Temperature dependence of NADPH oxidase in human eosinophils.
The phagocyte NADPH oxidase helps kill pathogens by producing superoxide anion, O2-. This enzyme is electrogenic because it translocates electrons across the membrane, generating an electron current, Ie. Using the permeabilized patch voltage-clamp technique, we studied the temperature dependence of Ie in human eosinophils stimulated by phorbol myristate acetate (PMA) from room temperature to >37 degrees C. 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 37 degrees C was surprisingly weak: the Arrhenius activation energy Ea was only 14 kcal mol(-1) (Q10, 2.2). In contrast, steady-state NADPH oxidase activity was strongly temperature dependent at 20-30 degrees C, with Ea 25.1 kcal mol(-1) (Q10, 4.2). The maximum Ie measured at 34 degrees C was -30.5 pA. Above 30 degrees C, the temperature dependence of both Ie and O2- production was less pronounced. Above 37 degrees C, Ie was inhibited reversibly. After rapid temperature increases, a secondary increase in Ie ensued, suggesting that high temperature promotes assembly of additional NADPH oxidase complexes. Evidently, about twice as many NADPH oxidase complexes are active near 37 degrees C than at 20 degrees C. Thus, the higher Q10 of steady-state Ie 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 37 degrees C.
Morgan
Deri
D
Department of Molecular Biophysics and Physiology, Rush Presbyterian St Luke's Medical Center, Chicago, IL 60612, USA.
Cherny
Vladimir V
VV
Murphy
Ricardo
R
Xu
Wei
W
Thomas
Larry L
LL
DeCoursey
Thomas E
TE
D000328:Adult; D000465:Algorithms; D004594:Electrophysiology; D004804:Eosinophils / Q000201:enzymology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010101:Oxygen Consumption / Q000187:drug effects; D018408:Patch-Clamp Techniques; D013481:Superoxides / Q000378:metabolism; D013696:Temperature; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D013481:Superoxides; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate