Solubilization and cleavage of human neutrophil (N) affinity-labeled receptors for leukotriene B4 (LTB4)

LTB4 chemotactic receptors in purified N plasma membranes (PMs) have been affinity-labeled with [3H]-C-1 aminopropylamide-LTB4 ([3H]APA-LTB4) by disuccinimidyl suberate (DSS) cross-linking. Intact Ns were pretreated with diisopropylfluorophosphate, suspended at 107/ml in Hanks' solution-10 mM HEPES (pH 7.4), incubated for 30 min at 40C with 30 nM [3H]APA-LTB4 and 25 min with 1 mM bis[2-(succinimidooxycarbonyloxy)-ethyl] sulfone, an impermeant analog of DSS, and sonified for 30 sec at 40C. The 10,000 g supernatant of the sonicate was centrifuged at 40,000 g for 30 min at 40C on a discontinuous gradient of 10-50 g % sucrose, from which a mean of 78% of the radiolabel was recovered with PM markers. The extent and specificity of labeling of intact N receptors were similar to those of receptors in PMs. The radioactively-labeled receptors appeared as a single band of 35-40 kd in sodium dodecyl sulfate (SDS) 10 g % polyacrylamide gel electrophoresis. Cleavage of radiolabeled receptors with 1 mg/ml of cyanogen bromide in 70% formic acid for 18 hr at room temperature or with 30 mM HCl under N2 for 4 hr at 1050C converted a mean of 18-32% of the radioactivity to a band of 14 kd in SDS-15 g % PAGE. N receptors for LTB4, thus, are localized in the PM and can be isolated for structural studies