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Infect Immun. 1984 December; 46(3): 631-634

Binding of pseudomonal leukocidin to rabbit polymorphonuclear leukocytes.

ABSTRACT

The leukocytotoxic toxin pseudomonal leukocidin, produced by Pseudomonas aeruginosa, was radioiodinated with chloramine-T reagent. Binding of [125I]leukocidin to rabbit polymorphonuclear leukocytes was found to be concentration dependent at 37 degrees C. A Scatchard plot of binding data was linear, indicating that leukocidin binds to a single population of sites. The dissociation constant, KD, was calculated from the Scatchard plot to be 2.5 X 10(-7) M, and the number of binding sites per leukocyte was approximately 4.4 X 10(5). Unlabeled leukocidin or antileukocidin antibody reduced the binding of [125I]leukocidin to the leukocytes. A leukocidin-binding protein was extracted from rabbit polymorphonuclear leukocytes with Triton X-100 and purified by leukocidin-Sepharose 4B affinity column chromatography. Approximately 60 micrograms of binding protein was obtained from 8.1 mg of material extracted from the leukocytes. The binding protein had a molecular weight of about 50,000 as shown by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and staining with silver nitrate. Under nondenaturing conditions, its molecular weight was also about 50,000, as shown by gel filtration-Sephadex G-200 chromatography. The 50,000-dalton protein purified in this way from rabbit polymorphonuclear leukocytes competitively inhibited the binding of leukocidin to leukocytes and inactivated leukocidin activity. With equimolar amounts of the 50,000-dalton protein and leukocidin, up to 90% inactivation of leukocidin was observed.