Studies on the superoxide releasing site in plasma membranes of neutrophils with ESR spin-labels.

Superoxide (O2-)-generating membranes of pig blood neutrophils were studied by the ESR spin-label method. Neutrophils were spin-labeled with doxylstearic acids, consisting of nitroxide free radicals bonded to the 5, 7, 12, or 16 position of stearic acid (5-, 7-, 12-, or 16-DS), to detect the reduction of their nitroxide radicals at different positions in the membrane. The spin-labeled cells were then stimulated with phorbol myristate acetate (PMA). Stimulation of the labeled cells resulted in a marked decrease in the spin concentration of 5-DS due to the reduction by O2-, but not in those of the other three DS labels. This reduction of 5-DS was completely inhibited by copper salicylate (CS), a hydrophobic and permeable O2(-)-scavenger, but not by superoxide dismutase (SOD). CS was not inhibitory on the respiratory burst, i.e., O2(-)-generating activity of neutrophils. On the contrary, if the spin-labels were present in the extracellular medium, SOD inhibited the reduction of all four DS labels due to O2- released from PMA-stimulated cells. These results suggest that the O2(-)-releasing site is not located at the outer surface of the plasma membrane but in an inner hydrophobic environment a short distance (around 4-5 A) from its outer surface.