Temperature dependence of NADPH oxidase in human eosinophils

The phagocyte NADPH oxidase helps kill pathogens by producing superoxide anion, O-2(-). This enzyme is electrogenic because it translocates electrons across the membrane, generating an electron current, I-e. Using the permeabilized patch voltage-clamp technique, we studied the temperature dependence of I-e in human eosinophils stimulated by phorbol myristate acetate (PMA) from room temperature to > 37degreesC. For comparison, NADPH oxidase activity was assessed by cytochrome c reduction. The intrinsic temperature dependence of the assembled, functioning NADPH oxidase complex measured during rapid temperature increases to 37degreesC was surprisingly weak: the Arrhenius activation energy E-a was only 14 kcal mol(-1) (Q(10), 2.2). In contrast, steady-state NADPH oxidase activity was strongly temperature dependent at 20-30degreesC, with E-a 25.1 kcal mol(-1) (Q(10), 4.2). The maximum I-e measured at 34degreesC was -30.5 pA. Above 30degreesC, the temperature dependence of both I-e and O-2(-) production was less pronounced. Above 37degreesC, I-e was inhibited reversibly. After rapid temperature increases, a secondary increase in I-e ensued, suggesting that high temperature promotes assembly of additional NADPH oxidase complexes. Evidently, about twice as many NADPH oxidase complexes are active near 37degreesC than at 20degreesC. Thus, the higher Q(10) of steady-state I-e reflects both increased activity of each NADPH oxidase complex and preferential assembly of NADPH oxidase complexes at high temperature. In summary, NADPH oxidase activity in intact human eosinophils is maximal precisely at 37degreesC.