation of rPorY and rPorX. For autophosphorylation assay, rPorY (1 μg) was incubated with [32P-γ]ATP for 1, 5, 10 and 20 min at 0 °C. For transphosphorylation assay, rPorX (5 μg) and excess cold ATP (1 mM) were added to aliquots of rPorY 5 min after start of autophosphorylation and further incubated at 37 °C for indicated periods. The relative intensities of the radiolabelled protein bands were indicated in the graph after normalization. (C) The effects of compounds on the autophosphorylation of rPorY and the transfer of a phosphoryl group from rPorY to rPorX were examined. Phosphorylation assays were performed in the presence or absence of 10 mM MgCl2 and 1 mM MnCl2.
