STRUCTURAL AND CATALYTIC PROPERTIES OF THE SOLUBILIZED SUPEROXIDE-GENERATING ACTIVITY OF HUMAN POLYMORPHONUCLEAR LEUKOCYTES - SOLUBILIZATION, STABILIZATION IN SOLUTION, AND PARTIAL CHARACTERIZATION

Purified human polymorphonuclear (PMN) leukocytes that had been incubated with opsonized zymosan particles or with phorbol myristate acetate to achieve optimal activation of the superoxide-generating activity were homogenized and centrifuged to recover the subcellular particles that contained 92-95% of the superoxide-generating activity of the homogenates. Centrifugation of the particle-associated superoxide-generating activity on 10-50% sucrose gradients resolved the predominant activity from established enzymatic and other markers for the lysosomal granules, mitochondria, nuclei, cytosol, and plasma membranes. Incubation of the particle-associated superoxide-generating activity with 0.25 g/100 mL deoxycholate in 0.34 M sucrose solubilized a mean of 12.4 and 13.8% of the total activity in the particles derived from PMN leukocytes that had been activated by opsonized zymosan and phorbol myristate acetate, respectively. The specific activity of the solubilized superoxide-generating activity was one-tenth to one-fifth that of the particle-associated superoxide-generating activity. Nonetheless, both preparations were similar with respect to the pH range of 6.4-7.6 for optimal activity, the Km value for NADPH which was lower than that for NADH, the resistance to inhibition by sodium azide, and the susceptibility to inhibition by p-(chloromercuri)-phenylsulfonic acid. The rapid loss of activity of solubilized superoxide-generating activity at 25 °C was reduced at 4 °C, and the half-time for decay at 4 °C was extended from 18 h to over 50 h by the addition of glycerol, ethylene glycol, or dimethyl sulfoxide. Filtration of solubilized superoxide-gen-erating activity on Sephacryl S-200 at 4 °C for 10 h resolved two peaks of Mr 150000 and >300000 that maintained the initial level of specific activity. Isoelectric focusing of solubilized superoxide-generating activity for 18-24 h at 4 °C in a glycerol gradient containing ethylene glycol defined a pI of 7.6-8.3. Human PMN leukocyte superoxide-generating activity thus can be solubilized and purified in quantities sufficient for studies of its biochemical characteristics. © 1979, American Chemical Society. All rights reserved.