SUBCELLULAR-LOCALIZATION OF THE SUPEROXIDE-FORMING ENZYME IN HUMAN NEUTROPHILS

The subcellular distribution of the superoxide (O2-)-forming enzyme in human neutrophils was investigated. Cells were activated by phorbol myristate acetate or by opsonized zymosan, and were then fractionated by zonal-rate sedimentation at 2 different speeds. At high speed, the specific granules were resolved from the azurophils and the membrane fraction, while at low speed, the azurophil granules could be separated from fast-sedimenting particle aggregates. Under both conditions, the major portion of the O2--forming activity (60-70% of the total) was associated with the membrane fraction which was characterized by the presence of alkaline phosphatase, alkaline phosphodiesterase I and acid aryl phosphatase. No significant O2--forming activity was found in either specific or azurophil granules. Some activity was present in the fastest sedimenting fractions which, as shown by EM, were heterogeneous and contained aggregated material which included membrane fragments. These fractionation results provide strong additional support for the current view that the activable O2--forming system is localized in the plasma membrane of human neutrophils.