Detergent solubilization of the formyl peptide chemotactic receptor. Strategy based on covalent affinity labeling.
The formyl peptide chemotactic receptor has been solubilized by digitonin treatment of purified human neutrophil membranes. Of several potential assay methods tested for their ability to separate receptor-bound from free ligand, only gel filtration through an acrylamide cross-linked agarose matrix yielded satisfactory results. Approximately 70% of the receptor initially present in the membrane was recovered in the digitonin extract. Binding of 125I-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys to the soluble receptor was rapid (t 1/2 at 22 degrees C less than 5 min), of high affinity (Kd = 2.2 nM) and saturable. The relative potencies of a small series of peptides as inhibitors of binding to the soluble receptor paralleled their potencies as inhibitors of the membrane-bound receptor. N-Formylation of the peptides was required for high affinity binding. Binding was maximal at pH 6.5 and was sulfhydryl-dependent; 20 microM p-chloromercuriphenylsulfonic acid decreased binding by 50%. 125I-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys was specifically cross-linked to the soluble receptor with ethylene glycol bis(succinimidyl succinate) and an apparent molecular weight of 55,000 to 70,000 and determined for the soluble receptor by sodium dodecyl sulfate polyacrylamide gel electrophoresis. A strategy for obtaining an active, detergent-soluble receptor preparation based on covalent affinity labeling is presented.
D000345:Affinity Labels; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009504:Neutrophils / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D044042:Receptors, Formyl Peptide
D000345:Affinity Labels; D009842:Oligopeptides; D011956:Receptors, Cell Surface; D044042:Receptors, Formyl Peptide