Isolation and partial characterization of the membrane protein constituents of human neutrophil receptors for chemotactic formylmethionyl peptides

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Abstract
Plasma membranes of human neutrophils were solubilized in buffer containing a nonionic detergent and applied to a formylmethionylleucylphenylalanine (fMet-Leu-Phe)-Sepharose column that was washed and eluted with the chemotactic peptide fMet-Leu-Phe. Analysis of the eluate by filtration on Bio-Gel P150 in sodium dodecyl sulfate (Na-DodSO4) buffer and by NaDodSO4-polyacrylamide gel electrophoresis revealed 3 predominant membrane proteins of approximate MW 94,000 (MP-1), 68,000 (MP-2), and 40,000 (MP-3), of which MP-2 accounted for 74-93% of the total protein. Purified MP-1 and MP-2 contained an above average content of hydrophobic amino acids, while MP-2 and MP-3 had an above average content of acid and/or amide amino acids and a below average content of basic amino acids. MP-2 and MP-3, but not MP-1, bound [3H]fMet-Leu-Phe in equilibrium dialysis chambers. Both MP-2 and MP-3 exhibited high-affinity sites with a valence of 0.2-0.3 and mean Ka values of 9 .times. 108 and 2 .times. 107 M-1, respectively, and low-affinity sites with a valence of 0.3-0.5 and mean Ka values of 3 .times. 107 and 2 .times. 106 M-1 (n = 3). The specificity of the binding of fMet-Leu-Phe was suggested by the failure of MP-2 and MP-3 to bind lipid chemotactic factors and to adhere to a Sepharose column to which had been coupled chemotactic fragments of the 5th component of complement. A series of synthetic formylmethionyl peptides exhibited the same rank order of potency as inhibitors of the binding of [3H]fMet-Leu-Phe by MP-2 and as stimuli of neutrophil chemotaxis. Membrane proteins isolated by fMet-Leu-Phe-Sepharose affinity chromatography may represent constituents of specific human neutrophil receptors for chemotactic peptides.

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