Hexokinase activity in airway cells is reduced by BrPy.
Addition of BrPy to H441 cells reduced total hexokinase activity in cell extracts in a dose-dependent manner with an IC50 of 1.2 ± 0.28 mM (Fig. 2A). The data did not follow a classic sigmoid curve, and there was an indication that the inhibition was biphasic. We were unable to unambiguously fit such a curve to the data. However, the IC50 obtained from the initial inhibition of hexokinase activity was lower at 0.04 ± 0.01 mM. As there was no statistical difference in hexokinase activity between pretreatment with 100 µM and pretreatment with 1 mM, it was decided to use the lower concentration of BrPy. At this concentration, total cellular hexokinase activity was reduced by 25.1 ± 11.6% in H441 cells cultured at air-liquid interface (n = 6 experiments; Fig. 2B).
Fig. 2.  Hexokinase activity is inhibited by 3-bromopyruvic acid (BrPy). A: effect of BrPy concentration on hexokinase activity in cell extracts from H441 cells exposed to 5 mM glucose. The dose-response data did not follow a classic sigmoid curve, and there was an indication that the inhibition was biphasic. Two curves could be fitted to the data to reflect initial inhibition (left-hand curve) with an IC50 of 0.04 ± 0.01 mM or overall inhibition (right-hand side) with an IC50 of 1.2 ± 0.28 mM (n = 4 experiments). B: total hexokinase activity in cell extracts from control (●) or BrPy (100 μM)-treated cells (○). Individual data points are shown with mean ± SD. ****Significantly different from control, P < 0.0001.